U.S. patent application number 15/460109 was filed with the patent office on 2017-09-21 for nucleic acid amplification and detection devices, systems and methods.
The applicant listed for this patent is Abbott Laboratories. Invention is credited to Ali Attarwalla, Michael Giraud, Matthew J. Hayes, Michael S. Hazell, Dean Khan, Sonal Sadaria Nana, Timothy J. Patno, Eric B. Shain, Eric D. Yeaton.
Application Number | 20170266668 15/460109 |
Document ID | / |
Family ID | 59847494 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170266668 |
Kind Code |
A1 |
Nana; Sonal Sadaria ; et
al. |
September 21, 2017 |
Nucleic Acid Amplification and Detection Devices, Systems and
Methods
Abstract
The instant disclosure provides nucleic acid amplification
systems and multi-reaction analysis systems useful in the efficient
processing of samples, including clinical samples. Integrated
systems that include nucleic acid amplification devices
functionally combined with multi-reaction analysis systems are also
included. Also provided are methods for monitoring multiple
concurrent nucleic acid amplification reactions that include the
use of devices and systems described herein.
Inventors: |
Nana; Sonal Sadaria;
(Chicago, IL) ; Shain; Eric B.; (Glencoe, IL)
; Hazell; Michael S.; (Cambridge, GB) ; Yeaton;
Eric D.; (Epsom, NH) ; Giraud; Michael;
(Dallas, TX) ; Hayes; Matthew J.; (Great Shelford,
GB) ; Patno; Timothy J.; (Barrington, IL) ;
Attarwalla; Ali; (Mount Prospect, IL) ; Khan;
Dean; (Forest Park, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories |
Abbott Park |
IL |
US |
|
|
Family ID: |
59847494 |
Appl. No.: |
15/460109 |
Filed: |
March 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62308632 |
Mar 15, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/12 20130101;
B01L 2300/1894 20130101; B01L 2300/0609 20130101; B01L 2300/0654
20130101; B01L 2300/1822 20130101; B01L 2300/0645 20130101; B01L
2300/16 20130101; B01L 7/52 20130101; B01L 2200/147 20130101 |
International
Class: |
B01L 7/00 20060101
B01L007/00 |
Claims
1. A thermal block for simultaneous nucleic acid amplification and
reaction analysis; the thermal block comprising: a) at least two
reaction vessel wells, each well comprising: i) a top opening
configured to receive a reaction vessel inserted vertically into
the well; ii) a side aperture configured to allow light to pass
laterally into the reaction vessel well, wherein upon insertion of
a reaction vessel into the well a majority of the sidewall of the
reaction vessel is in thermal contact with the well and a portion
of the sidewall is exposed to light by the aperture; b) a thermal
transfer surface opposite the side apertures of the two reaction
vessel wells; and c) a mounting hole positioned between the at
least two reaction vessel wells and having a center axis
perpendicular to the plane of the thermal transfer surface.
2. The thermal block of claim 1, wherein the block is bilaterally
symmetrical along a vertical axis.
3. The thermal block of claim 1, wherein the thermal transfer
surface is essentially flat.
4. The thermal block of claim 1, wherein the mounting hole is
positioned equidistant from the two reaction vessels.
5. The thermal block of claim 4, wherein the mounting hole is
centrally positioned between the top and bottom sides of the
thermal block.
6. The thermal block of claim 4, wherein the mounting hole is
centrally positioned between the right and left sides of the
thermal block.
7. The thermal block of claim 1, wherein the top surface of the
thermal block comprises a raised flange encircling the
circumference of each of the two reaction vessel wells.
8. The thermal block of claim 1, wherein the surface of the thermal
block opposite the thermal surface comprises a plurality of raised
ridges.
9. The thermal block of claim 8, wherein the plurality of raised
ridges emanate radially from the mounting hole.
10. The thermal block of claim 1, wherein the thermal block is
constructed of aluminum.
11. The thermal block of claim 1, wherein at least the two reaction
vessel wells are nickel plated.
12. The thermal block of claim 11, wherein at least a portion of
the thermal block other than the two reaction vessel wells is
nickel plated.
13. The thermal block of claim 12, wherein the entire thermal block
is nickel plated.
14. The thermal block of claim 1, wherein the two reaction vessel
wells comprise a lubrication coating.
15. The thermal block of claim 14, wherein the lubrication coating
is a dry lubrication coating.
16. The thermal block of claim 1, wherein the thermal block further
comprises a temperature detection area configured for the
functional attachment of a temperature detector positioned
proximally to each reaction vessel well.
17. The thermal block of claim 16, wherein the temperature
detection areas are positioned below the reaction vessel wells.
18. The thermal block of claim 1, wherein each reaction vessel well
further comprises a basal reservoir configured such that upon
insertion of the reaction vessel into the well the reaction vessel
does not contact the bottom of the well.
19. The thermal block of claim 1, wherein the thermal block has a
mass of 2 to 4 grams.
20. A nucleic acid amplification module, the module comprising: a)
a thermoelectric cooler unit comprising a mounting hole; b) a
thermal block of claim 1, wherein the thermal transfer surface is
in thermal contact with a first surface of the thermoelectric
cooler unit; and c) a heatsink configured to receive a mechanical
fastener, wherein the heatsink is in thermal contact with a second
surface of the thermoelectric cooler unit and the mounting holes
are aligned such that the thermal block, thermoelectric cooler
unit, and the heatsink are joined by a mechanical fastener
positioned through the mounting holes and affixed to the
heatsink.
21. The module of claim 20, further comprising a conductive pad
between the thermal block and the thermoelectric cooler unit or
between the thermoelectric cooler unit and the heatsink.
22. The module of claim 21, wherein the conductive pad is a
graphite pad.
23. The module of claim 20, wherein the module comprises conductive
pads both between the thermal block and the thermoelectric cooler
unit and between the thermoelectric cooler unit and the
heatsink.
24. The module of claim 20, wherein the mechanical fastener joins
the thermal block, thermoelectric cooler unit, and the heatsink by
a compression force.
25. The module of claim 24, wherein the mechanical fastener is a
compression screw.
26. The module of claim 24, wherein the compression force is
between 100 and 200 pounds per square inch (psi).
27. The module of claim 20, wherein the thermal block and the
thermoelectric cooler are supported by a support bar fastened to
the heatsink.
28. The module of claim 20, wherein the module further comprises a
heatsink fan configured to force air past the heatsink.
29. The module of claim 28, wherein the heatsink is joined to the
heatsink fan by a duct.
30. The module of claim 20, wherein the thermal block has an
operating thermal slew rate of greater than 5.degree. C. per
second.
31. The module of claim 20, wherein the module further comprises
one or more resistance thermometers (RTDs) in thermal contact with
the thermal block.
32. The module of claim 31, wherein the module comprises two RTDs
in thermal contact with the thermal block, wherein each of the two
RTDs are in proximity with a reaction vessel well of the thermal
block.
33. The module of claim 31, wherein the thermal contact between the
one or more RTDs and the thermal block is maintained by a
cantilever bar comprising one or more cantilever arms.
34. The module of claim 20, wherein the module comprises an
attached printed circuit board (PCB) for monitoring or controlling
at least one electrical component of the module.
35. The module of claim 34, wherein the PCB is conformal
coated.
36. The module of claim 31, wherein the module comprises at least
one RTD in thermal contact with the thermal block and electrically
connected to the PCB.
37. The module of claim 20, wherein the module comprises a RTD in
thermal contact with the thermal block or the heatsink, wherein the
RTD in thermal contact with the thermal block or the heatsink is
configured to monitor the temperature of the thermal block or
heatsink and trigger a cutoff of power to the thermal block or
heatsink if the temperature indicates a thermal error
condition.
38. The module of claim 20, wherein the heatsink is configured to
receive a second mechanical fastener and the nucleic acid
amplification module further comprises a second thermal block in
thermal contact with a second thermoelectric cooler unit in thermal
contact with the heatsink, wherein the second thermal block, the
second thermoelectric cooler unit and the heatsink are joined by a
second mechanical fastener positioned through mounting holes in the
second thermal block and the second thermoelectric cooler unit and
affixed to the heatsink.
39. The module of claim 38, wherein the first thermal block and the
second thermal block comprise separate electrical connections and
are controlled independently.
40. The module of claim 20, wherein the module further comprises
one or more reaction vessel clamping bars.
41. The module of claim 40, wherein the one or more reaction vessel
clamping bars provides a compression force on reaction vessels
within the reaction vessel wells of 5 N or more.
42-72. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn.119(e), this application claims
priority to the filing date of the U.S. Provisional Patent
Application Ser. No. 62/308,632, filed Mar. 15, 2016, the
disclosure of which application is herein incorporated by
reference.
BACKGROUND
[0002] The majority of clinical decisions are based on laboratory
and health test data. An increasing variety of tests and a demand
for clinical laboratories to process a greater volume of samples
creates strain on testing facilities, personnel and equipment. In
addition, health care testing needs are unpredictable on a
day-to-day and even hour-to-hour basis making the efficient
scheduling of test sample processing a challenging endeavor. In
addition, the frequent occurrence of tests requiring rush or STAT
processing in the clinical setting adds additional difficulties and
can result in inefficient testing resource allocation.
[0003] Clinical testing facilities utilizing nucleic acid
amplification based methods are not immune to such pressures. On
the contrary the variety of nucleic acid based diagnostic tests is
growing and demand from clinicians and patients for such tests is
also expanding. There is a burden on clinical testing facilities to
efficiently perform a wider range of different nucleic acid
amplification based tests while maintaining the versatility to
process rush samples out of order when they unexpectedly
arrive.
SUMMARY
[0004] Aspects of the instant disclosure include nucleic acid
amplification systems, thermal blocks for use therein,
multi-reaction analysis systems useful in the efficient processing
of samples, integrated systems that include nucleic acid
amplification devices functionally combined with multi-reaction
analysis systems and methods for monitoring multiple concurrent
nucleic acid amplification reactions that include the use of such
devices and systems.
[0005] Aspects of the instant disclosure include a thermal block
for simultaneous nucleic acid amplification and reaction analysis;
the thermal block comprising: a) two or more reaction vessel wells,
each well comprising: i) a top opening configured to receive a
reaction vessel inserted vertically into the well; ii) a side
aperture configured to allow light to pass laterally into the
reaction vessel well, wherein upon insertion of a reaction vessel
into the well a majority of the sidewall of the reaction vessel is
in thermal contact with the well and a portion of the sidewall is
exposed to light by the aperture; b) a thermal transfer surface
opposite the side apertures of the two reaction vessel wells; and
c) a mounting hole positioned between the two reaction vessel wells
and having a center axis perpendicular to the plane of the thermal
transfer surface.
[0006] In some instances, aspects of the thermal block include
where the block is bilaterally symmetrical along a vertical axis.
In some instances, aspects of the thermal block include where the
thermal transfer surface is essentially flat. In some instances,
aspects of the thermal block include where the mounting hole is
positioned equidistant from the two reaction vessels. In some
instances, aspects of the thermal block include where the mounting
hole is centrally positioned between the top and bottom sides of
the thermal block. In some instances, aspects of the thermal block
include where the mounting hole is centrally positioned between the
right and left sides of the thermal block. In some instances,
aspects of the thermal block include where the top surface of the
thermal block comprises a raised flange encircling the
circumference of each of the two reaction vessel wells. In some
instances, aspects of the thermal block include where the surface
of the thermal block opposite the thermal surface comprises a
plurality of raised ridges, including where the plurality of raised
ridges emanate radially from the mounting hole. In some instances,
aspects of the thermal block include where the thermal block is
constructed of aluminum. In some instances, aspects of the thermal
block include where at least the two reaction vessel wells are
nickel plated, at least a portion of the thermal block other than
the two reaction vessel wells is nickel plated and/or the entire
thermal block is nickel plated. In some instances, aspects of the
thermal block include where the two reaction vessel wells comprise
a lubrication coating, including wherein the lubrication coating is
a dry lubrication coating. In some instances, aspects of the
thermal block include a temperature detection area configured for
the functional attachment of a temperature detector positioned
proximally to each reaction vessel well, including where the
temperature detection areas are positioned below the reaction
vessel wells. In some instances, aspects of the thermal block
include where each reaction vessel well further comprises a basal
reservoir configured such that upon insertion of the reaction
vessel into the well the reaction vessel does not contact the
bottom of the well. In some instances, aspects of the thermal block
include where the thermal block has a mass of 2 to 4 grams.
[0007] Aspects of the instant disclosure include a nucleic acid
amplification module, the module comprising: a) a thermoelectric
cooler unit comprising a mounting hole; b) a thermal block (e.g.,
as described above), wherein the thermal transfer surface is in
thermal contact with a first surface of the thermoelectric cooler
unit; and c) a heatsink configured to receive a mechanical
fastener, wherein the heatsink is in thermal contact with a second
surface of the thermoelectric cooler unit and the mounting holes
are aligned such that the thermal block, thermoelectric cooler
unit, and the heatsink are joined by a mechanical fastener
positioned through the mounting holes and affixed to the
heatsink.
[0008] In some instances, aspects of the nucleic acid amplification
module include a conductive pad between the thermal block and the
thermoelectric cooler unit or between the thermoelectric cooler
unit and the heatsink, including wherein the conductive pad is a
graphite pad. In some instances, aspects of the nucleic acid
amplification module include where the module comprises conductive
pads both between the thermal block and the thermoelectric cooler
unit and between the thermoelectric cooler unit and the heatsink.
In some instances, aspects of the nucleic acid amplification module
include where the mechanical fastener joins the thermal block,
thermoelectric cooler unit, and the heatsink by a compression
force, including wherein the mechanical fastener is a compression
screw. In some instances, aspects of the nucleic acid amplification
module include where the compression force is between 100 and 200
pounds per square inch (psi). In some instances, aspects of the
nucleic acid amplification module include where the thermal block
and the thermoelectric cooler are supported by a support bar
fastened to the heatsink. In some instances, aspects of the nucleic
acid amplification module include a heatsink fan configured to
force air past the heatsink, including wherein the heatsink is
joined to the heatsink fan by a duct. In some instances, aspects of
the nucleic acid amplification module include where the thermal
block has an operating thermal slew rate of greater than 5.degree.
C. per second. In some instances, aspects of the nucleic acid
amplification module include one or more resistance thermometers
(RTDs) in thermal contact with the thermal block. In some
instances, aspects of the nucleic acid amplification module include
two RTDs in thermal contact with the thermal block, wherein each of
the two RTDs are in proximity with a reaction vessel well of the
thermal block. In some instances, aspects of the nucleic acid
amplification module include an attached printed circuit board
(PCB) for monitoring or controlling at least one electrical
component of the module, including wherein the PCB is conformal
coated. In some instances, aspects of the nucleic acid
amplification module include at least one RTD in thermal contact
with the thermal block and electrically connected to the PCB. In
some instances, aspects of the nucleic acid amplification module
include a RTD in thermal contact with the thermal block or the
heatsink, wherein the RTD in thermal contact with the thermal block
or the heatsink is configured to monitor the temperature of the
thermal block or heatsink and trigger a cutoff of power to the
thermal block or heatsink if the temperature indicates a thermal
error condition. In some instances, aspects of the nucleic acid
amplification module include where the heatsink is configured to
receive a second mechanical fastener and the nucleic acid
amplification module further comprises a second thermal block in
thermal contact with a second thermoelectric cooler unit in thermal
contact with the heatsink, wherein the second thermal block, the
second thermoelectric cooler unit and the heatsink are joined by a
second mechanical fastener positioned through mounting holes in the
second thermal block and the second thermoelectric cooler unit and
affixed to the heatsink. In some instances, aspects of the nucleic
acid amplification module include where the first thermal block and
the second thermal block comprise separate electrical connections
and are controlled independently. In some instances, aspects of the
nucleic acid amplification module includes one or more reaction
vessel clamping bars, including where the one or more reaction
vessel clamping bars provides a compression force on reaction
vessels within the reaction vessel wells of 5 N or more.
[0009] Aspects of the instant disclosure include a multi-reaction
analysis module for the optical analysis of a plurality of
amplification reaction vessels, the module comprising: a) an optics
detection unit comprising an optical signal processor and a
plurality of linearly arranged optical blocks, each optical block
comprising: i) an illumination component configured to illuminate a
reaction with excitation light; ii) a optics block aperture
configured to pass excitation light to the reaction vessel and
receive emission light from the reaction vessel; iii) a measurement
channel configured to pass emission light to the optical signal
processor; and iv) a reference channel configured to pass reference
light to the optical signal processor; b) a linear conveyer
configured to convey the optics detection unit linearly past each
reaction vessel of the plurality of amplification reactions,
wherein each optics block aperture of the plurality of linearly
arranged optical blocks is optically exposed to the sidewall of
each reaction vessel.
[0010] In some instances, aspects of the multi-reaction analysis
module include where the illumination component comprises one or
more light emitting diode (LED) emitters or two or more light
emitting diode (LED) emitters of different emission wavelengths,
including where the wavelengths of the two or more LED emitters are
at least 50 nm apart. In some instances, aspects of the
multi-reaction analysis module include where the illumination
component comprises frequency modulation. In some instances,
aspects of the multi-reaction analysis module include where the
illumination component comprises time division modulation. In some
instances, aspects of the multi-reaction analysis module include
where the optics detection unit comprises two or more optical
blocks or three optical blocks.
[0011] Aspects of the instant disclosure include an integrated
multi-reaction nucleic acid amplification and analysis system, the
system comprising: a) a nucleic acid amplification module and a
multi-reaction analysis module, wherein the spacing between the
side apertures of the reaction vessel wells is unequal to the
spacing between the optics block apertures of the optical blocks
such that no more than one reaction vessel side aperture and one
optics block aperture may be in alignment at any one time.
[0012] In some instances, aspects of the integrated multi-reaction
nucleic acid amplification and analysis system include where the
spacing between the side apertures is greater than the spacing
between the optics block apertures. In some instances, aspects of
the integrated multi-reaction system include where the spacing
between the side apertures is less than the spacing between the
optics block apertures. In some instances, aspects of the
integrated multi-reaction system include two thermal blocks affixed
to a single heatsink. In some instances, aspects of the integrated
multi-reaction system include wherein the multi-reaction analysis
module comprises three optical blocks. In some instances, aspects
of the integrated multi-reaction system include where each
illumination component comprises two or more LED emitters of
differing wavelengths, including wherein the wavelengths of the two
or more LED emitters are at least 50 nm apart.
[0013] Aspects of the instant disclosure include an integrated
multi-reaction nucleic acid amplification and analysis system, the
system comprising: a) a multi-reaction nucleic acid amplification
module comprising two or more thermal blocks comprising two or more
linearly arranged and evenly spaced reaction vessel wells each
having a side aperture configured to allow light to pass laterally
into the reaction vessel well; b) a traveling optics detection unit
comprising: i) an optical signal processor; ii) a plurality of
linearly arranged evenly spaced optical blocks, each optical block
comprising an illumination component and a optics block aperture
configured to pass excitation light to a reaction vessel and
receive emission light from the reaction vessel; and iii) a linear
conveyer configured to convey the traveling optics detection unit
linearly past the side aperture of each of the two or more reaction
vessel wells, wherein the spacing between the side apertures of the
reaction vessel wells is unequal to the spacing between the optics
block apertures of the optical blocks such that no more than one
side aperture and one optics block aperture may be in alignment at
any one time.
[0014] In some instances, aspects of the integrated multi-reaction
system include where the traveling optics detection unit comprises
three optical blocks. In some instances, aspects of the integrated
multi-reaction system include where the illumination component
comprises two or more LED emitters of differing wavelengths,
including where the wavelengths of the two or more LED emitters are
at least 50 nm apart.
[0015] Aspects of the instant disclosure include a method of
monitoring nucleic acid amplification in a plurality of
amplification reaction vessels, the method comprising: a) linearly
scanning a traveling optics detection unit having an excitation
component and a plurality of optics block apertures past the
plurality of amplification reaction vessels, wherein no more than
one optics block aperture is in optical alignment with a
amplification reaction vessel at any one time; b) receiving a scan
signal from the optics detection unit comprising background noise
and emission peaks; c) determining emission peaks within the
background noise according to a plurality of reaction vessel
windows; d) measuring an intensity value for each reaction vessel
window to monitor the amplification in each reaction vessel of the
plurality.
