U.S. patent application number 14/796239 was filed with the patent office on 2015-11-05 for microfluidic system for amplifying and detecting polynucleotides in parallel.
The applicant listed for this patent is Handylab, Inc.. Invention is credited to Sundaresh N. Brahmasandra, Karthik Ganesan, Kalyan Handique, Jeff Williams.
Application Number | 20150315631 14/796239 |
Document ID | / |
Family ID | 40580274 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150315631 |
Kind Code |
A1 |
Handique; Kalyan ; et
al. |
November 5, 2015 |
MICROFLUIDIC SYSTEM FOR AMPLIFYING AND DETECTING POLYNUCLEOTIDES IN
PARALLEL
Abstract
The present technology provides for an apparatus for detecting
polynucleotides in samples, particularly from biological samples.
The technology more particularly relates to microfluidic systems
that carry out PCR on nucleotides of interest within microfluidic
channels, and detect those nucleotides. The apparatus includes a
microfluidic cartridge that is configured to accept a plurality of
samples, and which can carry out PCR on each sample individually,
or a group of, or all of the plurality of samples
simultaneously.
Inventors: |
Handique; Kalyan;
(Ypsilanti, MI) ; Brahmasandra; Sundaresh N.; (Ann
Arbor, MI) ; Ganesan; Karthik; (Ann Arbor, MI)
; Williams; Jeff; (Chelsea, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Handylab, Inc. |
Franklin Lakes |
NJ |
US |
|
|
Family ID: |
40580274 |
Appl. No.: |
14/796239 |
Filed: |
July 10, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13692929 |
Dec 3, 2012 |
9080207 |
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14796239 |
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13035725 |
Feb 25, 2011 |
8323900 |
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13692929 |
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11985577 |
Nov 14, 2007 |
7998708 |
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13035725 |
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11728964 |
Mar 26, 2007 |
9040288 |
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11985577 |
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60786007 |
Mar 24, 2006 |
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60859284 |
Nov 14, 2006 |
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60859284 |
Nov 14, 2006 |
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60959437 |
Jul 13, 2007 |
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Current U.S.
Class: |
506/9 ;
506/26 |
Current CPC
Class: |
B01L 3/502723 20130101;
B01L 2400/0487 20130101; B01L 3/502715 20130101; B01L 2300/0887
20130101; G01N 21/6428 20130101; B01L 2300/0816 20130101; B01L
2300/087 20130101; C12Q 1/686 20130101; G01N 2021/6419 20130101;
B01L 9/527 20130101; F16K 99/0044 20130101; F16K 2099/0084
20130101; B01L 2300/1861 20130101; F16K 99/003 20130101; B01L
2300/021 20130101; F16K 99/0001 20130101; B01L 2300/0681 20130101;
Y02A 90/10 20180101; B01L 2400/0442 20130101; B01L 2400/0481
20130101; F16K 99/0061 20130101; B01L 2300/1822 20130101; B01L
2300/0867 20130101; B01L 2300/1827 20130101; C12Q 1/6806 20130101;
F16K 99/0032 20130101; B01L 3/502738 20130101; B01L 2400/0611
20130101; B01L 2400/0677 20130101; B01L 2400/0683 20130101; G01N
2021/6421 20130101; B01L 2200/147 20130101; B01L 2200/027 20130101;
B01L 2200/10 20130101; B01L 2200/16 20130101; B01L 2300/045
20130101; B01L 7/52 20130101; B01L 2200/148 20130101; G01N
2035/00881 20130101; G01N 2021/6441 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of carrying out amplification on a plurality of
samples, the method comprising: introducing each of the plurality
of samples into an amplification zone of a multi-lane microfluidic
cartridge; isolating each of the plurality of samples within the
multi-lane microfluidic cartridge; independently thermally cycling
each amplification zone; and amplifying polynucleotides contained
within each of the plurality of samples.
2. The method of claim 1, further comprising detecting the presence
of a polynucleotide or a polynucleotide probe in the plurality of
samples.
3. The method of claim 1, wherein independently thermally cycling
each amplification zone comprises heating each amplification zone
with a plurality of heaters.
4. The method of claim 1, wherein independently thermally cycling
each amplification zone comprises heating each amplification zone
with four heaters.
5. The method of claim 1, wherein independently thermally cycling
each amplification zone comprises heating each amplification zone
with two long heaters and two short heaters.
6. The method of claim 1, wherein isolating each of the plurality
of samples comprises closing at least one valve.
7. A method of carrying out amplification on a plurality of
samples, the method comprising: introducing a sample of a plurality
of samples into an amplification zone in a lane of a multi-lane
microfluidic cartridge; isolating the sample from the plurality of
samples within the multi-lane microfluidic cartridge; thermally
controlling the amplification zone independently from another
amplification zone in another lane of the multi-lane microfluidic
cartridge; and amplifying polynucleotides contained within the
sample within the amplification zone while thermal cycling the
amplification zone.
8. The method of claim 7, further comprising detecting the presence
of a polynucleotide or a polynucleotide probe in the sample.
9. The method of claim 7, wherein thermally controlling the
amplification zone comprises heating the amplification zone with a
plurality of heaters.
10. The method of claim 7, wherein thermally controlling the
amplification zone comprises heating the amplification zone with
four heaters.
11. The method of claim 7, wherein thermally controlling the
amplification zone comprises heating the amplification zone with
two long heaters and two short heaters.
12. The method of claim 7, wherein isolating the sample from the
plurality of samples comprises closing at least one valve.
13. The method of claim 7, further comprising: introducing a second
sample of a plurality of samples into a second amplification zone
in a second lane of a multi-lane microfluidic cartridge; isolating
the second sample from the plurality of samples within the
multi-lane microfluidic cartridge; thermally controlling the second
amplification zone independently from another amplification zone in
another lane of the multi-lane microfluidic cartridge; and
amplifying polynucleotides contained within the second sample
within the second amplification zone while thermal cycling the
second amplification zone.
14. The method of claim 13, isolating the second sample from the
plurality of samples comprises closing at least one valve.
15. A method of carrying out nucleic acid amplification on a
plurality of samples, the method comprising: introducing the
plurality of samples into a plurality of amplification zones of a
multi-lane microfluidic cartridge; isolating the plurality of
samples within the plurality of amplification zones; and
independently heating each amplification zone in order to amplify
polynucleotides contained within the sample within the
amplification zone.
16. The method of claim 15, further comprising detecting the
presence of a polynucleotide or a polynucleotide probe in the
plurality of samples.