[0016] In some instances, aspects of the method include where the
optics unit comprises a plurality of excitation components that are
time modulated. In some instances, aspects of the method include
where the optics unit comprises a plurality of excitation
components that are frequency modulated. In some instances, aspects
of the method include where each optics block aperture is part of
an optics block and the method further comprises cross-talk
subtraction. In some instances, aspects of the method include where
the method further comprises one or more calibration measurement
steps before or during the linear scanning step. In some instances,
aspects of the method include where the one or more calibration
measurement steps comprise aligning the plurality of optics block
apertures with a dark target, including where the dark target
comprises black polycarbonate. In some instances, aspects of the
method include where the one or more calibration measurement steps
comprises toggling the excitation component on or off. In some
instances, aspects of the method include where the one or more
calibration measurement steps comprises measuring a reference
channel and adjusting the power supplied to the excitation
component based on the measured reference channel. In some
instances, aspects of the method include where at least one of the
one or more calibration measurement steps is performed at least
once per scan. In some instances, aspects of the method include
where at least one of the one or more calibration measurement steps
is performed at least twice per scan. In some instances, aspects of
the method include where the measurement from the calibration
measurement step is applied to a value obtained from the scan
during a signal processing pathway.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 depicts one embodiment of a thermal block as
described herein.
[0018] FIG. 2 provides an alternative view of the thermal block
depicted in FIG. 1 and a corresponding reaction vessel for which
the thermal block is configured.
[0019] FIG. 3 depicts an embodiment of a partially mounted thermal
block as described herein with capped reaction vessels loaded into
the reaction vessel wells.
[0020] FIG. 4 depicts an embodiment of a thermoelectric cooler for
use in a nucleic acid amplification device as described herein.
[0021] FIG. 5 depicts one embodiment of a mounted pair of thermal
blocks fastened to a single heat sink as described herein.
[0022] FIG. 6 depicts one embodiment of an assembled nucleic acid
amplification device as described herein with associated
thermoregulatory components.
[0023] FIG. 7 provides a partially exploded view of the embodiment
of an assembled nucleic acid amplification device with associated
thermoregulatory components as depicted in FIG. 6.
[0024] FIG. 8 provides a functional block diagram of an integrated
amplification and detection unit as described herein.
[0025] FIG. 9 depicts the optical aperture (i.e., optics block
aperture) side of an embodiment of an optics detection unit as
described herein.
[0026] FIG. 10 provides an alternative view of the embodiment of
the optics detection unit depicted in FIG. 9.
[0027] FIG. 11 provides an internal view of an embodiment of an
optical block as described herein.
[0028] FIG. 12 provides an exploded view of the internal components
of an embodiment of an optical block as described herein.
[0029] FIG. 13 provides a schematic cut away representation of the
reference channel and the measurement channel light paths according
to an embodiment of an optical block as described herein.
[0030] FIG. 14 provides a cut away view of an embodiment of
integrated system that combines an amplification unit and a
multi-reaction analysis unit as described herein.
[0031] FIG. 15 depicts an embodiment of a linear conveyor as
described herein.
[0032] FIG. 16 demonstrates the peak pattern generated during a
lateral scan of a reaction vessel according to an embodiment of the
instant disclosure.
[0033] FIG. 17 depicts a dark target according to an embodiment of
the instant disclosure.
[0034] FIG. 18 provides a schematic representation of a dark target
measurement according to an embodiment of the instant
disclosure.
[0035] FIG. 19 depicts an embodiment of a reaction vessel clamping
and extraction device as described herein.
[0036] FIG. 20 depicts the circuit boards of an optics detection
unit according to an embodiment of the instant disclosure.
[0037] FIG. 21 provides a block diagram schematizing the
communication between components of an embodiment of an optics
detection unit as described here.
[0038] FIG. 22 depicts one embodiment of 2-way time division
multiplexing as described herein.
[0039] FIG. 23 provides a schematic representation of a
multi-reaction vessel scan performed by an integrated system
according to an embodiment as described herein.
[0040] FIG. 24 provides a schematic representation of a signal
processing pathway according to one embodiment as described
herein.
[0041] FIG. 25 provides a schematic representation of a signal
processing pathway according to one embodiment as described
herein.
[0042] FIG. 26 depicts positions along a signal processing pathway
that calibration measurements may be applied according to an
embodiment as described herein.
[0043] FIG. 27 depicts an embodiment of a nucleic acid
amplification system with attached cantilever bar having a
plurality of cantilever arms as described herein.
[0044] FIG. 28 depicts a frontal view of an embodiment of a nucleic
acid amplification system with attached cantilever bar having a
plurality of cantilever arms as described herein.
[0045] FIG. 29 depicts a profile view of an embodiment of a
cantilever bar having a cantilever arm as described herein.
DEFINITIONS
[0046] The term "analyte" as used herein refers to a target
molecule to be detected in a sample wherein detection of the
analyte may be indicative of a biological state of the organism
from which the sample was derived. For example, where an analyte is
a nucleic acid analyte, detection of the nucleic acid analyte may
be indicative of a biological state of the organisms from which the
sample was derived including e.g., where detection of viral nucleic
acid may indicate infection with a particular pathogen, etc.
[0047] The term "reaction vessel" as used herein generally
referrers to a container within which an amplification reaction is
performed. Such reaction vessels may be obtained from commercial
sources, e.g., as off-the-shelf components, or may be custom
manufactured. Reaction vessels useful in nucleic acid amplification
reactions will generally be capable of rapidly transferring heat
across the vessel, e.g., through the use of highly conductive
materials (e.g., thermally conductive plastics) or physical
modifications of the vessel (e.g., thin walls). Common reaction
vessels include but are not limited to e.g., tubes, vials,
multi-well plates, and the like. Reaction vessels may be
constructed of a variety of materials including but not limited to
e.g., polymeric materials. In some instances, a reaction vessel of
the instant disclosure may be a reaction vessel and or reaction
vessel system as described in e.g., Attorney Docket No. ADDV-056WO,
which claims priority to U.S. Ser. No. 62/308,620, the disclosures
of which are incorporated herein by reference in their entireties.
In some instances, a component of a device or system or a method as
described herein may be configured for use with or to be applicable
with a reaction vessel and/or reaction vessel system as described
in e.g., Attorney Docket No. ADDV-056WO, which claims priority to
U.S. Ser. No. 62/308,620. The tem "polymeric materials" as used
herein includes, e.g., plastics, resins, etc., and other materials
generated by the joining of unit structures, e.g., in linear or
branched form. Useful polymeric materials may include but are not
limited to e.g., those commonly used in research and industrial
settings, including but not limited to: acetal, cyclic olefin
copolymer, ethylene propylene diene monomer rubber, ethylene
propylene rubber, ethylene-chlorotrifluoroethylene copolymer
(Halar.RTM.), ethylene-tetrafluoroethylene (Tefzel), fluorinated
ethylene propylene (Teflon.RTM.), fluorinated polyethylene, high
impact polystyrene, high-density polyethylene, low-density
polyethylene, modified polyphenylene ether, Permanox,
polyaryletherketone (PAEK) family polymeric materials (e.g.,
polyether ether ketone (PEEK) and the like), polycarbonate,
polyetherimide, polyethylene teraphthalate, polyethylene
terephthalate copolymer, polyfluoroalkoxy (Teflon.RTM.), polymethyl
methacrylate (acrylic), polymethylpentene, polypropylene,
polypropylene copolymer, polystyrene, polysulfone,
polyvinylidenedifluoride, ResMer.TM., styrene acrylonitrile,
tetrafluoroethylene, tetrafluoroethylene (Teflon.RTM.), Thermanox,
thermoplastic elastomer, thermoplastic polyester polyurethane,
Tritan.TM., cyclic olefin polymer (COP), and the like.
[0048] The term "assessing" includes any form of measurement, and
includes determining if an element is present or not. The terms
"determining", "measuring", "evaluating", "assessing" and
"assaying" are used interchangeably and include quantitative and
qualitative determinations. Assessing may be relative or absolute.
"Assessing the identity of" includes determining the most likely
identity of a particular compound or formulation or substance,
and/or determining whether a predicted compound or formulation or
substance is present or absent. "Assessing the quality of" includes
making a qualitative or quantitative assessment of quality e.g.,
through the comparisons of a determined value to a reference or
standard of known quality.
[0049] The term "bodily fluid" as used herein generally refers to
fluids derived from a "biological sample" which encompasses a
variety of sample types obtained from an individual or a population
of individuals and can be used in a diagnostic, monitoring or
screening assay. The definition encompasses blood and other liquid
samples of biological origin. The definition also includes samples
that have been manipulated in any way after their procurement, such
as by mixing or pooling of individual samples, treatment with
reagents, solubilization, or enrichment for certain components,
such as nucleated cells, non-nucleated cells, pathogens, etc.
[0050] The term "biological sample" encompasses a clinical sample,
and also includes cells in culture, cell supernatants, cell
lysates, serum, plasma, biological fluid, and tissue samples. The
term "biological sample" includes urine, saliva, cerebrospinal
fluid, interstitial fluid, ocular fluid, synovial fluid, blood
fractions such as plasma and serum, and the like.
[0051] The terms "control", "control assay", "control sample" and
the like, refer to a sample, test, or other portion of an
experimental or diagnostic procedure or experimental design for
which an expected result is known with high certainty, e.g., in
order to indicate whether the results obtained from associated
experimental samples are reliable, indicate to what degree of
confidence associated experimental results indicate a true result,
and/or to allow for the calibration of experimental results. For
example, in some instances, a control may be a "negative control"
assay such that an essential component of the assay is excluded
such that an experimenter may have high certainty that the negative
control assay will not produce a positive result. In some
instances, a control may be "positive control" such that all
components of a particular assay are characterized and known, when
combined, to produce a particular result in the assay being
performed such that an experimenter may have high certainty that
the positive control assay will not produce a positive result.
Controls may also include "blank" samples, "standard" samples
(e.g., "gold standard" samples), validated samples, etc.
[0052] The term "inputting", as used herein, is used to refer to
any way of entering information into a computer, such as, e.g.,
through the use of a user interface. For example, in certain cases,
inputting can involve selecting a reference spectrum or a spectral
characteristic or library thereof that is already present on a
computer system. In other cases, inputting can involve adding a
spectrum or a spectral characteristic to a computer system, e.g.,
by measuring the spectrum of a sample on a device capable of
interfacing with a computer. Inputting can also be done using a
user interface.
[0053] By "data processing unit", as used herein, is meant any
hardware and/or software combination that will perform the
functions required of it. For example, any data processing unit
herein may be a programmable digital microprocessor such as
available in the form of an electronic controller, mainframe,
server or personal computer (desktop or portable). Where the data
processing unit is programmable, suitable programming can be
communicated from a remote location to the data processing unit, or
previously saved in a computer program product (such as a portable
or fixed computer readable storage medium, whether magnetic,
optical or solid state device based).
DETAILED DESCRIPTION
[0054] The instant disclosure provides nucleic acid amplification
systems and multi-reaction analysis systems useful in the efficient
processing of samples, including clinical samples. Integrated
systems that include nucleic acid amplification devices
functionally combined with multi-reaction analysis systems are also
included. Also provided are methods for monitoring multiple
concurrent nucleic acid amplification reactions that include the
use of devices and systems described herein.
[0055] Before the present invention is described in greater detail,
it is to be understood that this invention is not limited to
particular embodiments described, as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0056] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0057] Certain ranges are presented herein with numerical values
being preceded by the term "about." The term "about" is used herein
to provide literal support for the exact number that it precedes,
as well as a number that is near to or approximately the number
that the term precedes. In determining whether a number is near to
or approximately a specifically recited number, the near or
approximating un-recited number may be a number which, in the
context in which it is presented, provides the substantial
equivalent of the specifically recited number.
[0058] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0059] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0060] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0061] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
Methods
[0062] The instant disclosure provides methods of amplifying a
target nucleic acid, i.e., nucleic acid analyte, present in a
sample and detecting a target nucleic acid by monitoring a nucleic
acid amplification reaction. Methods of the instant disclosure
include the use of devices and systems described herein, including
nucleic acid amplification devices and multi-reaction
monitoring/analysis devices and integrated systems thereof, in
amplifying and/or monitoring the amplification of a target nucleic
acid as described herein.
Real-Time Polymerase Chain Reaction
[0063] Such monitoring to detect the presence of a nucleic acid
analyte and/or quantify the initial amount of a nucleic acid
analyte in a sample, e.g., a biological sample, is the basis for
what is commonly referred to as real-time polymerase chain reaction
(real-time PCR).
[0064] The PCR process is a nucleic acid amplification method
whereby a target nucleic acid sequence is amplified by a factor of
2.sup.n by repeating (1) a denaturing temperature (e.g., of
95.degree. C.) that serves to denature the two strands of a double
stranded nucleic acid template; (2) an annealing temperature (e.g.,
on the order of 55.degree. C. to 65.degree. C.) that serves to
anneal one or more complementary nucleic acids to a single strand
of the denatured nucleic acid; and (3) an extension temperature
that provides the permissive temperature for a nucleic acid
polymerase to extend the complementary nucleic acid according to
the sequence of the template, alternately n times (referred as a
"thermal cycle").
[0065] In real-time PCR, the amount of nucleic acid is measured at
a plurality of time points during the amplification reaction to
determine the actual or relative amount of target nucleic acid
analyte initially present in the sample. Real-time PCR may be
quantitative, semi-quantitative or qualitative. Real-time PCR is
generally carried out in a thermal cycler with the capacity to
illuminate each amplification sample with a beam of light of at
least one specified wavelength and detect the fluorescence emitted
by an excited fluorophore that is either incorporated into the
amplicon or unquenched during amplification. Non-specific
fluorochromes (e.g., DNA binding dyes such as e.g., SYBR Green) or
specific fluorescent hybridization probes may be used. Using
different-colored labels, fluorescent probes can be used in
multiplex assays for monitoring several target sequences in the
same tube.
[0066] One method of using fluorescently labeled probes relies on a
DNA-based probe with a fluorescent reporter at one end and a
quencher of fluorescence at the opposite end of the probe. The
close proximity of the reporter to the quencher prevents detection
of its fluorescence. When bound to a target sequence, breakdown of
the probe by the 5' to 3' exonuclease activity of the polymerase
breaks the reporter-quencher proximity and thus allows unquenched
emission of fluorescence, which can be detected after excitation
with a particular wavelength of light. An increase in the product
targeted by the reporter probe at each PCR cycle therefore causes a
proportional increase in fluorescence due to the breakdown of the
probe and release of the reporter. Any convenient polymerase with
5' to 3' exonuclease activity may find use in such assays including
but not limited to wild-type Taq polymerase and modified or
engineered polymerases including but not limited to e.g., those
available from commercial suppliers such as e.g., New England
Biolabs (Ipswich, Mass.), Life Technologies (Carlsbad, Calif.),
Sigma Aldrich (St. Louis, Mo.) and Kapa Biosystems, Inc.
(Wilmington, Mass.) such as e.g., KAPA2G DNA Polymerases.
[0067] In some instances, the methods as described herein and/or
the devices and systems may be programed, developed and/or used in
conjunction with a method for multi-assay processing and analysis
as described in e.g., Attorney Docket No. ADDV-057WO, which claims
priority to U.S. Ser. No. 62/308,625, and/or a sample processing
device and/or method related thereto as described in e.g., Attorney
Docket No. ADDV-057WO, which claims priority to U.S. Ser. No.
62/308,625, the disclosures of which are incorporated herein by
reference in their entireties.
Multi-Reaction Monitoring
[0068] Methods of the instant disclosure include multi-reaction
monitoring where the amplification of a plurality, e.g., two or
more, ongoing amplification reactions are analyzed in real-time to
quantitatively, semi-quantitatively, or qualitatively determine
whether a target nucleic acid is present in a sample. The number of
reactions monitored in a multi-reaction monitoring method of the
instant disclosure may vary and may range from 2 to 100 or more
where the number of reactions is scalable with the number of
multi-reaction monitoring devices used in the method.
[0069] In some instances, the number of samples monitored in a
multi-reaction monitoring method utilizing a single multi-reaction
analysis device, as described herein, may be limited by the range
and/or rate of travel of an optics device attached to a conveyor
which passes the optics device past the samples to facilitate
monitoring. As such, the number of reaction vessels monitored by a
single multi-reaction analysis device, as described herein, will
vary and may range from 1 to 48 or more including but not limited
to e.g., 1 to 44, 1 to 40, 1 to 36, 1 to 32, 1 to 28, 1 to 24, 1 to
20, 1 to 16, 1 to 12, 1 to 8, 1 to 4, 2 to 48, 2 to 44, 2 to 40, 2
to 36, 2 to 32, 2 to 28, 2 to 24, 2 to 20, 2 to 16, 2 to 12, 2 to
8, 2 to 4, 4 to 48, 4 to 44, 4 to 40, 4 to 36, 4 to 32, 4 to 28, 4
to 24, 4 to 20, 4 to 16, 4 to 12, 4 to 8, 8 to 48, 8 to 44, 8 to
40, 8 to 36, 8 to 32, 8 to 28, 8 to 24, 8 to 20, 8 to 16, 8 to 12,
12 to 48, 12 to 44, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 12 to
24, 12 to 20, 12 to 16, 16 to 48, 16 to 44, 16 to 40, 16 to 36, 16
to 32, 16 to 28, 16 to 24, 16 to 20, 20 to 48, 20 to 44, 20 to 40,
20 to 36, 20 to 32, 20 to 28, 20 to 24, 24 to 48, 24 to 44, 24 to
40, 24 to 36, 24 to 32, 24 to 28, 6 to 26, 8 to 24, 10 to 22, 10 to
20, 10 to 18, 10 to 16, 10 to 14, 24, 20, 18, 16, 14, 12, etc.
[0070] Methods of multi-reaction monitoring, as described herein,
generally include scanning a traveling optics detection unit,
configured to optically assess a nucleic acid amplification
reaction, past a plurality of reaction vessels in a manner
sufficient to collect real-time measurements for the quantitative,
semi-quantitative or qualitative assessment of amplification. In
many embodiments, the plurality of reaction vessels is linearly
aligned such that scanning linearly is sufficient to pass the
traveling optics detection unit past all vessels of the
plurality.
[0071] According to the methods described herein, the optical
analysis may be performed through aligned apertures, including
e.g., where, during the scanning, only one reaction vessel is
analyzed at a time through a pair of aligned apertures. For
example, in some instances, a traveling optics detection unit
having a plurality of linearly arranged optical blocks is scanned
past a plurality of amplification reaction vessels present in a
nucleic acid amplification device and aligned apertures of the
optical blocks and the nucleic acid amplification device allows for
the optical monitoring of amplification in the reaction
vessels.
[0072] In one embodiment, scanning of the optics block, emitting an
excitation light, along the sidewall of the reaction vessel allows
for the collection of an emissions peak defined by the path of the
excitation light along the sidewall of the reaction vessel. For
example, FIG. 16 depicts the passage of an analysis point across
the cross section of an ultra-thin wall reaction vessel, allowing
for the collection of the emission peak shown. Accordingly, in
embodiments where the sidewall of the reaction vessel is scanned,
either along the entire width of the reaction vessel or along the
width of the reaction vessel exposed by an aperture or a portion
thereof, more data is collected as compared to e.g., where a single
point on a portion of the reaction vessel is analyzed. Following
the collection of a scan, which may include a plurality of emission
peaks and other non-signal elements, including e.g., noise, the
scan may be further processed, including but not limited to through
various signal processing pathways including e.g., for the
detection and/or filtering and/or measurement of the peaks in the
scan.
[0073] In some instances, methods for monitoring multiple
amplification reactions as described herein may include various
procedures directed at reducing the noise in the detection system,
where "noise" encompasses any disturbance, including random
disturbances and persistent disturbances, which obscures or reduces
the clarity of a signal. Thus, noise may be electronic noise,
optical noise, and the like. Noise also encompasses crosstalk
between channels of a system where the channels are configured to
function independently.
[0074] Methods for reducing noise in the systems described herein
include but are not limited to time division multiplexing,
frequency division multiplexing, spatial separation, and the like.
In some instances, methods of the instant disclosure further
include post-signal acquisition noise reduction, including e.g.,
where noise is filtered out using one or more filtering
algorithms.
[0075] Time division multiplexing noise reduction, as it applies to
the devices and systems described herein, generally involves
separating or segmenting elements of a detection process across
time so as to reduce the simultaneous generation and/or collection
of signals, e.g., by different channels of the system, particularly
where two channels may share a light path or have shared components
of a light path.
[0076] In some instances, the methods as described herein include
time division multiplexing of the elements of an illumination
component used in multi-reaction analysis. For example, excitation
elements, including e.g., LED emitters as described in more detail
herein, may be sequentially toggled so as to prevent the
simultaneous activation of two different LEDs of the system thereby
preventing the simultaneous emission of excitation light from two
different LEDs, including e.g., two LEDs of different wavelengths.
Such methods may or may not involve "dark periods" also referred to
as "dummy" periods, i.e., periods where all or both illumination
elements are off. Dark periods may, in some instances, be utilized
between the toggling of illumination elements to further prevent
the simultaneous activation of both elements or the simultaneous
presence of light from two different elements. Toggling of
illumination elements may occur at any convenient frequency (i.e.,
interval) provided the frequency (i.e., interval) is sufficient for
the detection needs of the system and the frequencies of multiple
illumination elements are purposefully uncoordinated to prevent
simultaneous activation of elements.
[0077] As an example of 2-way time division multiplexing, in one
embodiment depicted in FIG. 22, during a cycle (1/fcy) a first LED
(LED1) of a first wavelength is toggled on for a time (Tx) equal to
one quarter of the cycle. Correspondingly, later in the cycle a
second LED (LED2) of a second wavelength is toggled on for a period
of time equal to Tx. In addition, measurement and calibration
windows may be configured to correspond with the toggling of each
LED. For example, measurement and calibration of the channel
corresponding to LED1 (Meas/Call) may be configured such that the
measurement window for the channel (2200) corresponds only with the
time LED1 is toggled on. Likewise the calibration window in the
channel corresponding to LED1 (2201) may follow the measurement
window and take place e.g., only when LED1 is toggled off. Similar
measurement and calibration windows (Meas/Cal2) may be spaced
throughout the cycle for the channel corresponding to LED2 such
that measurement for the channel is only taken e.g., when LED2 is
toggled on and calibration is taken when e.g., LED2 is toggled off.
In some instances, the measurement windows for each channel are
smaller than the time the LED of the channel is toggled on and may
include time before (T1) and after (T2) measurement when the LED
may be toggled on and no measurement is taken. Likewise,
calibration windows may similarly make use of time periods before
and after calibration that are smaller, e.g., by T1 and T2, than
the actual dark period between LED toggling. As will be readily
understood, calibration measurements need not be limited to time
periods where LEDs are off and calibration measurements may, in
some instances, be made when one or more LED elements are toggled
on.
[0078] 2-way time division multiplexing may find use in reducing
noise within optical blocks that contain two or more toggle-able
illumination elements, including e.g., toggle-able LED emitters.
Where a system involves more than two channels, including e.g., two
or more optics blocks each with two or more illumination elements
(e.g., three optics blocks with two LED emitters each, three optics
blocks with four LED emitters each, etc.), multiple 2-way time
division multiplexing cycles can be utilized. In some instances
where multiple 2-way time division multiplexing cycles are utilized
the cycles may have generally the same scheme but with a different
overall cycle times. In other instances, where multiple 2-way time
division multiplexing cycles are utilized the cycles may have the
same scheme and the same overall cycle times.
[0079] Frequency division multiplexing noise reduction, as it
applies to the devices and systems described herein, generally
involves modulation between shared optical paths (e.g., within an
optical block) to provide optical and/or electrical rejection
between blocks. Accordingly, the components of an optical block may
be similarly modulated with respect to one another but differently
modulated with respect to components of another optical block of
the system (i.e., modulation may differ from block to block). For
example, two channels sharing a first optical block may both
modulate at frequency X, while two channels sharing a second
optical block may both modulate at frequency Y, where frequencies X
and Y are not equal. In some instances, the frequencies of
different optical blocks of the system may be chosen to be as
separated as possible.
[0080] Any convenient frequency modulation may find use in
frequency division multiplexing as described herein, including but
not limited to e.g., 0.1 kHz, 0.2 kHz, 0.3 kHz, 0.4 kHz, 0.5 kHz,
0.6 kHz, 0.7 kHz, 0.8 kHz, 0.9 kHz, 1 kHz, 1.1 kHz, 1.2 kHz, 1.3
kHz, 1.4 kHz, 1.5 kHz, 1.6 kHz, 1.7 kHz, 1.8 kHz, 1.9 kHz, 2 kHz,
2.1 kHz, 2.2 kHz, 2.3 kHz, 2.4 kHz, 2.5 kHz, 2.6 kHz, 2.7 kHz, 2.8
kHz, 2.9 kHz, 3 kHz, etc. For example, in some instances, a first
channel may be frequency modulated at 1 kHz, a second channel at
1.6 kHz and a third channel at 2.3 kHz. In addition, where
frequency modulation is employed at a particular frequency, system
components receiving the modulated frequency will demodulate at a
corresponding frequency.
[0081] In addition, where frequency modulation is employed, the
detectors for a particular optical block will demodulate at a
frequency corresponding to the modulation frequency of the channels
of the block. As such, by demodulating the detectors of the block
at a particular frequency, the detectors not only reject stray
signal from the other channels of the other blocks, which are
modulated at a different frequency, but also are more immune to
ambient disturbances, including e.g., stray light.
[0082] Spatial separation noise reduction, as it applies to the
devices and systems described herein, generally involves particular
physical spacing of system components to prevent noise, including
optical crosstalk and/or electrical crosstalk, transfer between
system components. When spatial separation noise reduction is
employed considerations may be made to limit spatial separation so
not to needlessly increase the size of the overall system, the time
required to make measurements in the system, etc.
[0083] In some instances, the methods as described herein include
physical spacing of system components to physically prevent
simultaneous activation and/or measurement by different components
thereby preventing the transfer of noise, including optical noise
and/or electrical noise, from one component to another.
[0084] For example, in some instances, spatial separation noise
reduction may be employed in a multi-reaction monitoring method, as
described herein, by permanently spacing the individual units of
arrayed detection components and the individual units arrayed
reaction vessels unequally such that, following a measurement,
either the arrayed detection components or the arrayed reaction
vessels must be physically moved to align for the next measurement.
Accordingly, in certain embodiments, the spacing between individual
units of arrayed detection components may be less than the spacing
between the individual units of arrayed reaction vessels. In other
embodiments, the spacing between individual units of arrayed
detection components may be greater than the spacing between the
individual units of arrayed reaction vessels.
[0085] In one embodiment, as depicted in FIG. 23, a scan of arrayed
reaction vessels (R1-R6) with spacing "p" by an optical detection
unit (2300) with three optical blocks (A1-A3) spaced "p-d" apart
makes use of spatial separation to prevent simultaneous
measurements of any two reaction vessels. For example, as the
optical detection unit proceeds through the scan along the x-axis
of the diagram in FIG. 23, at no time is more than one optical
block (A1-A3) in alignment with a reaction vessel (R1-R6). Thus,
the unequal spacing between the optical blocks as compared to the
spacing between the reaction vessels prevents simultaneous
alignment of two optical block/reaction vessel pairs thus
preventing simultaneous measurements during the scan.
[0086] As shown the detail in FIG. 23, the optical block A1 aligned
with reaction vessel RV2 (2301) makes a measurement of RV2 and
proceeds to the next position (2302) where optical block A2 is
aligned with reaction vessel RV3, yet neither A1 and RV2 nor A3 and
Rv4 are in alignment when A2 is aligned with RV3. Following the
measurement at RV3 (2302) the optical unit proceeds to the next
position (2303) where RV4 is aligned with A3 and a measurement may
be made, however when RV4 and A3 are in alignment, neither A1 nor
A2 are aligned with any other reaction vessels. Accordingly, spaced
measurements may be made essentially as depicted across the scan
shown on the x-axis of FIG. 23, where from first to last: optics
block A3 reads RV1; optics block A2 reads RV1; optics block A3
reads RV2; optics block A1 reads RV1; optics block A2 reads RV2;
optics block A3 reads RV3; optics block A1 reads RV2; optics block
A2 reads RV3; optics block A3 reads RV4; optics block A1 reads RV3;
optics block A2 reads RV4; optics block A3 reads RV5; optics block
A1 reads RV4; optics block A2 reads RV5; optics block A3 reads RV6;
optics block A1 reads RV5; optics block A2 reads RV6; and optics
block A1 reads RV6. Accordingly, each optics block reads each
reaction vessel, however the spatial arrangement of the optics
blocks relative to the reaction vessels during the scan assures
that only one optics block reads only one reaction vessel at any
one time during a scan.
[0087] Various different spacing schemes may be employed to make
use of spatial separation noise reduction as described herein,
depending, at least part, on the size of the components of the
system and the conveyance means employed to physically move
components of the system.
[0088] In some instances, other spatial orientations of components
of the described systems may be employed to decrease noise of the
system. For example, in some instances optical measurements between
an optics block and a reaction vessel may be made at an angle that
decreases the introduction of noise into the measurements. For
example, as depicted in FIG. 14, measurement may be made between a
reaction vessel that is essentially vertical (at left) and an
optics block (cutaway at right) that is at some angle away from
horizontal, including e.g., where the analysis aperture of the
optics block (i.e., the optics block aperture) is angled up from
horizontal towards the reaction vessel. In some embodiments, e.g.,
as depicted in FIG. 14, the optics block and the thermal block(s)
may be configured in relation to one another such that the angled
side wall of the reaction vessel and the optics block aperture are
parallel. In such instances, the optical path of the optics block
may be essentially perpendicular to the angled sidewall of the
reaction vessel as it rests in the thermal block. For example, in
some embodiments, due to the angle of the reaction vessel side
wall, the optics block may be correspondingly angled including
e.g., where the optics block is angled between 1.degree. or less to
15.degree. or more from vertical including but not limited to e.g.,
1.degree., 2.degree., 3.degree., 4.degree., 5.degree., 6.degree.,
7.degree., 8.degree., 9.degree., 10.degree., 11.degree.,
12.degree., 13.degree., 14.degree., or 15.degree. from vertical,
where vertical may be determined e.g., based on the system as a
whole or the primary vertical axis of the reaction vessel as it
rests in the thermal block.
[0089] In some instances, the methods as described herein include
calibration of components of a described device or system. Such
calibration measurements may be performed before, during or after,
and combinations thereof, an analysis as described herein. Such
calibration measurements may be applied to the system before,
during or after, and combinations thereof, an analysis as described
herein. In some instances, a calibration measurement is performed
before a scan, including but not limited to before each scan. In
other instances, a calibration measurement is performed after a
scan including but not limited to after each scan. In some
instances, a calibration measurement is performed before and after
a scan including but not limited to before and after each scan. In
some instances, calibration is performed during a scan including
but not limited to during each scan. In instances where a
calibration measurement is taken more than once, including e.g.,
when a calibration measurement is taken before and after a scan
and/or during a scan the subsequent calibration measurement(s) may
be used to adjust the applied calibration including adjusting the
calibration applied to the system based on the preceding
calibration measurement.
[0090] In some instances, a calibration may be associated with
(e.g., applied to) each measurement or with a set of measurements,
including e.g., each set of measurements of a scan. Calibration
components may be built into the analysis components of the devices
as described herein.
[0091] For example, in one embodiment of an optics block depicted
in FIG. 13 an optics block of the instant disclosure may include a
reference channel where such a reference channel serves in
calibrating measurements made from the optics block. As shown in
FIG. 13 an optics block may include a measurement channel and a
reference channel. The light path of the measurement channel
originates at the LED illumination component and is directed to the
reaction vessel (RV) as excitation light. The RV returns emission
light into the optics block and such emission light is directed to
the measurement channel for measurement. In the reference channel,
stray light passes through the reference channel aperture and is
measured by an optical signal processor.
[0092] In some instances, an optics block may be calibrated by
measuring the reference channel and the measurement channel in the
absence of the emission light from a reaction vessel or in the
presence of a reference light and then adjusting components of the
optics block based on the measuring. For example, where the
measurements from the reference channel and the measurement channel
are unequal one or more components of the measurement channel may
be adjusted until the reference channel and measurement channels
are equal.
[0093] In some instances, at the beginning of a run, including but
not limited to the beginning of every run, the LED drive currents
may be set based on LED intensity values set for each channel
during LED gain calibration. Such drive current adjustment may
provide for consistent LED brightness run to run and may compensate
for LED degradation where present. In some instances, once a
current drive value is set for a run the value may be held constant
for the length of the run and/or until current drive calibration is
performed anew.
[0094] In one embodiment, at the beginning of a run the background
signal of each reference channel is measured and each measurement
is added to each LED reference intensity value to determine the set
point for the reference channels. Once the set points are
determined for each channel the LED drive currents are set to their
default levels and a control loop is initiated to adjust the LED
drive currents until the scan intensity to equal to the LED
reference intensity plus the reference background signal.
[0095] In some instances, a "dark target" may be employed for
calibration according to the methods as described herein, where an
optical measurement is taken with the optics block aperture aligned
to the dark target and a calibration is applied to the system based
on the dark target measurement.
[0096] In embodiments of the methods described herein, before,
during and/or after a scan, the detection unit may align with the
dark target and a reading at the dark target may be taken.
Accordingly, the dark target reading is analyzed to determine if
adjustment of the applied calibration is necessary. In other
embodiments, the dark target reading is used to calibrate one or
more components of the optics block. In some instances, the
detection unit must travel to the dark target to make a calibration
reading. In other instances, the dark target is positioned in
alignment with the optics block aperture of the optical unit when
the optical unit is "at home" or "at rest" in its default
position.
[0097] In some instances, two or more dark targets may be employed.
For example, in some instances two dark targets may be used
including but not limited to e.g., where a first dark target is
present at one end of a linear optical scan and a second dark
target is present at the other end of the linear optical scan. In
such instances, a dark target measurement may be taken before,
after or before and after each scan.
[0098] In instances where two or more calibration measurements are
taken, use of the multiple measurements may vary. In some
instances, the first and subsequent measurement(s), may be used in
a statistical computation and the result of the statistical
computation may be applied to the system. For example, the first
and subsequent measurement(s), e.g., the first and second
measurement, may be averaged and the resultant average may be
applied to the system. In another example, the first and subsequent
measurements may be complied and the median of the measurements may
be applied to the system.
[0099] In other instances where two or more calibration
measurements are taken, the first and subsequent measurement(s) may
be applied differently to the system. For example, in some
instances, the first measurement may be applied to one or more
hardware components of the system, e.g., an electrical current of a
component of the system may be adjusted based on the first
measurement, and subsequent measurements may be applied to the data
resulting from the instrument, e.g., based on a comparison of the
first measurement to the subsequent measurement(s). In some
instances, a calibration measurement may be made at the start of a
run and the components of the system may be adjusted based on the
calibration, then subsequent calibration measurements may be
applied to the resultant data including e.g., where the subsequent
measurement is compared to the first measurement and if the two are
different the data is adjusted based on the difference between the
first and second measurements.
[0100] Any component useful in normalizing the measurements between
the reference channel and measurement channel may find use in
applying a calibration as described herein. For example, in some
instances, the power supplied to the illumination component,
including e.g., one or more LED emitters of the illumination
component, may be adjusted, e.g., increased or decreased, according
to a calibration measurement made on the system. Such calibrations
may be applied before or after a reaction vessel measurement
including e.g., during a dark period. In other instances, such a
calibration may be applied before or after a scan. Combinations of
calibrations, e.g., applied both during scanning and before/after
scans, may also find use in the methods as described herein.
[0101] In some instances, even when efforts are taken to limit
noise and/or assure calibration of components, background noise may
be collected with emission peak signals of the instant disclosure.
Background noise present in a signal that also contains collected
emission peaks may be minimized during collection and/or may be
removed following to collection. For example, in some instances,
background noise may be minimized during collection by collecting
signal during a scan only when an optical block is in alignment
with a reaction vessel. In some instances, the period of time an
optical block is in alignment with a reaction vessel may be
referred to as a reaction vessel window.
[0102] In some instances, the applied reaction vessel window may be
smaller or larger than the actual time period that the optical
block aperture is in alignment with a reaction vessel aperture. For
example, in some instances, a reaction vessel window calibration
may be performed to determine the actual time period the optical
block aperture is in alignment with the reaction vessel aperture to
determine the actual reaction vessel window and the applied
reaction vessel window may be an expanded or contracted window
based on the actual reaction vessel window. Determining and/or
calibrating the actual reaction vessel window may be performed by
any convenient means including but not limited to e.g., loading one
or more reaction vessels with a fluorescent control (e.g., a
control fluorescent dye, control fluorescent beads, control
fluorescent nucleic acid, etc.), scanning the one or more reaction
vessels with the optics block and measuring the control to
determine the start and end of each control peak. In some
instances, the applied reaction vessel window will be larger than
the actual reaction vessel window including but not limited to
e.g., one or more encoder counts larger than the actual reaction
vessel window as determined by scanning a reaction vessel loaded
with a fluorescent control.
[0103] In other instances, background noise may be removed
following to collection e.g., through the use of one or more signal
processing algorithms. For example, in instances where signal
collection is not limited to reaction vessel windows, as described
above, emission peaks may be determined in the scan data through
the use of one or more peak finding algorithms or signal processing
filters. Such post-collection signal processing methods may be used
in combination with other methods of signal detection and/or
reducing noise as described herein.
[0104] Following any noise reduction or noise removal and/or
post-processing of the collected signal, a determination about the
amplification state of the reaction may be made. Such
determinations are generally based on a number of data points for
the reaction collected over time, e.g., used in curve fitting,
where e.g., the fitted curve can be used to calculate the starting
amount of the target nucleic acid in the sample and/or whether the
target nucleic acid is present in the sample. In some instances,
the inability to generate a curve may indicate the absence of a
particular nucleic acid analyte in a sample.
[0105] Signal processing steps of the instant disclosure will
include one or more of signal acquisition (i.e., signal scan),
detector background subtraction, match filtering, peak detection,
intensity normalization, scale factor application, system
background subtraction, crosstalk calibration, relative
fluorescence unit calculation, and/or assay determination.
[0106] In one embodiment, the signal processing steps of the system
includes the signal processing pathway provided in FIG. 24 which
includes signal acquisition (i.e., signal scan), detector
background subtraction, match filtering, peak detection, intensity
normalization and scale factor application to generate scaled data.
Such processing may be performed, e.g., on an optical PCB including
e.g., an optical master board.
[0107] In some instances, the signal processing pathway may further
include system background subtraction, crosstalk calibration,
relative fluorescence unit calculation, and assay determination. In
some instances, such additional processing may or may not be
performed on an optical master board and may e.g., take place on a
processor separate from the optical master board.
[0108] In some instances, signal processing of the instant
disclosure includes one or more signal acquisition (i.e., signal
scan) steps. By signal scan step is meant the signal (produced from
light emitted from the sample) detected on the measurement channel
before going through any signal processing other than an amplifier.
Such may represent the input to the entire signal processing
pathway as measured on the measurement channel detectors,
optionally after scaling with amplifiers. Each channel generally
has a separate signal scan.
[0109] In some instances, signal processing of the instant
disclosure includes one or more detector background subtraction
steps. By detector background subtraction is meant the average
signal when the illumination component (e.g., the respective LED(s)
for a channel) are toggled off. Detector background may be measured
at the beginning of each scan and detector background may be
measured separately for each channel. In some instances, at the
beginning of each scan, the background for each channel is measured
as the average of the signal on the measurement channel when the
LEDs are turned off in front of the optical dark target. In some
instances, this background value may be subtracted from each
obtained (i.e., scanned) data point.
[0110] In some instances, signal processing of the instant
disclosure includes one or more match filtering steps. By match
filtering is meant essentially noise filtering (e.g., filtering of
high frequency noise) to match the measured data to the shape of a
typical signal peak. In some instances, a single match filter is
defined per module. In some instances, match filtering may be
performed following detector background subtraction. In some
instances, a match filter as used herein may be defined by a
plurality of match filter taps or coefficients. The output of match
filtering is filtered data.
[0111] In some instances, signal processing of the instant
disclosure includes one or more peak detection steps. In some
instances, following match filtering a peak detection algorithm may
be applied to locate the maximum value within each reaction vessel
window where a reaction vessel window is a window of encoder counts
around the location of a signal peak for a given channel and
position which defines the range of encoder counts in which the
system searches for peaks. In one embodiment, each position has six
reaction vessel windows, one per channel and because channels 1 and
4, 2 and 5, and 3 and 6 each share an optics sub-block, the
reaction vessel (RV) windows for these channel pairs are the same.