17. The method of claim 15, wherein independently heating each
amplification zone comprises heating each amplification zone with a
plurality of heaters.
18. The method of claim 15, wherein independently heating each
amplification zone comprises heating each amplification zone with
four heaters.
19. The method of claim 15, wherein independently heating each
amplification zone comprises heating each amplification zone with
two long heaters and two short heaters.
20. The method of claim 15, wherein isolating the plurality of
samples comprises closing at least one valve.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/692,929, filed Dec. 3, 2012 and scheduled
to issue as U.S. Pat. No. 9,080,207 on Jul. 14, 2015, which is a
continuation of U.S. patent application Ser. No. 13/035,725, filed
Feb. 25, 2011, issued as U.S. Pat. No. 8,323,900 on Dec. 4, 2012,
which is a continuation of U.S. patent application Ser. No.
11/985,577, filed Nov. 14, 2007, issued as U.S. Pat. No. 7,998,708
on Aug. 16, 2011, which is a continuation-in-part of U.S. patent
application Ser. No. 11/728,964, filed Mar. 26, 2007, issued as
U.S. Pat. No. 9,040,288 on May 26, 2015, which claims the benefit
of U.S. Provisional Patent Application No. 60/786,007, filed Mar.
24, 2006, and U.S. Provisional Patent Application No. 60/859,284,
filed Nov. 14, 2006. U.S. patent application Ser. No. 11/985,577
claims the benefit of U.S. Provisional Patent Application No.
60/859,284, filed Nov. 14, 2006, and U.S. Provisional Patent
Application No. 60/959,437, filed Jul. 13, 2007. The disclosures of
U.S. patent application Ser. No. 13/692,929, U.S. patent
application Ser. No. 13/035,725, U.S. patent application Ser. No.
11/985,577, U.S. patent application Ser. No. 11/728,964, U.S.
Provisional Patent Application No. 60/859,284, and U.S. Provisional
Patent Application No. 60/959,437 are considered part of the
disclosure of this application, and are incorporated by reference
herein in their entirety.
TECHNICAL FIELD
[0002] The technology described herein generally relates to systems
for detecting polynucleotides in samples, particularly from
biological samples. The technology more particularly relates to
microfluidic systems that carry out PCR on nucleotides of interest
within microfluidic channels, and detect those nucleotides.
BACKGROUND
[0003] The medical diagnostics industry is a critical element of
today's healthcare infrastructure. At present, however, diagnostic
analyses no matter how routine have become a bottleneck in patient
care. There are several reasons for this. First, many diagnostic
analyses can only be done with highly specialist equipment that is
both expensive and only operable by trained clinicians. Such
equipment is found in only a few locations--often just one in any
given urban area. This means that most hospitals are required to
send out samples for analyses to these locations, thereby incurring
shipping costs and transportation delays, and possibly even sample
loss. Second, the equipment in question is typically not available
`on-demand` but instead runs in batches, thereby delaying the
processing time for many samples because they must wait for a
machine to fill up before they can be run.
[0004] Understanding that sample flow breaks down into several key
steps, it would be desirable to consider ways to automate as many
of these as possible. For example, a biological sample, once
extracted from a patient, must be put in a form suitable for a
processing regime that typically involves using PCR to amplify a
vector of interest. Once amplified, the presence of a nucleotide of
interest from the sample needs to be determined unambiguously.
Sample preparation is a process that is susceptible to automation
but is also relatively routinely carried out in almost any
location. By contrast, steps such as PCR and nucleotide detection
have customarily only been within the compass of specially trained
individuals having access to specialist equipment.
[0005] There is a need for a method and apparatus of carrying out
PCR and detection on prepared biological samples, and preferably
with high throughput. In particular there is a need for an
easy-to-use device that can deliver a diagnostic result on several
samples in a short time.
[0006] The discussion of the background to the technology herein is
included to explain the context of the technology. This is not to
be taken as an admission that any of the material referred to was
published, known, or part of the common general knowledge as at the
priority date of any of the claims.
[0007] Throughout the description and claims of the specification
the word "comprise" and variations thereof, such as "comprising"
and "comprises", is not intended to exclude other additives,
components, integers or steps.
SUMMARY
[0008] The present technology addresses systems for detecting
polynucleotides in samples, particularly from biological samples.
In particular, the technology relates to microfluidic systems that
carry out PCR on nucleotides of interest within microfluidic
channels, and detect those nucleotides.
[0009] An apparatus, comprising: a receiving bay configured to
receive a microfluidic cartridge; at least one heat source
thermally coupled to the cartridge and configured to carry out PCR
on a microdroplet of polynucleotide-containing sample, in the
cartridge; a detector configured to detect presence of one or more
polynucleotides in the sample; and a processor coupled to the
detector and the heat source, configured to control heating of one
or more regions of the microfluidic cartridge.
[0010] A method of carrying out PCR on a plurality of
polynucleotide-containing samples, the method comprising:
introducing the plurality of samples in to a microfluidic
cartridge, wherein the cartridge has a plurality of PCR reaction
chambers configured to permit thermal cycling of the plurality of
samples independently of one another; moving the plurality of
samples into the respective plurality of PCR reaction chambers; and
amplifying polynucleotides contained with the plurality of samples,
by application of successive heating and cooling cycles to the PCR
reaction chambers.
[0011] The details of one or more embodiments of the technology are
set forth in the accompanying drawings and further description
herein. Other features, objects, and advantages of the technology
will be apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows an exemplary apparatus, a microfluidic
cartridge, and a read head, as further described herein;
[0013] FIG. 2 shows an exemplary sample-preparation kit;
[0014] FIG. 3 shows a schematic diagram of an apparatus;
[0015] FIG. 4 shows a cross-section of a pipetting head and a
cartridge in position in a microfluidic apparatus.
[0016] FIG. 5 shows introduction of a PCR-ready sample into a
cartridge, situated in an instrument;
[0017] FIGS. 6A-6E show exemplary embodiments of an apparatus;
[0018] FIG. 7 shows an exploded view of an apparatus;
[0019] FIG. 8 shows a block diagram of control circuitry;
[0020] FIG. 9 shows a plan view of an exemplary multi-lane
microfluidic cartridge;
[0021] FIG. 10A shows an exemplary multi-lane cartridge;
[0022] FIG. 10B shows a portion of an exemplary multi-lane
cartridge;
[0023] FIGS. 11A-C show exploded view of an exemplary microfluidic
cartridge;
[0024] FIG. 12 shows an exemplary highly-multiplexed microfluidic
cartridge;
[0025] FIGS. 13-16 show various aspects of exemplary highly
multiplexed microfluidic cartridges; and
[0026] FIGS. 17A-C show various aspects of a radially configured
highly multiplexed microfluidic cartridge.
[0027] FIG. 18 shows an exemplary microfluidic network in a lane of
a multi-lane cartridge;
[0028] FIGS. 19A-19D show exemplary microfluidic valves;
[0029] FIG. 20 shows an exemplary bubble vent;
[0030] FIG. 21 shows a cross-section of a microfluidic cartridge,
when in contact with a heater substrate;
[0031] FIGS. 22A-22C shows various cut-away sections that can be
used to improve cooling rates during PCR thermal cycling;
[0032] FIG. 23 shows a plot of temperature against time during a
PCR process, as performed on a microfluidic cartridge as described
herein;
[0033] FIG. 24 shows an assembly process for a cartridge as further
described herein:
[0034] FIGS. 25A and 25B show exemplary deposition of wax droplets
into microfluidic valves;
[0035] FIG. 26 shows an exemplary heater unit;
[0036] FIGS. 27A and 27B show a plan view of heater circuitry
adjacent to a PCR reaction chamber;
[0037] FIG. 27C shows thermal images of heater circuitry in
operation;
[0038] FIG. 28 shows an overlay of an array of heater elements on
an exemplary multi-lane microfluidic cartridge, wherein various
microfluidic networks are visible;
[0039] FIG. 29 shows a cross-sectional view of an exemplary
detector;
[0040] FIG. 30 shows a perspective view of a detector in a
read-head;
[0041] FIG. 31A, 31B shows a cutaway view of an exemplary detector
in a read-head;
[0042] FIG. 32 shows an exterior view of an exemplary multiplexed
read-head with an array of detectors therein;
[0043] FIG. 33 shows a cutaway view of an exemplary multiplexed
read-head, as in FIG. 18;
[0044] FIG. 34 shows exemplary pre-amplifier circuitry for a
fluorescence detector;
[0045] FIG. 35A shows effects of aperturing on fluorescence
intensity; FIG. 35B shows a detector in cross section with an
exemplary aperture;
[0046] FIG. 36 shows an exemplary layout for electronics and
software components, as further described herein;
[0047] FIG. 37 shows an exemplary apparatus, a microfluidic
cartridge, and a read head, as further described herein;
[0048] FIGS. 38-39 show positioning of a cartridge in an exemplary
apparatus;
[0049] FIGS. 40 and 41 show removal of a heater unit from an
exemplary apparatus;
[0050] FIGS. 42A and 42B show an exemplary heater unit and heater
substrate;
[0051] FIGS. 43A and 43B show an exemplary apparatus having a
detector mounted in a sliding lid;
[0052] FIGS. 44A-44C show a force member;
[0053] FIGS. 45A-45D show a force member associated with a
detector;
[0054] FIG. 46 shows a block diagram of exemplary electronic
circuitry in conjunction with a detector as described herein;
[0055] Additional figures are illustrated within the examples, and
are further described therein.
[0056] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
Overview of Apparatus
[0057] The present technology relates to a system and related
methods for amplifying, and carrying out diagnostic analyses on,
polynucleotides (e.g., a DNA, RNA, mRNA, or rRNA) from biological
samples. For example, the system and methods can determine whether
a polynucleotide indicative of the presence of a particular
pathogen (such as a bacterium or a virus) can be present. The
polynucleotide may be a sample of genomic DNA, or may be a sample
of mitochondrial DNA. The nucleotides are typically provided to the
system having been isolated or released from particles such as
cells in the sample. The system includes a disposable microfluidic
cartridge containing multiple sample lanes in parallel and a
reusable instrument platform (a PCR analyzer apparatus) that can
actuate on-cartridge operations, can detect (e.g., by fluorescence
detection) and analyze the products of the PCR amplification in
each of the lanes separately, in all simultaneously, or in groups
simultaneously, and, optionally, can display the results on a
graphical user interface.
[0058] A system, microfluidic cartridge, heater unit, detector,
kit, methods, and associated computer program product, are now
further described.
[0059] By cartridge is meant a unit that may be disposable, or
reusable in whole or in part, and that is configured to be used in
conjunction with some other apparatus that has been suitably and
complementarily configured to receive and operate on (such as
deliver energy to) the cartridge.
[0060] By microfluidic, as used herein, is meant that volumes of
sample, and/or reagent, and/or amplified polynucleotide are from
about 0.1 .mu.l to about 999 .mu.l, such as from 1-100 .mu.l, or
from 2-25 .mu.l. Similarly, as applied to a cartridge, the term
microfluidic means that various components and channels of the
cartridge, as further described herein, are configured to accept,
and/or retain, and/or facilitate passage of microfluidic volumes of
sample, reagent, or amplified polynucleotide.
[0061] FIG. 1 shows a perspective view of an exemplary apparatus
100 consistent with those described herein, as well as various
components thereof, such as exemplary cartridge 200 that contains
multiple sample lanes, and exemplary read head 300 that contains
detection apparatus for reading signals from cartridge 200. The
apparatus 100 of FIG. 1 is able to carry out real-time PCR on a
number of samples in cartridge 200 simultaneously. Preferably the
number of samples is 12 samples, as illustrated with exemplary
cartridge 200, though other numbers of samples such as 4, 8, 10,
16, 20, 24, 25, 30, 32, 36, 40, and 48 are within the scope of the
present description. In preferred operation of the apparatus, a
PCR-ready solution containing the sample, and, optionally, one or
more analyte-specific reagents (ASR's) is prepared, as further
described elsewhere (see, e.g., U.S. patent application publication
2006-0166233, incorporated herein by reference), prior to
introduction into cartridge 200. An exemplary kit for preparing a
PCR-ready sample, for use with the system described herein, the kit
comprising buffers, lysis pellets, and affinity pellets, is shown
in FIG. 2.
System Overview
[0062] A schematic overview of a system 981 for carrying out
analyses described herein is shown in FIG. 3. The geometric
arrangement of the components of system 981 shown in FIG. 3, as
well as their respective connectivities, is exemplary and not
intended to be limiting.
[0063] A processor 980, such as a microprocessor, is configured to
control functions of various components of the system as shown, and
is thereby in communication with each such component. In
particular, processor 980 is configured to receive data about a
sample to be analyzed, e.g., from a sample reader 990, which may be
a barcode reader, an optical character reader, or an RFID scanner
(radio frequency tag reader). For example, the sample identifier
can be a handheld bar code reader. Processor 980 can be configured
to accept user instructions from an input 984, where such
instructions may include instructions to start analyzing the
sample, and choices of operating conditions.