In some instances, a RV window halfwidth value may be used to
define the width of all windows for all positions of a module. In
some instances, the RV window serves to reduce data processing
overhead by limiting the amount of data that needs to be checked
for peaks.
[0112] In some instances, signal processing of the instant
disclosure includes one or more intensity normalization steps
including e.g., LED intensity normalization. In some instances,
following peak detection, the detected peaks are divided by LED
intensity used in the scan to correct for LED drift during an assay
run. For example, at the beginning of each scan in front of the
optical dark target, the LED Intensity for each channel is measured
then, after the peaks are detected, the peaks are divided by the
LED Intensity for the respective channel. In some instances, LED
intensity normalization is performed on a scan by scan basis.
[0113] In some instances, signal processing of the instant
disclosure includes one or more scale factor application steps. In
some instances, following LED intensity normalization, the
normalized data is multiplied by a scaling factor to generate
scaled data. Scale factor application may be performed on a channel
by channel basis where each channel has a calibrated scale factor,
e.g., where the calibrated scale factor is determined such that the
normalized/scaled data are comparable across their respective
dynamic intensity ranges to saturation.
[0114] In some instances, signal processing of the instant
disclosure includes the generation of scaled data. Scaled data,
when produced from intensity normalized data, will correct for any
variation in LED intensity from calibration during a protocol and
allow comparison of data across a run and between channels of a
run. In some instances, scaled data may be further processed. In
one embodiment, scaled data is further processed through a signal
processing pathway provided in FIG. 25 which includes system
background subtraction on the scaled data to generate system
background subtracted data, crosstalk correction application,
relative fluorescence unit conversion, and analysis.
[0115] In some instances, signal processing of the instant
disclosure includes one or more system background subtraction
steps. In some instances, system background subtraction includes
the subtraction of system background from the calibration for each
of the channels and positions from the corresponding scaled data
values for each scan. Such system background values may be
generated in a variety of methods including but not limited to
e.g., recording scaled data values for each channel in each
position using reaction vessels loaded with a control buffer. In
some instances, system background values may be determined
periodically, including but not limited to e.g., once daily, once
before starting a run, etc. In some instances, additional
determinations of system background values may be performed
including e.g., to compensate for potential variations in buffer,
potential variations in reaction vessel material, etc.
[0116] In some instances, signal processing of the instant
disclosure includes one or more crosstalk calibration or crosstalk
correction steps. Such crosstalk correction corrects for crosstalk
between the various dyes and channels. In some instances, e.g.,
where dyes with emission overlap are utilized in a method as herein
described in a device having a plurality of optical blocks a
crosstalk matrix may be generated and channel-to-channel crosstalk
may be corrected for. For example, a reaction vessel containing a
single dye at a known concentration may be scanned and the average
scaled data, with system background subtracted, may be measured for
each channel and the resultant values may be used to generate a
crosstalk matrix. In some instances, a crosstalk matrix may be used
to correct each measured value to remove any signal intensity due
to crosstalk from another channel or dye and/or all other channels
or dyes.
[0117] The emitted fluorescence intensity of various dyes is
variable with temperature. Accordingly, in some instances, a
crosstalk calibration matrix may take into account predicted
changes in dye intensity over the applicable temperature range
(e.g., from 30.degree. C. to 65.degree. C.). For example, in some
instances, based on the assay being run and/or the temperature
protocol used during amplification, a crosstalk correction factor
may be used to adjust the crosstalk calibration. Predicted changes
in dye intensity may be determined by any convenient method,
including e.g., empirical testing of dye intensity over temperature
ranges with or without associated curve fitting of such data.
During data collection and/or subsequent signal processing, the
known temperatures of a particular assay during cycling may be
employed to select a crosstalk correction factor at particular
temperatures for each dye used in the assay. A matrix of crosstalk
correction factors across temperatures may allow for adjusting the
crosstalk calibration based on specific dye combinations at certain
temperatures.
[0118] Once reference calibration values are determined, e.g., from
one or more calibration measurements as described herein,
calibration may be applied to various points of one or more signal
processing pathways where convenient and/or appropriate. In one
embodiment, various calibrations may be applied as indicated in
FIG. 26.
Devices and Systems
[0119] The instant disclosure generally provides devices and
systems for the amplification of nucleic acids in a reaction vessel
and the analysis and monitoring of such amplification by optical
measurements taken through the sidewall of the reaction vessel. The
devices and systems disclosed herein may provide an amplification
function or an analysis/monitoring function or may be integrated,
providing both an amplification function and an analysis/monitoring
function. Also provided are components of such devices and systems,
including but not limited to e.g., a thermal block component, an
optical block component, an illumination component and the
like.
Nucleic Acid Amplification Devices
[0120] The instant disclosure provides thermal control devices
useful in the amplification of nucleic acids. Such devices may be
referred to as nucleic acid amplification devices and are often
commonly referred to as thermocyclers.
[0121] Where electricity is employed to control thermal cycling, at
a minimum, a thermocycler useful in nucleic acid amplification with
include a thermal block, a thermoelectric cooler and a control
unit, such components configured together to regulate the
temperature of a reaction vessel in a controlled manner so as to
cycle the reaction through multiple rounds of heating and cooling
through a defined series of temperature steps.
[0122] A nucleic acid amplification device of the instant
disclosure may include thermoregulatory components in addition to
the thermal block and thermoelectric cooler including but not
limited to e.g., a heatsink, a fan, a duct, a vent, etc. Two or
more thermoregulatory components of a nucleic acid amplification
device will generally be in thermal contact with one another. By
"thermal contact" is meant that heat may flow from one component to
the other, i.e., the components are not separated by a thermal
insulator. Two components in thermal contact with one another may
be in direct contact, i.e., direct physical contact. Alternatively,
two components in thermal contact with one another may be in
indirect contact including e.g., where one or more conductive
materials between the two components places the two components in
thermal contact with one another. For example, a thermal block and
a thermoelectric cooler in thermal contact with one another may be
in directed physical contact or may be in indirect contact,
thermally joined by one or more conductive materials.
[0123] Conductive materials useful in thermally joining
thermoregulatory components of the nucleic acid amplification
devices as described herein include solids, semi-solids,
semi-fluids and liquids that are sufficiently thermo-conductive to
allow heat to readily flow between the components. A useful
conductive material will generally, but not necessarily, have a
thermal resistance that is less than that of the two or more
components that it thermally joins. Conductive materials useful in
thermally joining two components of the devices described herein
include conductive metals and conductive non-metal solids as
described herein. However, such conductive materials useful in
thermally joining two components are not limited to solids any may
include e.g., thermal greases, thermal adhesives, thermal gels,
thermal coatings, thermal composites, encapsulated thermal
materials (e.g., phase-change materials (e.g., encapsulated thermal
liquids, etc.), and the like. In some instances, an adhesive used
to join two components of a subject device or system (e.g., an
epoxy) may serve as a conductive material between the two
components.
[0124] The actual configuration of the thermal joining of two
components with a conductive material will vary and may include
e.g., where the entire interface between the two components is
occupied or covered by the conductive material, where a portion of
the interface between the two components is occupied or covered by
the conductive material, where a majority of the interface between
the two components is occupied or covered by the conductive
material, where half of the interface between the two components is
occupied or covered by the conductive material, where a minority of
the interface between the two components is occupied or covered by
the conductive material, etc.
[0125] In some instances, a conductive material that thermally
joins two components may be in the form of a pad where the pad
covers the entire interface between the two components. In other
instances, a conductive material that thermally joins two
components may be in the form of a pad where the pad covers only a
portion of the interface between the two components. In instances
where a pad is used that covers only a portion of the interface
between the two components, in certain embodiments, multiple, i.e.,
a plurality of, pads may be used where the pads may be positioned
such that there is space between the pads. In other instances, a
plurality of pads may be arrayed such that there is essentially no
space between the pads. Accordingly, where multiple pads are
utilized the plurality of pads may collectively cover a portion of
the interface between the components, a majority of the interface
between the two components, a minority of the interface between the
two components, or the entire interface between the two
components.
[0126] Conductive solid materials useful in constructing
thermoregulatory components and/or as a conductive material that
thermally joins two or more thermoregulatory components include but
are not limited to metals and/or metal alloys. Considerations that
may be made considering the selection of a particular metal for use
in a thermoregulatory component or as a conductive material that
thermally joins two or more thermoregulatory components include but
are not limited to the thermal conductivity of the metal and/or the
thermal expansion of the metal. Such thermal properties of common
metals that may find use in one or more components or to thermally
join two or more components are as follows (thermal conductivity in
British thermal unit per hour foot degree Fahrenheit
(Btu/h.times.ft.times..degree. F.); thermal expansion in inch per
inch per .degree. F..times.10.sup.6): Aluminum (136; 13.1),
Antimony (120; NA), Brass (Yellow) (69.33; 11.2), Copper (231;
9.8), Gold (183; 7.9), Iron, Cast (46.33; 6), Lead, solid (20.39;
16.4), Nickel (52.4; 5.8), Platinum (41.36; 4.9), Silver (247.87;
10.8), Steel, mild (26.0-37.5; 6.7), Steel, Stainless 304 (8.09;
9.6), Steel, Stainless 430 (8.11; 6), Tin, solid (38.48; 13),
Titanium 99.0% (12.65; 4.7), Tungsten (100.53; 2.5), Zinc (67.023;
22.1), Zirconium (145; 3.2). In some instances, useful metals or
alloys include but are not limited to Aluminum, 2024, Temper-T351
(conductivity 143 W/m.times.deg. C.), Aluminum, 2024, Temper-T4
(conductivity 121 W/m.times.deg. C.), Aluminum, 5052, Temper-H32
(conductivity 138 W/m.times.deg. C.), Aluminum, 5052, Temper-O
(conductivity 144 W/m.times.deg. C.), Aluminum, 6061, Temper-O
(conductivity 180 W/m.times.deg. C.), Aluminum, 6061, Temper-T4
(conductivity 154 W/m.times.deg. C.), Aluminum, 6061, Temper-T6
(conductivity 167 W/m.times.deg. C.), Aluminum, 7075, Temper-T6
(conductivity 130 W/m.times.deg. C.), Aluminum, A356, Temper-T6
(conductivity 128 W/m.times.deg. C.), Aluminum, Pure (conductivity
220 W/m.times.deg. C.), Beryllium, Pure (conductivity 175
W/m.times.deg. C.), Brass, Red, 85% Cu-15% Zn (conductivity 151
W/m.times.deg. C.), Brass, Yellow, 65% Cu-35% Zn (conductivity 119
W/m.times.deg. C.), Copper, Alloy, 11000 (conductivity 388
W/m.times.deg. C.), Copper, Aluminum bronze, 95% Cu-5% Al
(conductivity 83 W/m.times.deg. C.), Copper, Brass, 70% Cu-30% Zn
(conductivity 111 W/m.times.deg. C.), Copper, Bronze, 75% Cu-25% Sn
(conductivity 26 W/m.times.deg. C.), Copper, Constantan, 60% Cu-40%
Ni (conductivity 22.7 W/m.times.deg. C.), Copper, Drawn Wire
(conductivity 287 W/m.times.deg. C.), Copper, German silver, 62%
Cu-15% Ni-22% Zn (conductivity 24.9 W/m.times.deg. C.), Copper,
Pure (conductivity 386 W/m.times.deg. C.), Copper, Red brass, 85%
Cu-9% Sn-6% Zn (conductivity 61 W/m.times.deg. C.), Gold, Pure
(conductivity 318 W/m.times.deg. C.), Invar, 64% Fe-35% Ni
(conductivity 13.8 W/m.times.deg. C.), Iron, Cast (conductivity 55
W/m.times.deg. C.), Iron, Pure (conductivity 71.8 W/m.times.deg.
C.), Iron, Wrought, 0.5% C (conductivity 59 W/m.times.deg. C.),
Kovar, 54% Fe-29% Ni-17% Co (conductivity 16.3 W/m.times.deg. C.),
Lead, Pure (conductivity 35 W/m.times.deg. C.), Magnesium, Mg--Al,
Electrolytic, 8% AI-2% Zn (conductivity 66 W/m.times.deg. C.),
Magnesium, Pure (conductivity 171 W/m.times.deg. C.), Molybdenum
(conductivity 130 W/m.times.deg. C.), Nichrome, 80% Ni-20% Cr
(conductivity 12 W/m.times.deg. C.), Nickel, Ni--Cr, 80% Ni-20% Cr
(conductivity 12.6 W/m.times.deg. C.), Nickel, Ni--Cr, 90% Ni-10%
Cr (conductivity 17 W/m.times.deg. C.), Nickel, Pure (conductivity
99 W/m.times.deg. C.), Silver, Pure (conductivity 418
W/m.times.deg. C.), Solder, Hard, 80% Au-20% Sn (conductivity 57
W/m.times.deg. C.), Solder, Hard, 88% Au-12% Ge (conductivity 88
W/m.times.deg. C.), Solder, Hard, 95% Au-3% Si (conductivity 94
W/m.times.deg. C.), Solder, Soft, 60% Sn-40% Pb (conductivity 50
W/m.times.deg. C.), Solder, Soft, 63% Sn-37% Pb (conductivity 51
W/m.times.deg. C.), Solder, Soft, 92.5% Pb-2.5% Ag-5% In
(conductivity 39 W/m.times.deg. C.), Solder, Soft, 95% Pb-5% Sn
(conductivity 32.3 W/m.times.deg. C.), Steel, Carbon, 0.5% C
(conductivity 54 W/m.times.deg. C.), Steel, Carbon, 1.0% C
(conductivity 43 W/m.times.deg. C.), Steel, Carbon, 1.5% C
(conductivity 36 W/m.times.deg. C.), Steel, Chrome, Cr0%
(conductivity 73 W/m.times.deg. C.), Steel, Chrome, Cr1%
(conductivity 61 W/m.times.deg. C.), Steel, Chrome, Cr20%
(conductivity 22 W/m.times.deg. C.), Steel, Chrome, Cr5%
(conductivity 40 W/m.times.deg. C.), Steel, Chrome-Nickel, 18%
Cr-8% Ni (conductivity 16.3 W/m.times.deg. C.), Steel, Invar, 36%
Ni (conductivity 10.7 W/m.times.deg. C.), Steel, Nickel, Ni0%
(conductivity 73 W/m.times.deg. C.), Steel, Nickel, Ni20%
(conductivity 19 W/m.times.deg. C.), Steel, Nickel, Ni40%
(conductivity 10 W/m.times.deg. C.), Steel, Nickel, Ni80%
(conductivity 35 W/m.times.deg. C.), Steel, SAE 1010 (conductivity
59 W/m.times.deg. C.), Steel, SAE 1010, Sheet (conductivity 63.9
W/m.times.deg. C.), Steel, Stainless, 316 (conductivity 16.26
W/m.times.deg. C.), Steel, Tungsten, WO % (conductivity 73
W/m.times.deg. C.), Steel, Tungsten, W1% (conductivity 66
W/m.times.deg. C.), Steel, Tungsten, W10% (conductivity 48
W/m.times.deg. C.), Steel, Tungsten, W5% (conductivity 54
W/m.times.deg. C.), Tin, Cast, Hammered (conductivity 62.5
W/m.times.deg. C.), Tin, Pure (conductivity 64 W/m.times.deg. C.),
Titanium (conductivity 15.6 W/m.times.deg. C.), Tungsten
(conductivity 180 W/m.times.deg. C.), Zinc, Pure (conductivity
112.2 W/m.times.deg. C.), and the like.
[0127] Conductive solid materials useful in constructing
thermoregulatory components and/or as a conductive material that
thermally joins two or more thermoregulatory components include but
are not limited to nonmetals. Considerations that may be made
considering the selection of a particular nonmetal for use in a
thermoregulatory component or as a conductive material that
thermally joins two or more thermoregulatory components include but
are not limited to the thermal conductivity of the nonmetal and/or
the thermal expansion of the nonmetal. Useful nonmetal materials
include but are not limited to e.g., carbon based materials and
carbon containing materials (including engineered any synthetic
materials) including but not limited to diamond, graphite,
graphene, carbon nanotube, carbon fiber, etc. Thermal properties of
such carbon based and carbon containing materials are well-known
and include e.g., those described in e.g., Dasgupta et al. Journal
of Composite Materials, 26 (1992) 2736-2758; Sweeting, Composites
Part A: Applied Science and Manufacturing, 35 (2004) 933-938;
Wetherhold et al., Journal of Composite Materials, 28 (1994)
1491-1498; Balandin, Nature Materials 10, (2011) 569-581; the
disclosures of which are incorporated herein by reference in their
entirety.
[0128] Physical joining of the components, including
thermoregulatory components, of the subject nucleic acid
amplification device may be achieved by a variety of means
depending on the particular components to be joined. In certain
instances, any convenient method of joining may find use including
but not limited to fastening mediated by a mechanical fastener
including e.g., a bolt (e.g., an anchor bolt), a captive fastener,
a clamp (or cramp), a clasp, a clip, a flange, a grommet, a latch,
a nail, a peg, a pin, a retaining ring, a rivet, a snap fastener, a
staple, a strap, a threaded fastener (e.g., a captive threaded
fastener, a nut, a screw, a threaded insert, a threaded rod, etc.),
a tie, a toggle bolt, a wedge anchor, and the like. Such mechanical
fasteners may join two components by various forces and
combinations of forces including but not limited to e.g.,
frictional forces (e.g., sheer force), compression forces, tensile
forces, and combinations thereof. Physical joining of the
components the subject nucleic acid amplification device need not
necessarily employ a fastener and may, e.g., involve the use of an
adhesive, a weld, and the like. In some instances, a faster may be
used in combination with a non-fastener means including but not
limited to e.g., a faster and an adhesive, a fastener and a weld,
etc. In other instances, a fastener may be used alone, i.e.,
essentially without any other fastening means, including e.g.,
without adhesive.
[0129] Fasteners useful in the devices described herein may be made
out of various materials including but not limited to thermal
conductive materials and non-thermal conductive materials. As such,
useful materials for fasteners include metals and nonmetals,
including conductive nonmetals and nonconductive nonmetals. In some
instances, useful nonmetal fasteners include but are not limited to
e.g., polymeric materials, e.g., plastics, ceramics (e.g., alumina
(Al.sub.2O.sub.3), zirconia (ZrO.sub.2), etc.), and the like.
[0130] In some instances, including where at least one of the two
components to be joined is a thermoregulatory component the
thermodynamic properties of the component (including e.g., thermal
expansion) may be taken into account. For example, in some
instances, a compression fitting, including e.g., a compression
fitting mediated by a mechanical fastener, may join two
thermoregulatory components that differ in their thermal expansion
properties with sufficient compressive force, e.g., to prevent
movement of the components under various temperature conditions and
changes. However, is some instances, care should be taken to assure
such compressive force is not excessive to e.g., to prevent warping
of the components or cracking of the components, etc., under
various temperature conditions and changes. In some instances, a
conductive pad, as described herein, may find use in joining two
thermoregulatory components having different thermal expansion
properties.
[0131] When one or more fasteners are is used to join two or more
thermoregulatory components under a compression force the overall
compressive force between the two components will vary and may
range e.g., from 20 to 500 pounds per square inch (psi) including
but not limited to e.g., 20 to 500 psi, 30 to 500 psi, 40 to 500
psi, 50 to 500 psi, 60 to 500 psi, 70 to 500 psi, 80 to 500 psi, 90
to 500 psi, 100 to 500 psi, 100 to 450 psi, 100 to 400 psi, 100 to
350 psi, 100 to 300 psi, 100 to 250 psi, 100 to 200 psi, 110 to 200
psi, 120 to 200 psi, 130 to 200 psi, 140 to 200 psi, 100 to 190
psi, 100 to 180 psi, 100 to 170 psi, 100 to 160 psi, 70 to 300 psi,
80 to 290 psi, 90 to 280 psi, 100 to 270 psi, 110 to 260 psi, 120
to 250 psi, 130 to 240 psi, 140 to 230 psi, 70 to 200 psi, 80 to
190 psi, 90 to 180 psi, 100 to 170 psi, 110 to 160 psi, 120 to 160
psi, 130 to 160 psi, 140 to 160 psi, and the like. Such forces may
be determined either theoretically through the use of mechanical
force modeling or empirically by use of a force sensor, a force
gauge or analytical testing (e.g., deformation testing, failure
testing, etc.).