[0064] Processor 980 can also be configured to communicate with an
optional display 982, so that, for example, information including
but not limited to the current status of the system, progress of
PCR thermocycling, and any warning message in case of malfunction
of either system or cartridge, as well as results of analysis, are
transmitted to the display. Additionally, processor 980 may
transmit one or more questions to be displayed on display 982 that
prompt a user to provide input in response thereto. Thus, in
certain embodiments, input 984 and display 982 are integrated with
one another.
[0065] Processor 980 can be optionally further configured to
transmit results of an analysis to an output device such as a
printer, a visual display, or a speaker, or a combination thereof,
the transmission being either directly through a directly dedicated
printer cable, or wirelessly, or via a network connection.
[0066] Processor 980 is still further optionally connected via a
communication interface such as a network interface to a computer
network 988. The communication interface can be one or more
interfaces selected from the group consisting of: a serial
connection, a parallel connection, a wireless network connection
and a wired network connection such as an ethernet, firewire, cable
connection, or one using USB connectivity. Thereby, when the system
is suitably addressed on the network, a remote user may access the
processor and transmit instructions, input data, or retrieve data,
such as may be stored in a memory (not shown) associated with the
processor, or on some other computer-readable medium that is in
communication with the processor. The computer network connection
may also permit extraction of data to a remote location, such as a
personal computer, personal digital assistant, or network storage
device such as computer server or disk farm. The apparatus may
further be configured to permit a user to e-mail results of an
analysis directly to some other party, such as a healthcare
provider, or a diagnostic facility, or a patient.
[0067] Although not shown in FIG. 3, in various embodiments, input
984 can include one or more input devices selected from the group
consisting of: a keyboard, a touch-sensitive surface, a microphone,
a track-pad, and a mouse. A suitable input device may further
comprise a reader of formatted electronic media, such as, but not
limited to, a flash memory card, memory stick, USB-stick, CD, or
floppy diskette. An input device may further comprise a security
feature such as a fingerprint reader, retinal scanner, magnetic
strip reader, or bar-code reader, for ensuring that a user of the
system is in fact authorized to do so, according to, for example,
pre-loaded identifying characteristics of authorized users. An
input device may additionally--and simultaneously--function as an
output device for writing data in connection with sample analysis.
For example, if an input device is a reader of formatted electronic
media, it may also be a writer of such media. Data that may be
written to such media by such a device includes, but is not limited
to, environmental information, such as temperature or humidity,
pertaining to an analysis, as well as a diagnostic result, and
identifying data for the sample in question.
[0068] Additionally, in various embodiments, the apparatus can
further comprise a data storage medium configured to receive data
from one or more of the processor, an input device, and a
communication interface, the data storage medium being one or more
media selected from the group consisting of: a hard disk drive, an
optical disk drive, or one or more removable storage media such as
a CD-R, CD-RW, USB-drive, and a flash card.
[0069] Processor 980 is further configured to control various
aspects of sample diagnosis, as follows in overview, and as further
described in detail herein. The system is configured to operate in
conjunction with a complementary cartridge 994, such as a
microfluidic cartridge. The cartridge is itself configured, as
further described herein, to receive one or more samples 996
containing one or more polynucleotides in a form suitable for
amplification and diagnostic analysis. The cartridge has dedicated
regions within which amplification, such as by PCR, of the
polynucleotides is carried out when the cartridge is situated in
the apparatus.
[0070] The microfluidic cartridge is received by a receiving bay
992 configured to selectively receive the cartridge. For example,
the receiving bay and the microfluidic cartridge can be
complementary in shape so that the microfluidic cartridge is
selectively received in, e.g., a single orientation. The
microfluidic cartridge can have a registration member that fits
into a complementary feature of the receiving bay. The registration
member can be, for example, a cut-out on an edge of the cartridge,
such as a corner that is cut-off, or one or more notches that are
made on one or more of the sides. By selectively receiving the
cartridge, the receiving bay can help a user to place the cartridge
so that the apparatus can properly operate on the cartridge. The
receiving bay can also be configured so that various components of
the apparatus that can operate on the microfluidic cartridge (heat
sources, detectors, force members, and the like) are positioned to
properly operate on the microfluidic cartridge. In some
embodiments, the apparatus can further include a sensor coupled to
the processor, the sensor configured to sense whether the
microfluidic cartridge is selectively received.
[0071] The receiving bay is in communication with a heater unit 998
that itself is controlled by processor 980 in such a way that
specific regions of the cartridge, such as individual sample lanes,
are independently and selectively heated at specific times during
amplification and analysis. The processor can be configured to
control application of heat to the individual sample lanes,
separately, in all simultaneously, or in groups simultaneously.
[0072] The heat source can be, for example, a contact heat source
such as a resistive heater or a network of resistive heaters, or a
Peltier device, and the like. The contact heat source can be
configured to be in direct physical contact with one or more
distinct locations of a microfluidic cartridge received in the
receiving bay. In various embodiments, each contact source heater
can be configured to heat a distinct location having an average
diameter in 2 dimensions from about 1 millimeter (mm) to about 15
mm (typically about 1 mm to about 10 mm), or a distinct location
having a surface area of between about 1 mm.sup.2 about 225
mm.sup.2 (typically between about 1 mm.sup.2 and about 100
mm.sup.2, or in some embodiments between about 5 mm.sup.2 and about
50 mm.sup.2).
[0073] In various embodiments, the heat source can be situated in
an assembly that is removable from the apparatus, for example, to
permit cleaning or to replace the heater configuration.
[0074] In various embodiments, the apparatus can include a
compliant layer at the contact heat source configured to thermally
couple the contact heat source with at least a portion of a
microfluidic cartridge received in the receiving bay. The compliant
layer at can have a thickness of between about 0.05 and about 2
millimeters and a Shore hardness of between about 25 and about
100.
[0075] In various embodiments, the apparatus can further include
one or more force members (not shown in FIG. 3) configured to apply
force to thermally couple the at least one heat source at least a
portion of a microfluidic cartridge received in the receiving
bay.
[0076] In various embodiments, the one or more force members are
configured to apply force to a plurality of locations in the
microfluidic cartridge. The force applied by the one or more force
members can result in an average pressure at an interface between a
portion of the receiving bay and a portion of the microfluidic
cartridge of between about 5 kilopascals and about 50 kilopascals,
for example, the average pressure can be at least about 7
kilopascals, and still more preferably at least about 14
kilopascals. At least one force member can be manually operated. At
least one force member can be mechanically coupled to a lid at the
receiving bay, whereby operation of the lid operates the force
member. The application of force is important to ensure consistent
thermal contact between the heater wafer and the PCR reactor and
microvalves in the microfluidic cartridge.
[0077] In various embodiments, the apparatus can further include a
lid at the receiving bay, the lid being operable to at least
partially exclude ambient light from the receiving bay. The lid can
be, for example, a sliding lid. The lid can include the optical
detector. A major face of the lid at the optical detector or at the
receiving bay can vary from planarity by less than about 100
micrometers, for example, less than about 25 micrometers. The lid
can be configured to be removable from the apparatus. The lid can
include a latching member that ensures that the lid is securely
closed before amplification reactions are applied to the samples in
the cartridge.
[0078] The processor is also configured to receive signals from and
control a detector 999 configured to detect a polynucleotide in a
sample in one or more of the individual sample lanes, separately or
simultaneously. The processor thereby provides an indication of a
diagnosis from the cartridge 994. Diagnosis can be predicated on
the presence or absence of a specific polynucleotide in a
particular sample. The diagnosis can be transmitted to the output
device 986 and/or the display 982, as described hereinabove.
[0079] The detector can be, for example, an optical detector that
includes a light source that selectively emits light in an
absorption band of a fluorescent dye, and a light detector that
selectively detects light in an emission band of the fluorescent
dye, wherein the fluorescent dye corresponds to a fluorescent
polynucleotide probe or a fragment thereof. Alternatively, for
example, the optical detector can include a bandpass-filtered diode
that selectively emits light in the absorption band of the
fluorescent dye and a bandpass filtered photodiode that selectively
detects light in the emission band of the fluorescent dye; or for
example, the optical detector can be configured to independently
detect a plurality of fluorescent dyes having different fluorescent
emission spectra, wherein each fluorescent dye corresponds to a
fluorescent polynucleotide probe or a fragment thereof; or for
example, the optical detector can be configured to independently
detect a plurality of fluorescent dyes at a plurality of different
locations on a microfluidic cartridge, wherein each fluorescent dye
corresponds to a fluorescent polynucleotide probe or a fragment
thereof in a different sample.
[0080] A suitable processor 980 can be designed and manufactured
according to, respectively, design principles and semiconductor
processing methods known in the art.
[0081] The system in FIG. 3 is configured so that a cartridge with
capacity to receive multiple samples can be acted upon by the
system to analyze multiple samples--or subsets
thereof--simultaneously, or to analyze the samples consecutively.
It is also consistent that additional samples can be added to a
cartridge, while previously added samples are being amplified and
analyzed.
[0082] The system shown in outline in FIG. 3, as with other
exemplary embodiments described herein, is advantageous at least
because it does not require locations within the system suitably
configured for storage of reagents. Neither does the system, or
other exemplary embodiments herein, require inlet or outlet ports
that are configured to receive reagents from, e.g., externally
stored containers such as bottles, canisters, or reservoirs.
Therefore, the system in FIG. 3 is self-contained and operates in
conjunction with a microfluidic cartridge, wherein the cartridge
has locations within it configured to receive mixtures of sample
and PCR reagents.
[0083] The system of FIG. 3 may be configured to carry out
operation in a single location, such as a laboratory setting, or
may be portable so that it can accompany, e.g., a physician, or
other healthcare professional, who may visit patients at different
locations. The system is typically provided with a power-cord so
that it can accept AC power from a mains supply or generator. An
optional transformer (not shown) built into the system, or situated
externally between a power socket and the system, transforms AC
input power into a DC output for use by the system. The system may
also be configured to operate by using one or more batteries and
therefore is also typically equipped with a battery recharging
system, and various warning devices that alert a user if battery
power is becoming too low to reliably initiate or complete a
diagnostic analysis.
[0084] The system of FIG. 3 may further be configured, for
multiplexed cartridge analysis. In one such configuration, multiple
instances of a system, as outlined in FIG. 3, are operated in
conjunction with one another to accept and to process multiple
cartridges, where each cartridge has been loaded with a different
sample. Each component shown in FIG. 3 may therefore be present as
many times as there are cartridges, though the various components
may be configured in a common housing.
[0085] In still another configuration, a system is configured to
accept and to process multiple cartridges, but one or more
components in FIG. 3 is common to multiple cartridges. For example,
a single device may be configured with multiple cartridge receiving
bays, but a common processor and user interface suitably configured
to permit concurrent, consecutive, or simultaneous, control of the
various cartridges. In such an embodiment a single detector, for
example, can scan across all of the multiple cartridges. It is
further possible that such an embodiment, also utilizes a single
sample reader, and a single output device.
[0086] In still another configuration, a system as shown in FIG. 3
is configured to accept a single cartridge, but wherein the single
cartridge is configured to process more than 1, for example, 2, 3,
4, 5, or 6, samples in parallel, and independently of one
another.
[0087] It is further consistent with the present technology that a
cartridge can be tagged, e.g., with a molecular bar-code indicative
of one or more of the samples, to facilitate sample tracking, and
to minimize risk of sample mix-up. Methods for such tagging are
described elsewhere, e.g., in U.S. patent application publication
Ser. No. 10/360,854, incorporated herein by reference.
[0088] In various embodiments, the apparatus can further include an
analysis port. The analysis port can be configured to allow an
external sample analyzer to analyze a sample in the microfluidic
cartridge; for example, the analysis port can be a hole or window
in the apparatus which can accept an optical detection probe that
can analyze a sample in situ in the microfluidic cartridge. In some
embodiments, the analysis port can be configured to direct a sample
from the microfluidic cartridge to an external sample analyzer; for
example, the analysis port can include a conduit in fluid
communication with the microfluidic cartridge that direct a liquid
sample to a chromatography apparatus, an optical spectrometer, a
mass spectrometer, or the like.
[0089] Apparatus 100 may optionally comprise one or more
stabilizing feet that cause the body of the device to be elevated
above a surface on which system 100 is disposed, thereby permitting
ventilation underneath system 100, and also providing a user with
an improved ability to lift system 100. There may be 2, 3, 4, 5, or
6, or more feet, depending upon the size of system 100. Such feet
are preferably made of rubber, or plastic, or metal, and in some
embodiments may elevate the body of system 100 by from about 2 to
about 10 mm above a surface on which it is situated. The
stabilizing function can also be provided by one or more runners
that run along one or more edges--or are inwardly displaced from
one or more edges--of the underside of the apparatus. Such runners
can also be used in conjunction with one or more feet. In another
embodiment, a turntable situated on the underside permits the
apparatus to be rotated in a horizontal or near-horizontal plane
when positioned on, e.g., a benchtop, to facilitate access from a
number of angles by a user.