[0132] One application of nucleic acid amplification devices is in
the monitoring of an amplification reaction during amplification to
identify the presence of a target or template nucleic acid, which
may in some instances be referred to herein as a "nucleic acid
analyte" or simply "analyte". In some instances, a nucleic acid
amplification device may find use in monitoring of an amplification
reaction during amplification to quantify the amount of a target
nucleic acid analyte in a sample, e.g., a biological sample.
Accordingly, the nucleic acid amplification devices described
herein may find use in the amplification required for real-time PCR
applications as described herein.
Thermal Blocks
[0133] A thermal block of the instant disclosure provides a thermal
mass to or from which heat is flowed in order to provide accurate
control of the temperature of one or more reaction vessels in
thermal contact with the block. This instant disclosure provides a
multi-well block capable of simultaneous control of the temperature
of multiple reaction vessels. In one embodiment, the thermal block
is capable of simultaneous temperature control of two reaction
vessels. However, thermal blocks are not limited to two reaction
vessels and may, in certain instances, allow for the simultaneous
temperature control of three or more, four or more, five or more,
six or more, seven or more, eight or more, nine or more, ten or
more, reaction vessels provided the thermal block is configured to
contact each reaction vessel with essentially the same
temperature.
[0134] Reaction vessels generally seat into a reaction vessel well
of the thermal block where a reaction vessel well may be a
depression in the thermal block that conforms to the outer shape of
the reaction vessel. In some instances, a reaction vessel well may
at least partially extend from the thermal block. In yet other
instances, a reaction vessel well may make use of a combination of
depression features and extension features to conform to the outer
shape of the reaction vessel including e.g., where the top surface
of the thermal block includes a raised flange or lip around the
edge of the reaction vessel well such that the reaction vessel well
is not flush with the top surface of the thermal block. In other
instances, the reaction vessel well is flush with the top surface
of the thermal block. In either instance, where the reaction vessel
well is formed from a depression in the top of the thermal block or
an extension from the top of the thermal block or a combination
thereof the reaction vessel may be loaded vertically (i.e., from
the top) into the reaction vessel well.
[0135] The reaction vessel well may be dimensioned to conform to
the shape of the reaction vessel such that a majority of the
reaction vessel remains in thermal contact with the reaction vessel
well. Accordingly, where a reaction vessel is e.g., a commercially
available reaction vessel (e.g., a commercially available PCR tube)
or is dimensioned to match a commercially available reaction vessel
the reaction vessel well may conform to the shape of a commercially
available reaction vessel such that, upon vertical insertion into
the reaction vessel, the majority of the reaction vessel and the
reaction vessel well remain in thermal contact. In instances where
a reaction vessel is of a custom design the reaction vessel well
may conform to the shape of the custom designed vessel such that,
upon vertical insertion into the reaction vessel, the majority of
the reaction vessel and the reaction vessel well remain in thermal
contact.
[0136] In certain instances, the reaction vessel well may be
configured such that the sides, e.g., only the sides and not the
bottom, of the reaction vessel well conforms to the shape of the
reaction vessel. In such instances, upon insertion into the
reaction vessel well, the sides of the reaction vessel well and not
the bottom will remain in thermal contact with the reaction vessel
well.
[0137] Although generally configured to conform to the shape of the
reaction vessel, the reaction vessel well may, in some instances,
have dimensions that purposefully do not conform to the shape of
the reaction vessel. For example, in some instances, the reaction
vessel well may have a circumference that conforms to the outer
circumference of the reaction vessel (including where the reaction
vessel is essentially conical, essentially a conical pyramid, or a
combination thereof) but a vertical depth that is larger than the
vertical dimension of the reaction vessel such that, upon insertion
of the reaction vessel into the reaction vessel well, an empty
space or cavity persists beneath the inserted reaction vessel. In
some instances, such empty space may serve as a "reservoir" under
an inserted reaction vessel, e.g., to collect any unintentional
liquid (i.e., spillage, condensation, etc.) that may be adhered to
the reaction vessel.
[0138] The reaction vessel wells of a thermal block of the instant
disclosure may be oriented in relative position to one another
within the thermal block such that the reaction vessel wells are
subjected to essentially equal temperatures during thermal cycling.
Accordingly, the reaction vessel wells may be evenly spaced from
the center of the block including e.g., where the reaction vessel
wells are equidistant from the vertical midline of the thermal
block. Such thermal blocks with evenly spaced reaction vessels may
be bilaterally symmetric, e.g., the thermal block has a vertical
line of symmetry such that the right and left halves of the thermal
block are mirror images.
[0139] The thermal block of the instant disclosure, although
bilaterally symmetric, may be asymmetric other respects. For
example, the thermal block may have a thermal transfer side of a
different configuration or surface shape as compared to the
non-thermal transfer side, where the "thermal transfer side" is
defined as the side of the block where primary heat transfer takes
place. In such instances, a thermal block of the instant disclosure
may have a thermal transfer side that is essentially planar or flat
and a non-thermal transfer side that is nonplanar or not flat,
including but not limited to e.g., where the non-thermal transfer
side comprises structural elements including but not limited to
e.g., ridges which may provide structural rigidity to the thermal
block.
[0140] In many embodiments, thermal transfer side of the thermal
block is opposite non-thermal transfer side. As such, in many
embodiments, the thermal block of the instant disclosure may be six
sided with: (1) a thermal transfer side, (2) a non-thermal transfer
side opposite the thermal transfer side, (3) a top side having
openings for vertical insertion of the reaction vessel(s) into
reaction vessel well(s), a (4) right side, a (5) left side, and (6)
a bottom. In many instances, the right side, left side and bottom
are thin and essentially featureless, with the exception of
providing attachment points for particular components (including
e.g., a support bar), making the a thermal transfer side, the
non-thermal transfer side and the a top side the primary functional
sides of the thermal block. Accordingly, in some instances, a
thermal block of the instant disclosure includes a top side that is
greater in area (as determined by the largest length and width
dimensions of the top) than the bottom of the thermal block (as
determined by the largest length and width dimensions of the
bottom).
[0141] In such instances, the non-thermal transfer side may serve
as the analysis side of the thermal block where the term "analysis
side" is defined as the side of the block from which analysis of
the reaction vessel(s) is performed. The analysis side of the
thermal block may include analysis openings for the optical
analysis of the reaction vessels. In some instances, the optical
openings include one or more apertures in the reaction vessel well
configured such that light may pass into, e.g., excitation light,
and out of, e.g., emission light, the reaction vessel by way of the
aperture. The position of an analysis aperture in a reaction vessel
will vary and may include, e.g., where the aperture is configured
for analysis at the side of the reaction vessel.
[0142] As will be understood through further description of the
multi-reaction analysis systems and integrated systems described
herein. The relative positioning of the thermal transfer side and
the analysis side opposite each other on the thermal block allows
for simultaneous thermal cycling and analysis of the reaction
vessels.
[0143] In many embodiments, the overall shape of the thermal block
provides for center mounting of the thermal block to other
components of the nucleic acid amplification device. For example,
in certain instances, the thermal block has a mounting hole
centrally positioned on a face (i.e., the thermal transfer surface
or surface opposite the thermal transfer surface) of the thermal
block. As such, the mounting hole may be perpendicular to the
planar axis of the thermal block (e.g., the mounting hole is
positioned having a center axis perpendicular to the plane of the
thermal transfer surface). In some instances, the central
positioning of the mounting hole may provide certain functional
benefits including but not limited to e.g., evenly distributed
compression forces across the thermal block upon affixing the
thermal block to other components with a compression fitting (e.g.,
limiting deformation of the block due to thermal expansion and
contraction effects during thermal cycling).
[0144] In some instances, the reaction vessel wells may be
positioned at equal distances from the central mounting hole, e.g.,
such that the thermal block is bilaterally symmetric on a vertical
axis through the mounting hole. The thermal block may include
structural features oriented around the central mounting hole
including e.g., structural ridges the emanate radially from the
central mounting hole. In some instances, the central mounting hole
is positioned half way between the right and left sides of the
thermal block. In some instances, the central mounting hole is
positioned half way between the top and the bottom of the thermal
block.
[0145] A thermal block of the instant disclosure may also have a
temperature detection area configured for the application of a
temperature sensor. Such a temperature detection are may be
essentially flat to allow the flush (i.e., flat against) attachment
of a flat sided temperature sensor to the thermal block. In some
instances, a temperature detection area may be positioned proximal
to a reaction vessel well such that the temperature reading at the
temperature detection area serves as a proxy for the temperature of
the reaction vessel. Accordingly, where a temperature detection
area is used as a proxy for the temperature of the reaction vessel
the area will be sufficiently close to the reaction vessel well
including but not limited to e.g., less than 2 cm from the reaction
vessel well. Any convenient position proximal to the reaction
vessel well may find use as a temperature detection area for
monitoring reaction temperature including but not limited to e.g.,
beneath the reaction vessel well, including but not limited to
within 2 cm from the bottom of the reaction vessel well.
[0146] In some instances, a thermal block may include a separate
temperature detection area positioned proximal to each reaction
vessel well such that the temperature of each reaction vessel may
be separately monitored. The size and shape of a temperature
detection area on a thermal block will vary. In some instances, the
temperature detection area may be dimensioned to receive a
temperature sensor. In some instances, the temperature sensor area
may include a recess that is essentially circular or half-circular.
In some instances, the temperature sensor area may include an
associated channel, including e.g., where the diameter of the
temperature sensor area is essentially equal to the width of the
channel (see e.g., the embodiment depicted in FIG. 1 having a
temperature sensor area (104) that is essentially equal in diameter
to the width of the corresponding channel (105)).
[0147] In some instances, the diameter of the temperature sensor
area may be wider or narrower than the width of the associated
channel. For example, in some instances, the temperature sensor
area may be wider than the associated channel. Such an embodiment
is depicted in FIG. 28 where e.g., the temperature sensor area
(2800) has a diameter that is wider than the width of the
associated channel (2801). In some instances, the temperature
sensor area is dimensioned to receive a temperature sensor of a
particular diameter or width and the associated channel has a width
that is smaller than the diameter or width of the temperature
sensor. In some instances, the temperature sensor employed has a
diameter or width less than both the diameter of the temperature
sensor area and any channel associated with the temperature sensor
area.
[0148] The thermal block may be constructed of any convenient and
appropriate thermally conductive material, including but not
limited to metal including but not limited to e.g., one or more
metal or metal alloy described herein including but not limited to
e.g., aluminum. In some instances, the thermal mass of the thermal
block may be kept relatively low, e.g., as in comparison to other
thermal regulatory components of the system, to provide for rapid
transitions between thermal cycling steps. The overall mass of the
thermal block may vary and may range anywhere from e.g., 1 g to 10
g including but not limited to e.g., 1 g to 5 g, 1 g to 4.5 g, 1 g
to 4.0 g, 1 g to 3.5 g, 1 g to 3.0 g, 1 g to 2.9 g, 1 g to 2.8 g, 1
g to 2.7 g, 1.5 g to 4 g, 1.5 g to 3.9 g, 1.5 g to 3.8 g, 1.5 g to
3.7 g, 1.5 g to 3.6 g, 1.5 g to 3.5 g, 1.5 g to 3.4 g, 1.5 g to 3.3
g, 1.5 g to 3.2 g, 1.5 g to 3.1 g, 1.5 g to 3.0 g, 1.5 g to 2.9 g,
1.5 g to 2.8 g, 2 g to 3.5 g, 2.5 g to 3.5 g, and the like.
[0149] One of more surfaces of the thermal block may be
functionally coated or plated including but not limited to
functional coating or plating to reduce corrosion, functional
coating or plating to friction corrosion, and the like. Such
functional coatings and/or platings will vary and may include e.g.,
metal plating, non-stick coating (e.g., dry lubricant), and the
like. Examples of useful functional coatings and/or platings may
include but are not limited to, electrolytic nickel plating,
electroless nickel plating (e.g., Nye-Croloy (Nickel Chrome
Plating), Sulfamate nickel, Kanigen, Nye-Kote), zinc
electroplating, High Velocity Oxy-Fuel (HVOF) Combustive Spray,
fluoropolymer-resin/lubricant blends, Xylan, molybdenum disulfide,
epoxy coatings (including air dry and thermal cure coatings),
phenolic coatings, phosphate ferrous metal coatings, polyurethane
coatings, PTFE coatings, PPS/Ryton coatings, FEP coatings,
PVDF/Dykor coatings, parylene coating, ECTFE/Halar coatings,
ceramic epoxy coatings, and the like. In some instances, the entire
thermal block may be coated or plated.
[0150] In some instances, a portion of the thermal block may be
coated or plated, including but not limited to a majority of the
thermal block, one or more surfaces of the thermal block, a
minority of the thermal block, the surface of the reaction vessel
well(s), the thermal transfer surface, the top surface, etc. In
some instances, particular surfaces of the thermal block will not
be coated or plated including but not limited to e.g., the thermal
transfer surface.
[0151] In some instances, thermal blocks of the instant disclosure
may be characterized in that, when combined with a particular TEC,
the thermal block may have an empirical thermal slew rate of
greater than 7.degree. C./sec including but not limited to e.g.,
greater than 8.degree. C./sec, greater than 9.degree. C./sec,
greater than 10.degree. C./sec, etc., in cooling, heating or both
cooling and heating. Such empirical thermal slew rates may be
determined by a variety of methods including but not limited to
e.g., "bang-bang" testing where heating and cooling are monitored
while the component is cycled between minimum and maximum
electrical currents. In some instances, empirical thermal slew
rates may be determined within a temperature range or for two
particular temperatures. For example, a thermal slew rate may be
determined e.g., for heating between 25.degree. C. and 95.degree.
C., for cooling between 25.degree. C. and 95.degree. C., for
heating and cooling between 25.degree. C. and 95.degree. C.,
etc.
[0152] In some instances, the thermal slew rate of a thermal block
as described herein may be expressed as an operating or applied
thermal slew rate which is defined as the slew rate of the thermal
block under normal operating conditions. In some instances, a
thermal block of the instant disclosure may have an operating
thermal slew rate of greater than 5.degree. C./sec.
[0153] One embodiment of a thermal block according to the instant
disclosure is depicted in FIG. 1 with the thermal transfer side of
the thermal block oriented away from the viewer and the analysis
side (100) oriented towards the viewer. The depicted thermal block
is configured with a mounting hole (101) positioned with the
central axis of the mounting hole oriented perpendicular to plane
of the analysis side (100) of the block. The depicted thermal block
contains two reaction vessel wells (102) each having a side
aperture (103). The depicted thermal block further displays
temperature monitoring areas (104) configured for the application
of temperature sensors used to monitor the temperature of the
thermal block in close proximity to the reaction vessels which can
serve as a proxy for the reaction vessel temperature. The
temperature monitoring areas (104) depicted further include
associated channels (105).
[0154] FIG. 2 depicts the thermal transfer side (200) of the
thermal block depicted in FIG. 1. From the thermal transfer side
the reaction vessel wells (102) and mounting hole can be clearly
seen (101). Also depicted is a reaction vessel (201) to which the
depicted reaction vessel wells have been configured to conform. The
reaction vessel is depicted in a vertical orientation above the
reaction vessel well to generally indicate the vertical direction
from which reaction vessels are loaded into the reaction vessel
wells as shown in the particular embodiment.
[0155] A thermal block (300) loaded with two capped reaction
vessels is depicted in FIG. 3. The reaction vessel caps (301) can
be seen protruding vertically from the reaction vessel which is
recessed into the reaction vessel well. The analysis side of the
thermal block is facing the viewer with a portion of a side
aperture (302) of a reaction vessel well visible. The thermal block
is depicted partially mounted with a mechanical fastener (303)
present in the mounting hole and a support bar (304) positioned
beneath the thermal block. Not pictured are the other
thermoregulatory elements to which a thermal block of this
particular embodiment would be mounted when in a complete nucleic
acid amplification device configuration. The thermal block further
depicts the presence of a temperature sensor (305) in the
temperature monitoring area.
Thermoelectric Coolers
[0156] The nucleic acid amplification devices described herein
generally include at least one thermoelectric cooler. A
thermoelectric cooler (TEC) generally acts as a solid-state heat
pump, functioning as a thermoelectric module that produces a
heating, cooling or stabilization effect by running electrical
energy through the device and transferring heat from one side of
the device to the other against the temperature gradient.
Thermoelectric coolers operate by the Peltier effect (or
thermoelectric effect).
[0157] TECs generally have two sides such that when direct current
(DC) flows through the device, heat is transferred from one side to
the other, resulting in one side cooling while the other heats.
Reversing the current results in an inversion of the thermal
gradient such that the previously cooling side heats and the
previously heating side cools. For sustained heating, cooling
and/or temperature stabilization, the TEC may be placed in thermal
contact with a heatsink.
[0158] In some embodiments, the TEC is made of ceramic materials
including but not limited to e.g., Alumina, Beryllium Oxide, and
Aluminum Nitride. In some instances, the TEC includes diced
ceramic.
[0159] In many embodiments, the TEC may include a central mounting
hole including where the central mounting hole is positioned with a
central axis perpendicular to the primary plane of the TEC. In some
instances, the central mounting hole of the TEC may be sized to
correspond with the central mounting hole of a thermal block to
which the TEC may be joined including but not limited to e.g.,
where the TEC and the thermal block have central mounting holes
that are essentially equal in diameter. As such, in some instances,
the central hole of the TEC will be the same or larger in diameter
as compared to the diameter of a mechanical fastener used to join
the TEC and the thermal block such that the mechanical fastener may
pass through the central mounting hole and sufficiently join the
TEC and the thermal block, e.g., a by compression force.
[0160] One embodiment of a TEC having a central mounding hole (400)
is depicted in FIG. 4. The depicted TEC can be seen to have two
sides or plates which, e.g., one of which would be positioned in
thermal contact with the thermal transfer side of a thermal block
and the other would be positioned in thermal contact with a
heatsink as described herein. The two plates of a TEC may be
different in the size in one dimension, as depicted, different in
size in more than one dimension, or may be essentially equal in all
dimensions.
[0161] Suitable thermoelectric coolers include but are not limited
to e.g., those available from commercial suppliers including but
not limited to e.g., Ferrotech Corp. (Bedford, N.H.), Marlow
Industries, Inc. (Dallas, Tex.), TE Technology, Inc. (Traverse
City, Mich.), and the like. In some instances, a TEC may be custom
ordered to contain a central mounting hole. In other instances, a
commercially available TEC may be modified to have a central
mounting hole.
[0162] In some instances, a TEC used in a nucleic acid
amplification device of the instant disclosure may be characterized
as having a rapid thermal slew rate. In some instances, a TEC of a
nucleic acid amplification device of the instant disclosure may
have an empirical thermal slew rate of greater than 7.degree.
C./sec including but not limited to e.g., greater than 8.degree.
C./sec, greater than 9.degree. C./sec, greater than 10.degree.
C./sec, etc., in cooling, heating or both cooling and heating. Such
empirical thermal slew rates may be determined by a variety of
methods including but not limited to e.g., "bang-bang" testing
where heating and cooling are monitored while the component is
cycled between minimum and maximum electrical currents. In some
instances, empirical thermal slew rates may be determined within a
temperature range or for two particular temperatures. For example,
a thermal slew rate may be determined e.g., for heating between
25.degree. C. and 95.degree. C., for cooling between 25.degree. C.
and 95.degree. C., for heating and cooling between 25.degree. C.
and 95.degree. C., etc.
[0163] In some instances, the thermal slew rate of a TEC as
described herein may be expressed as an operating or applied
thermal slew rate which is defined as the slew rate of the TEC
under normal operating conditions. In some instances, a TEC of the
instant disclosure may have an operating thermal slew rate of
greater than 5.degree. C./sec.
Thermal Flow Components
[0164] The nucleic acid amplification devices described herein may
also include additional thermal flow or heat transfer components
configured to passively direct or actively control the flow of
thermal energy within one or more components of the device. Thermal
flow components that may be included in a nucleic acid
amplification device as described herein may include but are not
limited to e.g., a heatsink, a fan, a duct, and the like.
[0165] In many embodiments, the nucleic acid amplification devices
described herein includes a heatsink. Any convenient heatsink
configuration may find use in the nucleic acid amplification
devices described herein. In some instances, a heatsink of the
instant disclosure may be configured with a plurality of fins. For
example, in some instances, a heatsink of the instant disclosure is
configured with a plurality metal fins such that heat transferred
to the heatsink is dissipated through the surface area of the fins.
A heat sink of the instant disclosure will generally be constructed
of a conductive material including but not limited to one or more
of the conductive metals described herein including but not limited
to e.g., copper.
[0166] Nucleic acid amplification devices, as described herein, may
include a TEC in thermal contact with a heatsink such that heat
generated by the TEC may be transferred to the heatsink and
dissipated. In some instances, a nucleic acid amplification device
of the instant disclosure may include a single heatsink in thermal
contact with two or more TECs, including but not limited to e.g.,
where each TEC is in separate thermal contact with a thermal
block.
[0167] In some instances, a heatsink of the instant disclosure is
designed with one or more sockets configured for the attachment of
a mechanical fastener. Such sockets may be configured for
attachment in a variety of ways including but not limited to e.g.,
where one or more of the sockets is threaded, one or more of the
sockets is configured with a compression fitting, one or more of
the sockets is configured with a snap fitting, one or more of the
sockets is notched, etc.
[0168] In some instances, a heatsink of the instant disclosure may
be configured with two or more sockets each for the attachment of a
thermal block and TEC pair. For example, a heatsink with two
sockets may be connected to two thermal block/TEC pairs, each with
a central mounting hole, for attachment to the heatsink using a
mechanical fastener inserted through the mounting holes and secured
to each socket. In some instances, thermal block/TEC pairs with
central mounting holes are affixed to threaded sockets of a
heatsink through the use of screw mechanical fasteners.
[0169] In one embodiment, as depicted in FIG. 5, a single heatsink
(500) can be seen with two separate thermal blocks (501) mounted to
the heatsink using separate mechanical fasteners (502). Parallel
lines on the top surface of the heatsink depict the edges of many
metal heat dissipation fins present in the heatsink. The TEC for
each thermal block is mounted between the heatsink and the thermal
block and is thus hidden from view. In this particular arrangement,
four reaction vessels are affixed to the single heatsink and can be
thermally cycled simultaneously.
[0170] In certain embodiments, e.g., as depicted in FIG. 6, a
nucleic acid amplification device of the instant disclosure may
further include a fan (600) and a duct (601) configured to draw or
push air through the heatsink (602) and further assist in
thermoregulation of the two thermal blocks (603) depicted in their
fully mounted arrangement. For example, the fan may be adjusted
on/off and/or the fan speed may be varied as needed to enhance the
heat dissipation function of the heatsink. The fan direction may
vary as desired and in some instances may be configured such that
air is drawn from the heatsink down through the duct and out a vent
beyond the fan. In some instances, the fan direction may be
configured such that air is pushed through the duct up and out
through the heatsink. Particular air flow directional
configurations, such as inside-out flow direction, may limit the
introduction of environmental contaminates into the device.
[0171] FIG. 7 provides a partially exploded view of the embodiment
depicted in FIG. 6 where the heatsink (700) can be seen exploded
out from the duct (701) and fan (702), which remain joined. In
addition, both thermal blocks (703 and 704) are exploded out from
the heatsink (700) allowing a view of the mounting sockets (705)
configured into the heatsink. One thermal block (703) is depicted
in thermal contact with its corresponding TEC while the other
thermal block (704) is exploded out from its TEC (706) and the
mechanical fastener (707) used to secure the thermal block and TEC
to the heatsink. Also depicted exploded out from the heatsink are
the thermal cutoff (708), discussed in more detail below, the
support bar (709) and the primary control board (710).
[0172] In addition to the thermoregulatory components utilized in
assisting thermal control of the thermal cycler, the devices and
systems described herein, including the analysis devices described
below, may include additional thermoregulatory components,
including but not limited to heatsinks, ducts, fans, vents and the
like for managing heat generated during normal processes. For
example, in some instances, a heat generating illumination unit may
have an attached heatsink to assist in dissipating heat generated
by the illumination unit. In other instances, a heat generating
circuit board or processor may be in association with a fan to
assist in dissipating heat generated by the circuit board or
processor.
Sensors, Circuitry and Control
[0173] The nucleic acid amplification devices of the instant
disclosure may include one or more sensors for receiving thermal
measurement data. In some instances, a thermal sensor, e.g., a
resistance temperature detector (RTD) may find use in measuring
and/or monitoring the temperature of a surface the nucleic acid
amplification device as described herein. For example, in some
instances, an RTD may find use in monitoring the reaction vessel
temperature, e.g., by measuring the temperature at a temperature
monitoring area of the thermal block that is in close proximity to
the reaction vessel.
[0174] RTDs appropriate for the applications, including the
devices, systems and methods as described herein, will vary and may
include but are not limited to e.g., 2 wire RTDs, 3 wire RTDs, 4
wire RTDs, thermistors, thermocouples, and the like.
[0175] Attachment of a RTD to a thermal block, e.g., at a
temperature monitoring area, such that the RTD and the thermal
block are and remain in thermal contact may be achieved by any
convenient method. For example, in some instances, the RTD may be
adhered to the thermal block using an adhesive, such as e.g., an
epoxy.
[0176] Metals that undergo thermal cycling rapidly expand and
contract. Accordingly, the maintenance of thermal contact between a
sensor and a thermally cycling metal to which the sensor is mounted
may be improved through the use of a cantilever bar mounted
independently of the thermal cycling surface. For example, a
cantilever arm may be mounted to a surface independent of the
thermal cycling surface in such a configuration that that the tip
of the cantilever arm applies pressure to the sensor such that the
sensor maintains contact with the thermal cycling surface despite
the rapid expansion and contraction of the metal of the thermal
cycling surface. In certain embodiments, the cantilever arm is a
separate element from the remaining components of the system and is
mounted to a portion of the system and holds the RDP in close
proximity to the thermal block.
[0177] Cantilever bars may be constructed of any convenient and
appropriate material, including e.g., those materials that have
flexible and/or elastic properties and/or those materials that
resist creep from continuous applied force. In some instances, a
cantilever bar may be constructed from a thermally and/or
electrically non-conductive material. Useful materials for
fashioning a cantilever bar, as described herein, include but are
not limited to e.g., polymeric materials such as plastics,
including e.g., polyaryletherketone (PAEK) family polymeric
materials (e.g., polyether ether ketone (PEEK). A cantilever bar
may be constructed from multiple different materials or may, in
some instances, be constructed from a single material, such as
e.g., only a PAEK family polymer such as e.g., PEEK. In some
instances, a cantilever bar may be constructed of a first material
and a cantilever arm extending from the cantilever bar may be
constructed of a second material, e.g., as separate components. In
some instances, the cantilever bar and a cantilever arm extending
from the cantilever bar may be constructed of the same material or
may be constructed as a single component. In some instances, a
material used in constructing a cantilever bar or a cantilever arm
of the present disclosure may have multiple beneficial properties
including e.g., where the material used both prevents heat flow
from the sensor and resists creep from continuous applied
force.
[0178] A cantilever bar may be configured to have one or more
cantilever arms extending from the cantilever bar, including but
not limited to e.g., 2 or more cantilever arms, 3 or more
cantilever arms, 4 or more cantilever arms, 2 cantilever arms, 3
cantilever arms, 4 cantilever arms, etc. A subject cantilever arm
may extend from the cantilever bar at any convenient and
appropriate angle including e.g., where the cantilever arm and the
cantilever bar are perpendicular to one another.
[0179] A cantilever arm will generally have a tip which contacts a
sensor and a base end that connects to or is borne from the
cantilever bar. A cantilever bar may have one or more features
(e.g., a hole, socket, flange, lip, etc.) for attaching the
cantilever bar to a device or a specific component of a device,
such as a component of the device that is independent of the
thermal cycling component(s) of the device. In some instances, a
cantilever arm may be directly attached to a device or a component
of a device, such as a component of the device that is independent
of the thermal cycling component(s) of the device, without the use
of a cantilever bar. In such instances, the cantilever arm may
include one or more features (e.g., a hole, socket, flange, lip,
etc.) for direct attachment to the component of the device.
[0180] In some instances, a sensor that is contacted by the tip of
a cantilever arm may be held in place (i.e., held in thermal
contact with the thermal cycling component) solely by a cantilever
arm. In some instances, despite the use of a cantilever arm, a
sensor may nonetheless be otherwise adhered to the thermal cycling
component by another means, e.g., through the use of an adhesive
(e.g., an epoxy). In some cases, a cantilever arm may prevent loss
of thermal contact between the sensor and the thermal cycling
component upon failure of an adhesive. In some cases, despite
structural failure of an adhesive used to attach a sensor to a
thermal cycling component, a cantilever arm may provide the
compression force necessary to maintain thermal contact between the
sensor and the thermal cycling component and the adhesive, although
structurally inadequate to maintain thermal contact between the
sensor and the thermal cycling component in the absence of the
cantilever arm, may provide a thermal conduit through which such
thermal contact is maintained.
[0181] Turning to the embodiment depicted in FIG. 27, a cantilever
bar (2700) may, in some instances, include four cantilever arms
(2701) each having a tip that contacts a RTD (2702) present in a
temperature monitoring area of the thermal block (2703). The left
most cantilever arm (2701) depicted in FIG. 27 is shown in cross
section, highlighting the shape of the cantilever arm which, in
concert with the PEEK polymer material used to construct the
cantilever arm, contributes to the elasticity or "spring action" of
the cantilever arm, resulting in constant pressure on the RTD at
the cantilever arm tip. As depicted, in some instances, the
cantilever arm tip may, but need not necessarily, be flat. The
cantilever bar may be affixed, e.g., via a fastener (2704),
independently from the thermal cycling component (i.e., the thermal
block), e.g., by attaching the cantilever bar to the heatsink
(2705) as depicted.
[0182] FIG. 28 provides a frontal view of an embodiment where
thermal contact between RTDs and the thermal block is maintained
through the use of a cantilever bar with a plurality of cantilever
arms. As depicted in FIG. 28, each thermal block (2802), having a
temperature monitoring area (2800) and associated channel (2801),
has two attached RTDs (hidden) that are held in place by cantilever
arms (2803) of a cantilever bar (2804). The subject cantilever bar
is attached at the heat sink (2805) independently of the thermal
blocks by three separate fasteners (2806). A profile view of one
embodiment of a cantilever arm (2900) is provided in FIG. 29,
displaying the cantilever bar (2901), fastener (2902) used to
attach the cantilever bar to the heatsink (2903), and the
associated thermal block (2904). As will be readily apparent to one
of ordinary skill in the relevant art, the use of the subject
cantilever arm and/or cantilever bar may not be limited to the
particular described application of maintaining thermal contact
between a RTD and a thermal block, but may also find use in
maintaining thermal contact between sensors and thermal cycling
components generally.
[0183] In some instances, one or more RTDs attached to the thermal
block may provide thermal feedback to a PCB to control the
temperature of the thermal block including e.g., to control the
thermal cycling temperature(s) of the thermal block. In some
instances, a thermal block having two attached RTDs, including but
not limited to e.g., a thermal block having two reaction vessel
wells each with an associated RTD, will be thermally regulated
according to feedback from both attached RTDs including but not
limited to e.g., where the temperature of the thermal block is
determined as the temperature calculated as the average from the
temperature measured at both RTDs.
[0184] In some instances, one or more RTDs of the thermal block may
be calibrated including e.g., calibrated such that the temperature
reading at the RTD is representative of the thermal block or
representative of the reaction vessel wells of the thermal block.
In addition, calibration of the RTD attached to the thermal block
may serve to assure the thermal controller is capable of bringing
the thermal block up to a correct temperature, down to a correct
temperature, etc. RTD calibration serves to provide a known output
with respect to a known temperature. In some instances, thermal
block RTD calibration is performed by inserting a calibrated
temperature sensor into the reaction vessel well and then recording
the calibrated sensor's value and the RTD's output and determining
if any difference exists between the two and if so to what degree
the two measurements differ. If differences are detected, one or
more components of the system may be adjusted or values processed
by the system (e.g., temperature readings from the RTDs) may be
adjusted to improve system functioning.
[0185] In other instances, an RTD may be used for general
monitoring of the functioning of the nucleic acid amplification
device or a component thereof. For example, in some instances, a
RTD may find use in monitoring the temperature of the heatsink,
e.g., as a means of detecting an abnormal temperature situation
occurring in the device. In some instances, a RTD may find use in
monitoring the temperature of one or more TECs, e.g., as a means of
detecting an abnormal temperature situation occurring in the
device. In some instances, a RTD may find use in monitoring the
temperature of one or more thermal blocks, e.g., as a means of
detecting an abnormal temperature situation occurring in the
device.
[0186] Abnormal temperature situations that could be detected by
system monitoring RTDs include but are not limited to e.g.,
insufficient heating, insufficient cooling, runaway heating,
runaway cooling, temperature miscalibration, etc. "Runaway"
conditions, including runaway heating and runaway cooling, as used
herein generally refer to a loss of thermal control where the
thermal output is the opposite of the desired condition, e.g.,
where the thermal output is continued heating when the system calls
for cooling and vice versa.
[0187] In some instance, a device as described herein may include a
"thermal cutoff" that deactivates the heating and/or cooling
components of a device when certain abnormal conditions are met or
a thermal error condition is detected including e.g., when a
runaway condition is detected. Such deactivation may involve the
cessation of power to the abnormally functioning component or a
disconnection of the abnormally functioning component from the
normal thermal control circuitry. Thermal cutoffs may be software
controlled or hardware controlled switches or physical fuses. For
example, in some embodiments, an RTD on a thermal block may, when a
thermal error condition is detected, trigger a shutdown of the
thermal block and associated components through a software
controlled mechanism. In other embodiments, a physical fuse, e.g.,
as present on a heatsink, may rupture when a thermal error
condition is reached triggering a shutdown of the heatsink and
components connected to the heatsink.
[0188] A thermal cutoff may be independently triggered, e.g., when
an internally monitored condition is met including e.g., a maximum
temperature, a minimum temperature, a prolonged temperature, etc.,
or may be externally triggered including e.g., triggered by a
circuit external to the thermal cutoff that receives monitoring
information from one or more sensors external to the thermal
cutoff. Any component of the herein described systems having a RTD
may be programed to respond to a thermal error condition including
for various purposes including but not limited to e.g., to indicate
to the user that an error has occurred, to trigger a shutdown of
one or more components of the system (e.g., to protect one or more
components of the system, to protect a sample being run in the
system, to protect parallel assays running in the system,
etc.).
[0189] RTDs of the instant disclosure may function to send
temperature monitoring data to one or more circuit boards to
monitor the functioning of the device and/or the progression of the
thermal cycling reaction. In some instances, data from the RTD is
processed by one or more circuit boards to indicate a deviation in
the system, including e.g., a thermal deviation from the programed
thermal cycling program. In some instances, a detected deviation
may signal a control unit to stop processing a particular sample or
all samples being processed. In other instances, including e.g.,
where a deviation is within a safe range, the deviation may trigger
circuitry to notify a user of the deviation. User notifications may
vary and, regardless of the particular type of signal indicated,
may include but are not limited to e.g., where the signal includes
the use of a visual indicator (e.g., an indicator light, a
notification on a graphical user interface, etc.), an or an audible
alarm (e.g., a buzzer), and the like.
[0190] Circuitry and/or data processing units of the instantly
described nucleic acid amplification devices include control
systems configured to control one or more components of the device
including but not limited to e.g., one or more TECs, one or more
fans, one or more sensors, etc. In some instances, circuitry of the
instant disclosure may include component specific, or device
specific circuit boards configured specifically to control a
particular component or a particular device. For example, in some
embodiments, a nucleic acid amplification device of the instant
disclosure may include a thermal control circuit board configured
(e.g., a thermal printed circuit board (thermal PCB)) where the
thermal control circuit board is physically mounted to the nucleic
acid amplification device as described herein or a component of the
device. In other instances, the PCB responsible for thermal control
may be mounted externally from the nucleic acid amplification
device and the nucleic acid amplification device may include a
mounted PCB that relays thermal information to the external thermal
control PCB. Such specific circuit boards may be linked, directed
or indirectly, to other components or circuits of the system, e.g.,
as depicted in the function block diagram of FIG. 8 representing an
integrated system, described in more detail below, that includes a
described nucleic acid amplification device.
[0191] Programming for circuits and/or components of the described
devices may be embedded programming or may be programming stored in
a computer readable storage medium including but not limited to an
external storage medium or a non-transitory computer readable
medium, and the like.
Multi-Reaction Analysis Devices
[0192] The instant disclosure includes multi-reaction analysis
devices configured for the analysis of multiple amplification
reaction vessels during the amplification reactions. For example,
multi-reaction analysis devices of the instant disclosure allow for
the monitoring of multiple real-time PCR reactions. Such
multi-reaction analysis devices include optical components,
conveyor components and signal detection/processing components
wherein such components are configured for the frequent monitoring
of multiple reaction vessels.
Optics
[0193] Multi-reaction analysis devices of the instant disclosure
include optical components sufficient for the optical analysis of
nucleic acid amplification reactions, including real-time PCR
reactions, as described herein. Such optical components will
include illumination components, including one or more excitation
components, and components for receiving emission light from the
reaction vessel.
[0194] As described herein a multi-reaction analysis device of the
instant disclosure includes an optics detection unit where an
optics detection unit includes an optical signal processor useful
in processing received optical signals into relevant electrical
signals that can be used in subsequent analyses and a plurality of
optical blocks containing optical components sufficient for
excitation of the sample and collection of emitted light from the
excited sample which is passed to the optical signal processor.
[0195] In one embodiment, an optics detection unit, as depicted in
FIG. 9, includes three optical blocks (900) in linear arrangement.
Each optical block includes a side facing optical aperture (901),
also referred to herein as a "optics block aperture", for passing
excitation light from the optics block to the reaction vessel and
passing emitted light from the reaction vessel back into the optics
block. The optics detection unit also includes an optical signal
processor (902) for processing the received emission light into
electrical signals useful to performing an analysis of the
amplification reaction as described herein.
[0196] FIG. 10 provides a rear view of the optics detection unit
depicted in FIG. 9, which shows the heatsinks (1000) positioned on
the rear of each optics block adjacent to the illuminator component
of each optics block. Such heatsinks in thermal contact with an
optics block and, specifically the illumination unit of the optics
block, serves to dissipate heat generated by the optics block,
including but not limited to e.g., heat generated by the
illumination unit of each optics block.
[0197] Illumination components of the instant disclosure include
any excitation light source sufficient to generate excitation light
that, after passing through the optics block, excites a fluorophore
present in the amplification reaction which, in turn, produces
emission light. Accordingly, the subject illumination components of
the instant disclosure will vary and may include but are not
limited to e.g., lamps, lasers, light emitting diodes (LED)
emitters, and the like.
[0198] In some instances, the illumination component contains one
or more LED emitters including but not limited to e.g., two or more
LED emitters, three or more LED emitters, four or more LED
emitters, one LED emitter, two LED emitters, three LED emitters,
four LED emitters, etc. In some instances, an illumination
component containing four LED emitters may contain two pairs of
identical LEDs or one pair of LEDs of a first wavelength and a
second pair of LEDs of a second wavelength. In instances where a
plurality of LED emitters is employed, any useful arrangement of
the LED emitters may find use in the illumination component
including but not limited to e.g., linear arrangement, staggered
arrangement, arrayed (e.g., "checker-board") arrangement, and the
like. Useful LED emitters of the subject disclosure will vary,
e.g., based on the particular assay to be performed by the device
the optical, electrical or physical constraints of the device and
the like.
[0199] LED emitters useful in an illumination component of the
subject optics block may include but are not limited to e.g., LED
emitters with a peak minimum wavelength (A) in nanometers (nm) of
between 350 and 750 nm, including but not limited to e.g., between
350 and 450, between 350 and 400, between 400 and 450, between 450
and 550, between 450 and 500, between 500 and 550, between 550 and
650, between 550 and 600, between 600 and 650, between 650 and 750,
between 650 and 700, between 700 and 750, about 400 nm, about 580
nm, about 470 nm, about 628 nm, about 528 nm, about 674 nm, and the
like.
[0200] LED emitters useful in an illumination component of the
subject optics block may include but are not limited to e.g., LED
emitters with a peak maximum wavelength (A) in nanometers (nm) of
between 350 and 750 nm, including but not limited to e.g., between
350 and 450, between 350 and 400, between 400 and 450, between 450
and 550, between 450 and 500, between 500 and 550, between 550 and
650, between 550 and 600, between 600 and 650, between 650 and 750,
between 650 and 700, between 700 and 750, about 400 nm, about 405
nm, about 592 nm, about 480 nm, about 648 nm, about 542 nm, about
689 nm, and the like.