[0090] FIG. 4 shows a schematic cross-sectional view of a part of
an apparatus as described herein, showing input of sample into a
cartridge 200 via a pipette 10 (such as a disposable pipette) and
an inlet 202. Inlet 202 is preferably configured to receive a
pipette or the bottom end of a PCR tube and thereby accept sample
for analysis with minimum waste, and with minimum introduction of
air. Cartridge 200 is disposed on top of and in contact with a
heater substrate 400. Read head 300 is positioned above cartridge
200 and a cover for optics 310 restricts the amount of ambient
light that can be detected by the read head.
[0091] FIG. 5 shows an example of 4-pipette head used for attaching
disposable pipette tips, prior to dispensing PCR-ready sample into
a cartridge.
Exemplary Systems
[0092] FIGS. 6A-6E show exterior perspective views of various
configurations of an exemplary system, as further described herein.
FIG. 6A shows a perspective view of a system 2000 for receiving
microfluidic cartridge (not shown), and for causing and controlling
various processing operations to be performed a sample introduced
into the cartridge. The elements of system 2000 are not limited to
those explicitly shown. For example, although not shown, system
2000 may be connected to a hand-held bar-code reader, as further
described herein.
[0093] System 2000 comprises a housing 2002, which can-be made of
metal, or a hardened plastic. The form of the housing shown in FIG.
6A embodies stylistic as well as functional features. Other
embodiments of the technology may appear somewhat differently, in
their arrangement of the components, as well as their overall
appearance, in terms of smoothness of lines, and of exterior
finish, and texture. System 2000 further comprises one or more
stabilizing members 2004. Shown in FIG. 6A is a stabilizing foot,
of which several are normally present, located at various regions
of the underside of system 2000 so as to provide balance and
support. For example, there may be three, four, five, six, or eight
such stabilizing feet. The feet may be moulded into and made of the
same material as housing 2002, or may be made of one or more
separate materials and attached to the underside of system 2000.
For example, the feet may comprise a rubber that makes it hard for
system 2000 to slip on a surface on which it is situated, and also
protects the surface from scratches. The stabilizing member of
members may take other forms than feet, for example, rails,
runners, or one or more pads.
[0094] System 2000 further comprises a display 2006, which may be a
liquid crystal display, such as active matrix, an OLED, or some
other suitable form. It may present images and other information in
color or in black and white. Display 2006 may also be a
touch-sensitive display and therefore may be configured to accept
input from a user in response to various displayed prompts. Display
2006 may have an anti-reflective coating on it to reduce glare and
reflections from overhead lights in an laboratory setting. Display
2006 may also be illuminated from, e.g., a back-light, to
facilitate easier viewing in a dark laboratory.
[0095] System 2000, as shown in FIG. 6A, also comprises a moveable
lid 2010, having a handle 2008. The lid 2010 can slide back and
forward. In FIG. 6A, the lid is in a forward position, whereby it
is "closed". In FIG. 6B, the lid is shown in a back position,
wherein the lid is "open" and reveals a receiving bay 2014 that is
configured to receive a microfluidic cartridge. Of course, as one
of ordinary skill in the art would appreciate, the technology
described herein is not limited to a lid that slides, or one that
slides back and forward. Side to side movement is also possible, as
is a configuration where the lid is "open" when positioned forward
in the device. It is also possible that the lid is a hinged lid, or
one that is totally removable.
[0096] Handle 2008 performs a role of permitting a user to move lid
2010 form one position to another, and also performs a role of
causing pressure to be forced down on the lid, when in a closed
position, so that pressure can be applied to a cartridge in the
receiving bay 2014. In FIG. 6C, handle 2008 is shown in a depressed
position, wherein force is thereby applied to lid 2014, and thus
pressure is applied to a cartridge received in the receiving bay
beneath the lid.
[0097] In one embodiment, the handle and lid assembly are also
fitted with a mechanical sensor that does not permit the handle to
be depressed when there is no cartridge in the receiving bay. In
another embodiment, the handle and lid assembly are fitted with a
mechanical latch that does not permit the handle to be raised when
an analysis is in progress.
[0098] A further configuration of system 2000 is shown in FIG. 6D,
wherein a door 2012 is in an open position. Door 2012 is shown in a
closed position in FIGS. 6A-C. The door is an optional component
that permits a user to access a heater module 2020, and also a
computer-readable medium input tray 2022. System 2000 can function
without a door that covers heater module 2020 and medium input
2022, but such a door has convenience attached to it. Although the
door 2012 is shown hinged at the bottom, it may also be hinged at
one of its sides, or at its upper edge. Door 2012 may alternatively
be a removable cover, instead of being hinged. Door 2012, may also
be situated at the rear, or side of system 2000 for example, if
access to the heater module and/or computer readable medium input
is desired on a different face of the system. It is also consistent
with the system herein that the heater module, and the computer
readable medium input are accessed by separate doors on the same or
different sides of the device, and wherein such separate doors may
be independently hinged or removable.
[0099] Heater module 2020 is preferably removable, and is further
described hereinbelow.
[0100] Computer readable medium input 2022 may accept one or more
of a variety of media. Shown in FIG. 2D is an exemplary form of
input 2022, a CD-Rom tray for accepting a CD, DVD, or mini-CD, or
mini-DVD, in any of the commonly used readable, read-writable, and
writable formats. Also consistent with the description herein is an
input that can accept another form of medium, such as a floppy
disc, flash memory such as memory stick, compact flash, smart
data-card, or secure-data card, a pen-drive, portable USB-drive,
zip-disk, and others. Such an input can also be configured to
accept several different forms of media. Such an input 2022 is in
communication with a processor (as described in connection with
FIG. 3, though not shown in FIGS. 6A-E), that can read data from a
computer-readable medium when properly inserted into the input.
[0101] FIG. 6E shows a plan view of a rear of system 2000. Shown
are an air vent 2024, or letting surplus heat escape during an
analysis. Typically, on the inside of system 2000, and by air vent
2024 and not shown in FIG. 6E, is a fan. Other ports shown in FIG.