[0201] In some instances, an illumination component of an optics
block as described herein may include two or more LED emitters. In
optical blocks having two or more LED emitters each LED emitter may
be defined as belonging to a channel including but not limited to
e.g., a first channel, a second channel, etc. In some instances,
three optical blocks each having two LED emitters may be referred
to as having six channels, including where all six channels have an
LED with a peak min/max of a different wavelength. In certain
instances, the LED emitters of a multi-channel, multi-block system
may be configured such that the average difference between the peak
min/max wavelengths of each pair of LED emitters that share an
optics block is maximized. For example, in such instances, six LED
emitters having six different peak min/max wavelengths of A, B, C,
X, Y, Z (where A<B<C<X<Y<Z) may be paired in optics
blocks as, e.g., A and X, B and Y, C and Z. In such instances,
where the average difference between the peak min/max wavelengths
of each pair of LED emitters that share an optics block is
maximized, overlap (i.e., crosstalk) between the emission
wavelength of the LED emitters in each block may be minimized.
[0202] In some instances, an optical block may contain two LED
emitters of different wavelengths where the distance between the
different wavelengths will vary and may range from 5 nm to 300 nm
or more including but not limited to e.g., at least 5 nm apart, at
least 10 nm apart, at least 15 nm apart, at least 20 nm apart, at
least 25 nm apart, at least 30 nm apart, at least 35 nm apart, at
least 40 nm apart, at least 45 nm apart, at least 50 nm apart, at
least 55 nm apart, at least 60 nm apart, at least 65 nm apart, at
least 70 nm apart, at least 75 nm apart, at least 80 nm apart, at
least 85 nm apart, at least 90 nm apart, at least 95 nm apart, at
least 100 nm apart, at least 105 nm apart, at least 110 nm apart,
at least 115 nm apart, at least 120 nm apart, at least 125 nm
apart, at least 130 nm apart, at least 135 nm apart, at least 140
nm apart, at least 145 nm apart, at least 150 nm apart, at least
155 nm apart, at least 160 nm apart, at least 165 nm apart, at
least 170 nm apart, at least 175 nm apart, at least 180 nm apart,
at least 185 nm apart, at least 190 nm apart, at least 195 nm
apart, at least 200 nm apart, not more than 300 nm apart, not more
than 290 nm apart, not more than 280 nm apart, not more than 270 nm
apart, not more than 260 nm apart, not more than 250 nm apart, not
more than 240 nm apart, not more than 230 nm apart, not more than
220 nm apart, not more than 210 nm apart, not more than 200 nm
apart, not more than 190 nm apart, not more than 180 nm apart, not
more than 170 nm apart, not more than 160 nm apart, not more than
150 nm apart, not more than 140 nm apart, not more than 130 nm
apart, not more than 120 nm apart, not more than 110 nm apart, not
more than 100 nm apart, etc.
[0203] In some instances, LED emitters of the subject disclosure
may be capable of being toggled (i.e., capable being turned on and
off, including turned on/off repeatedly). In some instances, the
wiring circuitry of an optics block having two or more LED emitters
is configured or the programming controlling such an optic block is
configured such that only one LED emitter may be toggled on at a
time. In such instances, when a first LED emitter of an optic block
is toggled on the second LED emitter of the optic block is toggled
off and vice versa.
[0204] In some instances, the toggling of LED emitters of an optic
block includes a time period where neither LED emitter of the optic
block is toggled on. In some instances, such a time period where
neither LED emitter of the optic block is toggled on is between
toggling the toggling off of a first emitter and the toggling on of
a second emitter.
[0205] LEDs of the disclosed multi-reaction analysis devices may
include physical or electrical components and/or configurations
allowing for use or one or more of the noise reduction methods
described herein, including but not limited to e.g., components
and/or configurations for time division multiplexing, components
and/or configurations for frequency division multiplexing,
components and/or configurations for spatial separation, and the
like.
[0206] Optics blocks of the instant disclosure will include a
variety of optical elements including but not limited to e.g.,
lenses, optical filters, mirrors (including e.g., dichroic
mirrors), apertures, etc. For example, in one embodiment depicted
in FIG. 11, an optics block of the instant disclosure includes an
optics block aperture (1100) allowing excitation light (generated
by the LED unit (1103)) to pass to the reaction vessel and emission
light to pass back to the optics block, a reference channel
aperture (1101) allowing light to pass from the optics block to the
signal processing unit in the reference channel, a measurement
channel aperture (1102) allowing light to pass from the optics
block to the signal processing unit in the measurement channel.
[0207] FIG. 12 provides an exploded view of the optical elements
according to one embodiment of an optics block the instant
disclosure. Such optical elements include illumination filters,
detection filters, lenses and minors. The arrangement of the
optical elements within the optics block may be sufficient to
generate measurement and reference light paths including e.g.,
measurement and reference light paths as depicted in FIG. 13. Along
the excitation light path to the measurement channel aperture, an
optics block of the instant disclosure may include a first lens
(1200) positioned proximal to the illumination component aperture,
referred to herein as a illumination aperture lens, through which
the excitation light from the illumination component passes.
Illumination light passes from the illumination aperture lens to a
illumination filter (1201) and to a first mirror (1202), referred
to herein as the excitation light mirror, which deflects the
excitation light upward towards a dichroic mirror (1203) and the
reference channel aperture (1204). Excitation light below a
wavelength threshold of the dichroic mirror is redirected through
the optics block aperture lens (1205) and out the optics block
aperture (1206), e.g., towards a reaction vessel.
[0208] Emission light, e.g., from a reaction vessel or a control
surface such as a dark target, proceeds through the optics block
aperture (1206) and the optics block aperture lens (1205). Emission
light above the threshold wavelength of the dichroic mirror (1203)
proceeds through the dichroic mirror towards the detection filter
(1207). Emission light passing through the detection filter (1207)
is redirected by a second mirror (1208), referred to herein as the
detection channel mirror, up towards the detection (i.e.,
measurement) channel aperture (1209). After passing through the
detection channel aperture lens (1210), emission light proceeds
through the detection channel aperture (1209), e.g., into a
measurement detector as part of or coupled to an optical signal
processor. Light in the reference channel, including e.g.,
illumination light configured to pass vertically through the
dichroic mirror (1203), stray light within the optical block, or
combinations thereof and the like, proceeds up through the
reference channel aperture lens (1211) and out the reference
channel aperture (1204), e.g., into a reference detector as part of
or coupled to an optical signal processor.
[0209] The arrangement of the optical elements within the optics
block is not limited to those arrangements specifically depicted
and may vary provided the necessary elements for producing the
described light paths are included and sufficient to generate, pass
and filter excitation and emission light as described herein.
[0210] Illumination filters useful in an optics block of the
instant disclosure include but are not limited to e.g.,
illuminations filters having a center wavelength (CWL) in
nanometers (nm) between 350 and 750 nm, including but not limited
to e.g., between 350 and 450, between 350 and 400, between 400 and
450, between 450 and 550, between 450 and 500, between 500 and 550,
between 550 and 650, between 550 and 600, between 600 and 650,
between 650 and 750, between 650 and 700, between 700 and 750,
about 409 nm, about 583 nm, about 475 nm, about 638 nm, about 535
nm, about 690 nm, and the like. Illumination filters useful in an
optics block of the instant disclosure also include but are not
limited to e.g., illuminations filters having a full width have
maximum (FWHM) in nm ranging from 5 nm to 100 nm, including but not
limited to e.g., about 5 nm to 10 nm, about 10 nm to 15 nm, about
15 nm to 20 nm, about 20 nm to 25 nm, about 25 nm to 30 nm, about
30 nm to 35 nm, about 35 nm to 40 nm, about 40 nm to 45 nm, about
45 nm to 50 nm, about 50 nm to 55 nm, about 55 nm to 60 nm, about
60 nm to 65 nm, about 65 nm to 70 nm, about 70 nm to 75 nm, about
75 nm to 80 nm, about 80 nm to 85 nm, about 85 nm to 90 nm, about
90 nm to 95 nm, about 95 nm to 100 nm, about 10 nm to 90 nm, about
10 nm to 80 nm, about 10 nm to 70 nm, about 10 nm to 60 nm, about
10 nm to 50 nm, about 10 nm to 40 nm, about 10 nm to 30 nm, about
10 nm to 20 nm, about 20 nm to 90 nm, about 30 nm to 90 nm, about
40 nm to 90 nm, about 50 nm to 90 nm, about 60 nm to 90 nm, about
70 nm to 90 nm, about 80 nm to 90 nm, 65 nm, 22 nm, 36 nm, 24 nm,
18 nm, 25 nm, and the like.
[0211] In some instances an illumination filter useful in an optics
block as described herein may be characterized in having a
particular combination of CWL and FWHM, including e.g.,
combinations of the CWL and the FWHM described above. For example,
in some instances an illumination filter of the subject disclosure
may be characterized as having a 409 nm CWL and a 65 nm FWHM, 583
nm CWL and a 22 nm FWHM, 475 nm CWL and a 36 nm FWHM, 638 nm CWL
and a 24 nm FWHM, 535 nm CWL and a 18 nm FWHM, 690 nm CWL and a 25
nm FWHM, and the like.
[0212] Detection filters useful in an optics block of the instant
disclosure include but are not limited to e.g., detection filters
having a center wavelength (CWL) in nanometers (nm) between 350 and
750 nm, including but not limited to e.g., between 350 and 450,
between 350 and 400, between 400 and 450, between 450 and 550,
between 450 and 500, between 500 and 550, between 550 and 650,
between 550 and 600, between 600 and 650, between 650 and 750,
between 650 and 700, between 700 and 750, about 490 nm, about 617
nm, about 524 nm, about 673 nm, about 565 nm, about 715 and the
like. Detection filters useful in an optics block of the instant
disclosure also include but are not limited to e.g., detection
filters having a full width have maximum (FWHM) in nm ranging from
5 nm to 100 nm, including but not limited to e.g., about 5 nm to 10
nm, about 10 nm to 15 nm, about 15 nm to 20 nm, about 20 nm to 25
nm, about 25 nm to 30 nm, about 30 nm to 35 nm, about 35 nm to 40
nm, about 40 nm to 45 nm, about 45 nm to 50 nm, about 50 nm to 55
nm, about 55 nm to 60 nm, about 60 nm to 65 nm, about 65 nm to 70
nm, about 70 nm to 75 nm, about 75 nm to 80 nm, about 80 nm to 85
nm, about 85 nm to 90 nm, about 90 nm to 95 nm, about 95 nm to 100
nm, about 10 nm to 90 nm, about 10 nm to 80 nm, about 10 nm to 70
nm, about 10 nm to 60 nm, about 10 nm to 50 nm, about 10 nm to 40
nm, about 10 nm to 30 nm, about 10 nm to 20 nm, about 20 nm to 90
nm, about 30 nm to 90 nm, about 40 nm to 90 nm, about 50 nm to 90
nm, about 60 nm to 90 nm, about 70 nm to 90 nm, about 80 nm to 90
nm, 42 nm, 22 nm, 24 nm, 20 nm, 18 nm, 36 nm, and the like.
[0213] In some instances an illumination filter useful in an optics
block as described herein may be characterized in having a
particular combination of CWL and FWHM, including e.g.,
combinations of the CWL and the FWHM described above. For example,
in some instances an illumination filter of the subject disclosure
may be characterized as having a 490 nm CWL and a 42 nm FWHM, 617
nm CWL and a 22 nm FWHM, 524 nm CWL and a 24 nm FWHM, 673 nm CWL
and a 20 nm FWHM, 565 nm CWL and a 18 nm FWHM, 715 nm CWL and a 36
nm FWHM, and the like.
[0214] In certain instances, the devices and systems described
herein may include an optical component external to the optical
analysis unit where such external components are useful in, e.g.,
making calibration measurements and/or measurements used in
determining proper functioning of the system or device.
[0215] In other instances, an external optical component may
include a dark target, wherein a "dark target" is a darkly colored
(i.e., black) component of the system or device from which a "dark
measurement" may be made. Such dark measurements may be employed
for calibration according to the methods as described herein, where
an optical measurement is taken with the optics block aperture
aligned to the dark target and a calibration is applied to the
system based on the dark target measurement. Any convenient and
appropriate optically dark element may be employed as a dark target
including but not limited to e.g., black polycarbonate, black
plastic, and the like.
[0216] In some instances, the dark target may contain a specific
angle or be mounted at a specific angle to further enhance a dark
measurement by preventing stray light, including e.g., reflected
light, from returning to the optics block during a dark target
measurement. A specific embodiment of a dark target configuration
is provided in FIG. 17 where the dark target is observed to have an
upper edge configured to have an angle (1700). The effect of a dark
target (1800) having an angled portion (1801) upon which the dark
measurement is made is depicted in the light-path diagram of FIG.
18. Accordingly, the dark target surface from which a dark target
measurement is made may, in some instances, not be perpendicular to
the emission light directed at the dark target.
Conveyors and Moveable Components
[0217] Multi-reaction analysis devices of the instant disclosure
include conveyors configured to scan an optical analysis unit past
a plurality of reaction vessels. In certain embodiments a linear
conveyer is paired with linearly arranged components, including
linearly arranged reaction vessels, linearly arranged optical
blocks, and the like. As described herein, in certain embodiments
the reaction vessels may remain stationary and the optical analysis
unit may travel past the reaction vessels, e.g., in a scanning or
linearly traveling motion.
[0218] For example, in one embodiment as depicted in FIG. 15, a
traveling optical analysis unit (1500) having a plurality of
linearly arranged optical blocks is attached a belt (1501) driven
conveyor so as to scan a plurality of linearly arranged thermal
blocks (1502) that contain linearly arranged reaction vessels. The
optical analysis unit (1500) travels on a track (1503), and in some
instances, may include one or more control and/or position
monitoring elements including a flag (1504) and flag sensors
(1505). Such flag and flag monitoring components provide a means
of, e.g., during automation, providing a processor with the
location of the optical analysis unit along the conveyor or
generating other useful data including e.g., the speed of travel of
the optical analysis device, etc.
[0219] Conveyor systems of the instant disclosure are not limited
to belt driven systems, e.g., as depicted in FIG. 15, and may also
include e.g., wheel/motor driven systems, actuator driven systems,
piston driven systems, lead-screw systems, etc. Any convenient
conveyor system may find use in the instant devices provided the
conveyor system is capable of passing, e.g., linearly passing, an
optical analysis unit past reaction vessels as described herein. In
yet other embodiments, a traveling linearly arranged set of
reaction vessels is passed, i.e., scanned, by a stationary optical
analysis unit.
[0220] In certain embodiments, multi-reaction analysis systems may
further include additional moveable components or moving elements.
For example, in some instances a multi-reaction analysis system as
described herein may include a reaction vessel clamping mechanism,
a reaction vessel extraction mechanism, etc.
[0221] One embodiment of a reaction vessel clamping and extraction
mechanism includes but is not limited to that depicted in FIG. 19,
where the top clamping bar (1900) is lifted and lowered through the
use of a mechanical linkage (1901) to clamp and unclamp reaction
vessels placed in the reaction vessel wells. The mechanism may
further include a reaction vessel extraction bar (1902) that may be
raised following completion of amplification and analysis of a set
of samples to facilitate extraction of the reaction vessels from
the reaction vessel wells. Any convenient mechanical linkages, and
combinations thereof, for the clamping bar and/or the extraction
bar may find use in reaction vessel clamping and/or reaction vessel
extraction as described including but not limited to e.g.,
multi-bar linkages, lifter plates, actuators, cam arms, pistons,
alignment pins, etc.
[0222] Clamping mechanisms may serve various purposes including but
not limited to e.g., securing the reaction vessels against movement
during various processes as described herein, shielding the
reaction vessels from light, e.g., to promote accurate optical
signal analysis, reducing thermal resistance between the reaction
vessel and the reaction vessel well, and the like. Extraction
mechanisms may serve various purposes including but not limited to
e.g., removing reaction vessels from reaction vessel wells where
thermal cycling causes the reaction vessels to adhere to the
reaction vessel wells.
[0223] In one embodiment, the compressive force of the clamping
mechanism is sufficient to reduce thermal interface resistance
between the reaction vessel and the reaction vessel well. In some
instances, sufficient compressive force reduces the thermal
resistance to less than 5.0 watts per degree Celsius (W/.degree.
C.) including but not limited to e.g., less than 4.9 W/.degree. C.,
less than 4.8 W/.degree. C., less than 4.7 W/.degree. C., less than
4.6 W/.degree. C., less than 4.5 W/.degree. C., less than 4.4
W/.degree. C., less than 4.3 W/.degree. C., less than 4.2
W/.degree. C., less than 4.1 W/.degree. C., less than 4.0
W/.degree. C., less than 3.9 W/.degree. C., less than 3.8
W/.degree. C., etc., as measured using the Yovanovich Method of air
interface. The compressive force sufficient to reduce the interface
thermal resistance will vary and may range from less than 2 newtons
(N) to more than 50 N including but not limited to e.g., 2 N to 50
N, 3 N to 50 N, 4 N to 50 N, 5 N to 50 N, 6 N to 50 N, 7 N to 50 N,
8 N to 50 N, 9 N to 50 N, 10 N to 50 N, 5 N to 45 N, 5 N to 40 N, 5
N to 35 N, 5N to 30 N, 5 N to 25 N, 5 N to 20 N, 5N to 15 N, 5 N to
10 N, 4 N to 45 N, 4 N to 40 N, 4 N to 35 N, 4 N to 30 N, 4 N to 25
N, 4 N to 20 N, 4N to 15 N, 4N to 10 N, 3 N to 45 N, 3 N to 40 N, 3
N to 35 N, 3N to 30 N, 3 N to 25 N, 3 N to 20 N, 3 N to 15 N, 3 N
to 10 N, etc. In some instances, the compressive force sufficient
to reduce the interface thermal resistance to less than 5.0
W/.degree. C. will vary and will range from less than 4 N to 20 N
or more including but not limited to e.g., 4 N to 50 N, 5 N to 50
N, 6 N to 50 N, 7 N to 50 N, 8 N to 50 N, 9 N to 50 N, 10 N to 50
N, 5 N to 45 N, 5 N to 40 N, 5 N to 35 N, 5N to 30 N, 5 N to 25 N,
5 N to 20 N, 5N to 15 N, 5 N to 10 N, 4 N to 45 N, 4 N to 40 N, 4 N
to 35 N, 4 N to 30 N, 4 N to 25 N, 4 N to 20 N, 4 N to 15 N, 4 N to
10 N, etc. Accordingly, the compressive force of a clamping
mechanism will vary and may range from 2 N or less to 20 N or more
including but not limited to e.g., 2 N or more, 3 N or more, 4 N or
more, 5 N or more, 6 N or more, 7 N or more, 8 N or more, 9 N or
more, 10 N or more, etc.
[0224] Clamping bar(s) of the instant disclosure may or may not be
heated, e.g., electrically heated. For example, in some instances,
the top clamp bar is heated. Such heating of clamp bars may serve
various purposes including but not limited to e.g., to heat the
reaction vessel cap and/or upper portion of the reaction vessel
including but not limited to e.g., the assist in thermoregulation
of the reaction vessel, to establish an even thermal gradient, to
prevent condensation of reaction components on cool surfaces of the
reaction vessel cap, to prevent condensation of reaction components
on cool surfaces of the reaction vessel, and the like. In some
instances, the system may not require such functions and, as such,
the clamping bar(s) of the system may not be heated.
Signal Detection and Processing
[0225] Multi-reaction analysis devices of the instant disclosure
include signal detection and signal and data processing units. For
example, when emission light is passed back through an optical
block as described herein, the emission light is directed to an
optical detector configured to measure the emission light, e.g.,
measure the intensity of the emission light. In general, useful
optical detectors for such purposed will, when light falls on the
detector, convert the light into electrical current. Accordingly,
useful optical detectors include photodiodes.
[0226] Photodiodes of signal detection devices as described herein
may be dedicated to a particular light path or channel or
wavelength or may be shared, e.g., shared between two or more
wavelengths. Sharing of a photodiode of the instant disclosure may
include one or more methods of reducing noise, e.g., cross-talk, as
described herein, including but not limited to e.g., time division
multiplexing, frequency division multiplexing, and the like.
Photodiodes may be configured as part a circuit board, including
but not limited where one or more photodiodes are operably attached
to an optics circuit board or a detector circuit board.