6E are as follows: a power socket 2026 for accepting a power cord
that will connect system 2000 to a supply of electricity; an
ethernet connection 2028 for linking system 2000 to a computer
network such as a local area network; an phone-jack connection 2032
for linking system 2000 to a communication network such as a
telephone network; one or more USB ports 2030, for connecting
system 2000 to one or more peripheral devices such as a printer, or
a computer hard drive; an infra-red port for communicating with,
e.g., a remote controller (not shown), to permit a user to control
the system without using a touch-screen interface. For example, a
user could remotely issue scheduling commands to system 2000 to
cause it to start an analysis at a specific time in the future.
[0102] Features shown on the rear of system 2000 may be arranged in
any different manner, depending upon an internal configuration of
various components. Additionally, features shown as being on the
rear of system 2000, may be optionally presented on another face of
system 2000, depending on design preference. Shown in FIG. 6E are
exemplary connections. It would be understood that various other
features, including inputs, outputs, sockets, and connections, may
be present on the rear face of system 2000, though not shown, or on
other faces of system 2000.
[0103] An exploded view of an exemplary embodiment of the apparatus
is shown in FIG. 7, particularly showing internal features of
apparatus 2000. Apparatus 2000 can comprise a computer readable
medium configured with hardware/firmware that can be employed to
drive and monitor the operations on a cartridge used therewith, as
well as software to interpret, communicate and store the results of
a diagnostic test performed on a sample processed in the cartridge.
Referring to FIG. 7, typical components of the apparatus 2000 are
shown and include, for example, control electronics 2005, removable
heater/sensor module 2020, detector 2009 such as a fluorescent
detection module, display screen or optionally combined display and
user interface 2006 (e.g., a medical grade touch sensitive liquid
crystal display (LCD)). In some embodiments, lid 2010, detector
2009, and handle 2008 can be collectively referred to as slider
module 2007. Additional components of apparatus 2000 may include
one or more mechanical fixtures such as frame 2019 to hold the
various modules (e.g., the heater/sensor module 2020, and/or the
slider module 2007) in alignment, and for providing structural
rigidity. Detector module 2009 can be placed in rails to facilitate
opening and placement of cartridge 2060 in the apparatus 2000, and
to facilitate alignment of the optics upon closing. Heater/sensor
module 2020 can be also placed on rails for easy removal and
insertion of the assembly.
[0104] Embodiments of apparatus 2000 also include software (e.g.,
for interfacing with users, conducting analysis and/or analyzing
test results), firmware (e.g., for controlling the hardware during
tests on the cartridge 812), and one or more peripheral
communication interfaces shown collectively as 2031 for peripherals
(e.g., communication ports such as USB/Serial/Ethernet to connect
to storage such as compact disc or hard disk, to connect input
devices such as a bar code reader and/or a keyboard, to connect to
other computers or storage via a network, and the like).
[0105] Control electronics 840, shown schematically in the block
diagram in FIG. 8, can include one or more functions in various
embodiments, for example for, main control 900, multiplexing 902,
display control 904, detector control 906, and the like. The main
control function may serve as the hub of control electronics 840 in
apparatus 2000 and can manage communication and control of the
various electronic functions. The main control function can also
support electrical and communications interface 908 with a user or
an output device such as a printer 920, as well as optional
diagnostic and safety functions. In conjunction with main control
function 900, multiplexer function 902 can control sensor data 914
and output current 916 to help control heater/sensor module 2020.
The display control function 904 can control output to and, if
applicable, interpret input from touch screen LCD 846, which can
thereby provide a graphical interface to the user in certain
embodiments. The detector function 906 can be implemented in
control electronics 840 using typical control and processing
circuitry to collect, digitize, filter, and/or transmit the data
from a detector 2009 such as one or more fluorescence detection
modules.
Microfluidic Cartridge
[0106] The present technology comprises a microfluidic cartridge
that is configured to carry out an amplification, such as by PCR,
of one or more polynucleotides from one or more samples. It is to
be understood that, unless specifically made clear to the contrary,
where the term PCR is used herein, any variant of PCR including but
not limited to real-time and quantitative, and any other form of
polynucleotide amplification is intended to be encompassed. The
microfluidic cartridge need not be self-contained and can be
designed so that it receives thermal energy from one or more
heating elements present in an external apparatus with which the
cartridge is in thermal communication. An exemplary such apparatus
is further described herein; additional embodiments of such a
system are found in U.S. patent application Ser. No. 11/940,310,
entitled "Microfluidic Cartridge and Method of Making Same", and
filed on even date herewith, the specification of which is
incorporated herein by reference.
[0107] By cartridge is meant a unit that may be disposable, or
reusable in whole or in part, and that is configured to be used in
conjunction with some other apparatus that has been suitably and
complementarily configured to receive and operate on (such as
deliver energy to) the cartridge.
[0108] By microfluidic, as used herein, is meant that volumes of
sample, and/or reagent, and/or amplified polynucleotide are from
about 0.1 .mu.l to about 999 .mu.l, such as from 1-100 .mu.l, or
from 2-25 .mu.l. Similarly, as applied to a cartridge, the term
microfluidic means that various components and channels of the
cartridge, as further described herein, are configured to accept,
and/or retain, and/or facilitate passage of microfluidic volumes of
sample, reagent, or amplified polynucleotide. Certain embodiments
herein can also function with nanoliter volumes (in the range of
10-500 nanoliters, such as 100 nanoliters).
[0109] One aspect of the present technology relates to a
microfluidic cartridge having two or more sample lanes arranged so
that analyses can be carried out in two or more of the lanes in
parallel, for example simultaneously, and wherein each lane is
independently associated with a given sample.
[0110] A sample lane is an independently controllable set of
elements by which a sample can be analyzed, according to methods
described herein as well as others known in the art. A sample lane
comprises at least a sample inlet, and a microfluidic network
having one or more microfluidic components, as further described
herein.
[0111] In various embodiments, a sample lane can include a sample
inlet port or valve, and a microfluidic network that comprises, in
fluidic communication one or more components selected from the
group consisting of: at least one thermally actuated valve, a
bubble removal vent, at least one thermally actuated pump, a gate,
mixing channel, positioning element, microreactor, a downstream
thermally actuated valve, and a PCR reaction chamber. The sample
inlet valve can be configured to accept a sample at a pressure
differential compared to ambient pressure of between about 70 and
100 kilopascals.
[0112] The cartridge can therefore include a plurality of
microfluidic networks, each network having various components, and
each network configured to carry out PCR on a sample in which the
presence or absence of one or more polynucleotides is to be
determined.
[0113] A multi-lane cartridge is configured to accept a number of
samples in series or in parallel, simultaneously or consecutively,
in particular embodiments 12 samples, wherein the samples include
at least a first sample and a second sample, wherein the first
sample and the second sample each contain one or more
polynucleotides in a form suitable for amplification. The
polynucleotides in question may be the same as, or different from
one another, in different samples and hence in different lanes of
the cartridge. The cartridge typically processes each sample by
increasing the concentration of a polynucleotide to be determined
and/or by reducing the concentration of inhibitors relative to the
concentration of polynucleotide to be determined.
[0114] The multi-lane cartridge comprises at least a first sample
lane having a first microfluidic network and a second lane having a
second microfluidic network, wherein each of the first microfluidic
network and the second microfluidic network is as elsewhere
described herein, and wherein the first microfluidic network is
configured to amplify polynucleotides in the first sample, and
wherein the second microfluidic network is configured to amplify
polynucleotides in the second sample.
[0115] In various embodiments, the microfluidic network can be
configured to couple heat from an external heat source to a sample
mixture comprising PCR reagent and neutralized polynucleotide
sample under thermal cycling conditions suitable for creating PCR
amplicons from the neutralized polynucleotide sample.
[0116] At least the external heat source may operate under control
of a computer processor, configured to execute computer readable
instructions for operating one or more components of each sample
lane, independently of one another, and for receiving signals from
a detector that measures fluorescence from one or more of the PCR
reaction chambers.
[0117] For example, FIG. 9 shows a plan view of a microfluidic
cartridge 100 containing twelve independent sample lanes 101
capable of simultaneous or successive processing. The microfluidic
network in each lane is typically configured to carry out
amplification, such as by PCR, on a PCR-ready sample, such as one
containing nucleic acid extracted from a sample using other methods
as further described herein. A PCR-ready sample is thus typically a
mixture comprising the PCR reagents and the neutralized
polynucleotide sample, suitable for subjecting to thermal cycling
conditions that create PCR amplicons from the neutralized
polynucleotide sample. For example, a PCR-ready sample can include
a PCR reagent mixture comprising a polymerase enzyme, a positive
control plasmid, a fluorogenic hybridization probe selective for at
least a portion of the plasmid and a plurality of nucleotides, and
at least one probe that is selective for a polynucleotide sequence.
Exemplary probes are further described herein. Typically, the
microfluidic network is configured to couple heat from an external
heat source with the mixture comprising the PCR reagent and the
neutralized polynucleotide sample under thermal cycling conditions
suitable for creating PCR amplicons from the neutralized
polynucleotide sample.
[0118] In various embodiments, the PCR reagent mixture can include
a positive control plasmid and a plasmid fluorogenic hybridization
probe selective for at least a portion of the plasmid, and the
microfluidic cartridge can be configured to allow independent
optical detection of the fluorogenic hybridization probe and the
plasmid fluorogenic hybridization probe.
[0119] In various embodiments, the microfluidic cartridge can
accommodate a negative control polynucleotide, wherein the
microfluidic network can be configured to independently carry out
PCR on each of a neutralized polynucleotide sample and a negative
control polynucleotide with the PCR reagent mixture under thermal
cycling conditions suitable for independently creating PCR
amplicons of the neutralized polynucleotide sample and PCR
amplicons of the negative control polynucleotide. Each lane of a
multi-lane cartridge as described herein can perform two reactions
when used in conjunction with two fluorescence detection systems
per lane. A variety of combinations of reactions can be performed
in the cartridge, such as two sample reactions in one lane, a
positive control and a negative control in two other lanes; or a
sample reaction and an internal control in one lane and a negative
control in a separate lane.
[0120] FIG. 10A shows a perspective view of a portion of an
exemplary microfluidic cartridge 200 according to the present
technology. FIG. 10B shows a close-up view of a portion of the
cartridge 200 of FIG. 10A illustrating various representative
components. The cartridge 200 may be referred to as a multi-lane
PCR cartridge with dedicated sample inlets 202. For example sample
inlet 202 is configured to accept a liquid transfer member (not
shown) such as a syringe, a pipette, or a PCR tube containing a PCR
ready sample. More than one inlet 202 is shown in FIGS. 10A, 10B,
wherein one inlet operates in conjunction with a single sample
lane. Various components of microfluidic circuitry in each lane are
also visible. For example, microvalves 204, and 206, and
hydrophobic vents 208 for removing air bubbles, are parts of
microfluidic circuitry in a given lane. Also shown is an ultrafast
PCR reactor 210, which, as further described herein, is a
microfluidic channel in a given sample lane that is long enough to
permit PCR to amplify polynucleotides present in a sample. Above
each PCR reactor 210 is a window 212 that permits detection of
fluorescence from a fluorescent substance in PCR reactor 210 when a
detector is situated above window 212. It is to be understood that
other configurations of windows are possible including, but not
limited to, a single window that straddles each PCR reactor across
the width of cartridge 200.
[0121] In preferred embodiments, the multi-sample cartridge has a
size substantially the same as that of a 96-well plate as is
customarily used in the art. Advantageously, then, such a cartridge
may be used with plate handlers used elsewhere in the art.
[0122] The sample inlets of adjacent lanes are reasonably spaced
apart from one another to prevent any contamination of one sample
inlet from another sample when a user introduces a sample into any
one cartridge. In an embodiment, the sample inlets are configured
so as to prevent subsequent inadvertent introduction of sample into
a given lane after a sample has already been introduced into that
lane. In certain embodiments, the multi-sample cartridge is
designed so that a spacing between the centroids of sample inlets
is 9 mm, which is an industry-recognized standard. This means that,
in certain embodiments the center-to-center distance between inlet
holes in the cartridge that accept samples from PCR tubes, as
further described herein, is 9 mm. The inlet holes can be
manufactured conical in shape with an appropriate conical angle so
that industry-standard pipette tips (2 .mu.l, 20 .mu.l, 200 .mu.l,
volumes, etc.) fit snugly therein. The cartridge herein may be
adapted to suit other, later-arising, industry standards not
otherwise described herein, as would be understood by one of
ordinary skill in the art.
[0123] In one embodiment, an exemplary microfluidic cartridge has
12 sample lanes. The inlet ports in this embodiment have a 6 mm
spacing, so that, when used in conjunction with an automated sample
loader having 4 heads, spaced equidistantly at 18 mm apart, the
inlets can be loaded in three batches of four inlets: e.g., inlets
1, 4, 7, and 10 together, followed by 2, 5, 8, and 11, then finally
3, 6, 9, and 12, wherein the 12 inlets are numbered consecutively
from one side of the cartridge to the other as shown.
[0124] A microfluidic cartridge as used