[0227] FIG. 20 provides a configuration of multi-reaction analysis
device circuit boards, including a detection printed circuit board
(PCB), an inner PCB and an outer PCB configured to attach to the
components of a multi-reaction analysis device as described herein
and provide the signal detection and processing functions of the
above described methods. Such PCBs, in some instances, may be
configured to perform one or more of the functions depicted in the
block diagram of FIG. 21.
[0228] Signal processing functions including e.g., noise filtering,
peak finding algorithms, and the like, may be performed with local
circuitry including e.g., where such functions or portions thereof
are performed on a local PCB including e.g., a PCB attached one or
more components of a multi-reaction analysis unit as described
herein. Alternatively, Signal processing functions including e.g.,
noise filtering, peak finding algorithms, and the like, may be
performed by remote circuitry including e.g., where such functions
or portions thereof are performed on a remote PCB or other signal
processing circuitry. Signals may be transferred from local to
remote circuitry through a variety of means including but not
limited to e.g., wired connections, wireless connections, etc.
Integrated Systems
[0229] The instant disclosure includes integrated systems that make
use of the devices described herein, including but not limited to
integrated multi-reaction nucleic acid amplification and analysis
systems. As applications of the described devices relate at least
in part to real-time PCR, a method requiring the monitoring of an
ongoing PCR reaction, integration of the described nucleic acid
amplification devices and multi-reaction analysis devices may
result, in many instances, in an integrated system for real-time
PCR analysis.
[0230] Integrated systems will provide for the simultaneous
functioning of both systems together where the systems are
connected by a physical connection, electrical connection or both.
For example, an integrated multi-reaction nucleic acid
amplification and analysis system may include a nucleic acid
amplification system physically mounted to a multi-reaction
analysis unit as described herein. In other instances, an
integrated multi-reaction nucleic acid amplification and analysis
system may include a nucleic acid amplification system electrically
connected (e.g., thought a wired connection) to a multi-reaction
analysis unit as described herein. Physical and/or electrical
connections in integrated systems promotes or allows for such
devices to function together either through physical coordination
and/or data or command transfer across devices.
[0231] The individual devices and components thereof may be
reconfigured into integrated devices in various ways provided such
reconfiguration does not render the element of the device or the
overall system incapable of performing the functions described
herein. In some instances, devices and systems, including
integrated systems and methods as described herein may make use of
a device or system or one or more components thereof as described
in e.g., Attorney Docket No. ADDV-054WO, which claims priority to
U.S. Ser. No. 62/308,617 and U.S. Ser. No. 62/357,772, the
disclosures of which are incorporated herein by reference in their
entireties. In some instances, devices and/or systems of the
instant disclosure may be configured to be used in conjunction with
or be connected to one or more sample preparation units as
described in e.g., Attorney Docket No. ADDV-055WO, which claims
priority to U.S. Ser. No. 62/308,618, the disclosures of which are
incorporated herein by reference in their entireties.
Computer Related Embodiments
[0232] In some instances, the components of the systems as
described herein may be connected by a wired data connection. Any
suitable and appropriate wired data connection may find use in
connecting the components of the described systems, e.g., as
described herein, including but not limited to e.g., commercially
available cables such as a USB cable, a coaxial cable, a serial
cable, a C2G or Cat2 cable, a Cat5/Cat5e/Cat6/Cat6a cable, a Token
Ring Cable (Cat4), a VGA cable, a HDMI cable, a RCA cable, an
optical fiber cable, and the like. In some instances, e.g., where
data security is less of a concern, wireless data connections may
be employed including but not limited to e.g., radio frequency
connections (e.g., PAN/LAN/MAN/WAN wireless networking, UHF radio
connections, etc.), an infrared data transmission connection,
wireless optical data connections, and the like.
[0233] The devices and systems of the instant disclosure may
further include a "memory" that is capable of storing information
such that it is accessible and retrievable at a later date by a
computer. Any convenient data storage structure may be chosen,
based on the means used to access the stored information. In
certain aspects, the information may be stored in a "permanent
memory" (i.e. memory that is not erased by termination of the
electrical supply to a computer or processor) or "non-permanent
memory". Computer hard-drive, CD-ROM, floppy disk, portable flash
drive and DVD are all examples of permanent memory. Random Access
Memory (RAM) is an example of non-permanent memory. A file in
permanent memory may be editable and re-writable.
[0234] Substantially any circuitry can be configured to a
functional arrangement within the devices and systems for
performing the methods disclosed herein. The hardware architecture
of such circuitry, including e.g., a specifically configured
computer, is well known by a person skilled in the art, and can
comprise hardware components including one or more processors
(CPU), a random-access memory (RAM), a read-only memory (ROM), an
internal or external data storage medium (e.g., hard disk drive).
Such circuitry can also comprise one or more graphic boards for
processing and outputting graphical information to display means.
The above components can be suitably interconnected via a bus
within the circuitry, e.g., inside a specific-use computer. The
circuitry can further comprise suitable interfaces for
communicating with general-purpose external components such as a
monitor, keyboard, mouse, network, etc. In some embodiments, the
circuitry can be capable of parallel processing or can be part of a
network configured for parallel or distributive computing to
increase the processing power for the present methods and programs.
In some embodiments, the program code read out from the storage
medium can be written into a memory provided in an expanded board
inserted in the circuitry, or an expanded unit connected to the
circuitry, and a CPU or the like provided in the expanded board or
expanded unit can actually perform a part or all of the operations
according to the instructions of the programming, so as to
accomplish the functions described.
[0235] In addition to the components of the devices and systems of
the instant disclosure, e.g., as described above, systems of the
disclosure may include a number of additional components, such as
data output devices, e.g., monitors and/or speakers, data input
devices, e.g., interface ports, keyboards, etc., actuatable
components, power sources, etc.
[0236] The instant disclosure includes computer readable medium,
including non-transitory computer readable medium, which stores
instructions for methods described herein. Aspects of the instant
disclosure include computer readable medium storing instructions
that, when executed by a computing device, cause the computing
device to perform one or more steps of a method as described
herein.
[0237] In certain embodiments, instructions in accordance with the
methods described herein can be coded onto a computer-readable
medium in the form of "programming", where the term "computer
readable medium" as used herein refers to any storage or
transmission medium that participates in providing instructions
and/or data to a computer for execution and/or processing. Examples
of storage media include a floppy disk, hard disk, optical disk,
magneto-optical disk, CD-ROM, CD-R, magnetic tape, non-volatile
memory card, ROM, DVD-ROM, Blue-ray disk, solid state disk, and
network attached storage (NAS), whether or not such devices are
internal or external to the computer. A file containing information
can be "stored" on computer readable medium, where "storing" means
recording information such that it is accessible and retrievable at
a later date by a computer.
[0238] The computer-implemented method described herein can be
executed using programming that can be written in one or more of
any number of computer programming languages. Such languages
include, for example, Java (Sun Microsystems, Inc., Santa Clara,
Calif.), Visual Basic (Microsoft Corp., Redmond, Wash.), and C++
(AT&T Corp., Bedminster, N.J.), as well as any many others.
[0239] Notwithstanding the appended claims, the disclosure is also
defined by the following clauses:
[0240] 1. A thermal block for simultaneous nucleic acid
amplification and reaction analysis; the thermal block
comprising:
[0241] a) at least two reaction vessel wells, each well comprising:
[0242] i) a top opening configured to receive a reaction vessel
inserted vertically into the well; [0243] ii) a side aperture
configured to allow light to pass laterally into the reaction
vessel well, wherein upon insertion of a reaction vessel into the
well a majority of the sidewall of the reaction vessel is in
thermal contact with the well and a portion of the sidewall is
exposed to light by the aperture;
[0244] b) a thermal transfer surface opposite the side apertures of
the two reaction vessel wells; and
[0245] c) a mounting hole positioned between the two reaction
vessel wells and having a center axis perpendicular to the plane of
the thermal transfer surface.
[0246] 2. The thermal block of clause 1, wherein the block is
bilaterally symmetrical along a vertical axis.
[0247] 3. The thermal block of any of clauses 1-2, wherein the
thermal transfer surface is essentially flat.
[0248] 4. The thermal block of any of clauses 1-3, wherein the
mounting hole is positioned equidistant from the two reaction
vessels.
[0249] 5. The thermal block of clause 4, wherein the mounting hole
is centrally positioned between the top and bottom sides of the
thermal block.
[0250] 6. The thermal block of clause 4, wherein the mounting hole
is centrally positioned between the right and left sides of the
thermal block.
[0251] 7. The thermal block of any of clauses 1-6, wherein the top
surface of the thermal block comprises a raised flange encircling
the circumference of each of the two reaction vessel wells.
[0252] 8. The thermal block of any of clauses 1-7, wherein the
surface of the thermal block opposite the thermal surface comprises
a plurality of raised ridges.
[0253] 9. The thermal block of clause 8, wherein the plurality of
raised ridges emanate radially from the mounting hole.
[0254] 10. The thermal block of any of clauses 1-9, wherein the
thermal block is constructed of aluminum.
[0255] 11. The thermal block of any of clauses 1-10, wherein at
least the two reaction vessel wells are nickel plated.
[0256] 12. The thermal block of clause 11, wherein at least a
portion of the thermal block other than the two reaction vessel
wells is nickel plated.
[0257] 13. The thermal block of clause 12, wherein the entire
thermal block is nickel plated.
[0258] 14. The thermal block of any of clauses 1-13, wherein the
two reaction vessel wells comprise a lubrication coating.
[0259] 15. The thermal block of clause 14, wherein the lubrication
coating is a dry lubrication coating.
[0260] 16. The thermal block of any of clauses 1-15, wherein the
thermal block further comprises a temperature detection area
configured for the functional attachment of a temperature detector
positioned proximally to each reaction vessel well.
[0261] 17. The thermal block of clause 16, wherein the temperature
detection areas are positioned below the reaction vessel wells.
[0262] 18. The thermal block of any of clauses 1-17, wherein each
reaction vessel well further comprises a basal reservoir configured
such that upon insertion of the reaction vessel into the well the
reaction vessel does not contact the bottom of the well.
[0263] 19. The thermal block of any of clauses 1-18, wherein the
thermal block has a mass of 2 to 4 grams.
[0264] 20. A nucleic acid amplification module, the module
comprising:
[0265] a) a thermoelectric cooler unit comprising a mounting
hole;
[0266] b) a thermal block of any of clauses 1-19, wherein the
thermal transfer surface is in thermal contact with a first surface
of the thermoelectric cooler unit; and
[0267] c) a heatsink configured to receive a mechanical fastener,
wherein the heatsink is in thermal contact with a second surface of
the thermoelectric cooler unit and the mounting holes are aligned
such that the thermal block, thermoelectric cooler unit, and the
heatsink are joined by a mechanical fastener positioned through the
mounting holes and affixed to the heatsink.
[0268] 21. The module of clauses 20, further comprising a
conductive pad between the thermal block and the thermoelectric
cooler unit or between the thermoelectric cooler unit and the
heatsink.
[0269] 22. The module of clause 21, wherein the conductive pad is a
graphite pad.
[0270] 23. The module of any of clauses 20-22, wherein the module
comprises conductive pads both between the thermal block and the
thermoelectric cooler unit and between the thermoelectric cooler
unit and the heatsink.
[0271] 24. The module of any of clauses 20-23, wherein the
mechanical fastener joins the thermal block, thermoelectric cooler
unit, and the heatsink by a compression force.
[0272] 25. The module of clause 24, wherein the mechanical fastener
is a compression screw.
[0273] 26. The module of any of clauses 24-25, wherein the
compression force is between 100 and 200 pounds per square inch
(psi).
[0274] 27. The module of any of clauses 20-26, wherein the thermal
block and the thermoelectric cooler are supported by a support bar
fastened to the heatsink.
[0275] 28. The module of any of clauses 20-27, wherein the module
further comprises a heatsink fan configured to force air past the
heatsink.
[0276] 29. The module of clause 28, wherein the heatsink is joined
to the heatsink fan by a duct.
[0277] 30. The module of any of clauses 20-29, wherein the thermal
block has an operating thermal slew rate of greater than 5.degree.
C. per second.
[0278] 31. The module of any of clauses 20-30, wherein the module
further comprises one or more resistance thermometers (RTDs) in
thermal contact with the thermal block.
[0279] 32. The module of clause 31, wherein the module comprises
two RTDs in thermal contact with the thermal block, wherein each of
the two RTDs are in proximity with a reaction vessel well of the
thermal block.
[0280] 33. The module of clauses 31 or 32, wherein the thermal
contact between the one or more RTDs and the thermal block is
maintained by a cantilever bar comprising one or more cantilever
arms.
[0281] 34. The module of any of clauses 20-33, wherein the module
comprises an attached printed circuit board (PCB) for monitoring or
controlling at least one electrical component of the module.
[0282] 35. The module of clause 34, wherein the PCB is conformal
coated.
[0283] 36. The module of any of clauses 31-35, wherein the module
comprises at least one RTD in thermal contact with the thermal
block and electrically connected to the PCB.
[0284] 37. The module of any of clauses 20-36, wherein the module
comprises a RTD in thermal contact with the thermal block or the
heatsink, wherein the RTD in thermal contact with the thermal block
or the heatsink is configured to monitor the temperature of the
thermal block or heatsink and trigger a cutoff of power to the
thermal block or heatsink if the temperature indicates a thermal
error condition.
[0285] 38. The module of any of clauses 20 to 37, wherein the
heatsink is configured to receive a second mechanical fastener and
the nucleic acid amplification module further comprises a second
thermal block in thermal contact with a second thermoelectric
cooler unit in thermal contact with the heatsink, wherein the
second thermal block, the second thermoelectric cooler unit and the
heatsink are joined by a second mechanical fastener positioned
through mounting holes in the second thermal block and the second
thermoelectric cooler unit and affixed to the heatsink.
[0286] 39. The module of clause 38, wherein the first thermal block
and the second thermal block comprise separate electrical
connections and are controlled independently.
[0287] 40. The module of any of clauses 20-39, wherein the module
further comprises one or more reaction vessel clamping bars.
[0288] 41. The module of clause 40, wherein the one or more
reaction vessel clamping bars provides a compression force on
reaction vessels within the reaction vessel wells of 5 N or
more.
[0289] 42. A multi-reaction analysis module for the optical
analysis of a plurality of amplification reaction vessels, the
module comprising:
[0290] a) an optics detection unit comprising an optical signal
processor and a plurality of linearly arranged optical blocks, each
optical block comprising: [0291] i) an illumination component
configured to illuminate a reaction with excitation light; [0292]
ii) a optics block aperture configured to pass excitation light to
the reaction vessel and receive emission light from the reaction
vessel; [0293] iii) a measurement channel configured to pass
emission light to the optical signal processor; and [0294] iv) a
reference channel configured to pass reference light to the optical
signal processor;
[0295] b) a linear conveyer configured to convey the optics
detection unit linearly past each reaction vessel of the plurality
of amplification reactions, wherein each optics block aperture of
the plurality of linearly arranged optical blocks is optically
exposed to the sidewall of each reaction vessel.
[0296] 43. The module of clause 42, wherein the illumination
component comprises one or more light emitting diode (LED)
emitters.
[0297] 44. The module of clause 43, wherein the illumination
component comprises two or more light emitting diode (LED) emitters
of different emission wavelengths.
[0298] 45. The module of clauses 44, wherein the wavelengths of the
two or more LED emitters are at least 50 nm apart.
[0299] 46. The module of any of clauses 42-45, wherein the
illumination component comprises frequency modulation.
[0300] 47. The module of any of clauses 42-46, wherein the
illumination component comprises time division modulation.
[0301] 48. The module of any of clauses 42-47, wherein the optics
detection unit comprises two or more optical blocks.
[0302] 49. The module of clause 48, wherein the optics detection
unit comprises three optical blocks.
[0303] 50. An integrated multi-reaction nucleic acid amplification
and analysis system, the system comprising:
[0304] a) a nucleic acid amplification module of any of clauses
20-40; and
[0305] b) a multi-reaction analysis module of any of clauses 41-48,
wherein the spacing between the side apertures of the reaction
vessel wells is unequal to the spacing between the optics block
apertures of the optical blocks such that no more than one side
aperture and one optics block aperture may be in alignment at any
one time.
[0306] 51. The system of clause 50, wherein the spacing between the
side apertures is greater than the spacing between the optics block
apertures.
[0307] 52. The system of clause 50, wherein the spacing between the
side apertures is less than the spacing between the optics block
apertures.
[0308] 53. The system of any of clauses 50-52, wherein the nucleic
acid amplification module comprises two thermal blocks affixed to a
single heatsink.
[0309] 54. The system of any of clauses 50-53, wherein the
multi-reaction analysis module comprises three optical blocks.
[0310] 55. The system of any of clauses 50-54, wherein each
illumination component comprises two or more LED emitters of
differing wavelengths.
[0311] 56. The system of clause 55, wherein the wavelengths of the
two or more LED emitters are at least 50 nm apart.
[0312] 57. An integrated multi-reaction nucleic acid amplification
and analysis system, the system comprising:
[0313] a) a multi-reaction nucleic acid amplification module
comprising two or more thermal blocks comprising two or more
linearly arranged and evenly spaced reaction vessel wells each
having a side aperture configured to allow light to pass laterally
into the reaction vessel well;
[0314] b) a traveling optics detection unit comprising: [0315] i)
an optical signal processor; [0316] ii) a plurality of linearly
arranged even spaced optical blocks, each optical block comprising
an illumination component and a optics block aperture configured to
pass excitation light to a reaction vessel and receive emission
light from the reaction vessel; and [0317] iii) a linear conveyer
configured to convey the traveling optics detection unit linearly
past the side aperture of each of the two or more reaction vessel
wells,
[0318] wherein the spacing between the side apertures of the
reaction vessel wells is unequal to the spacing between the optics
block apertures of the optical blocks such that no more than one
side aperture and one optics block aperture may be in alignment at
any one time.
[0319] 58. The system of clause 57, wherein the traveling optics
detection unit comprises three optical blocks.
[0320] 59. The system of any of clauses 57-58, wherein the
illumination component comprises two or more LED emitters of
differing wavelengths.
[0321] 60. The system of clause 59, wherein the wavelengths of the
two or more LED emitters are at least 50 nm apart.
[0322] 61. A method of monitoring nucleic acid amplification in a
plurality of amplification reaction vessels, the method
comprising:
[0323] a) linearly scanning a traveling optics detection unit
having an excitation component and a plurality of optics block
apertures past the plurality of amplification reaction vessels,
wherein no more than one optics block aperture is in optical
alignment with a amplification reaction vessel at any one time;
[0324] b) receiving a scan signal from the optics detection unit
comprising background noise and emission peaks;
[0325] c) determining emission peaks within the background noise
according to a plurality of reaction vessel windows;
[0326] d) measuring an intensity value for each reaction vessel
window to monitor the amplification in each reaction vessel of the
plurality.
[0327] 62. The method of clause 61, wherein the optics unit
comprises a plurality of excitation components that are time
modulated.
[0328] 63. The method of any of clauses 61-62, wherein the optics
unit comprises a plurality of excitation components that are
frequency modulated.
[0329] 64. The method of any of clauses 61-63, wherein each optics
block aperture is part of an optics block and the method further
comprises cross-talk subtraction.
[0330] 65. The method of any of clauses 61-64, wherein the method
further comprises one or more calibration measurement steps before
or during the linearly scanning step.
[0331] 66. The method of clause 65, wherein the one or more
calibration measurement steps comprise aligning the plurality of
optics block apertures with a dark target.
[0332] 67. The method of clause 66, wherein the dark target
comprises black polycarbonate.
[0333] 68. The method of any of clauses 65-67, wherein the one or
more calibration measurement steps comprises toggling the
excitation component on or off.
[0334] 69. The method of clauses 68, wherein the one or more
calibration measurement steps comprises measuring a reference
channel and adjusting the power supplied to the excitation
component based on the measured reference channel.
[0335] 70. The method of any of clauses 65-69, wherein at least one
of the one or more calibration measurement steps is performed at
least once per scan.
[0336] 71. The method of any of clauses 65-70, wherein at least one
of the one or more calibration measurement steps is performed at
least twice per scan.
[0337] 72. The method of any of clauses 65-71, wherein the
measurement from the calibration measurement step is applied to a
value obtained from the scan during a signal processing
pathway.
[0338] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
[0339] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *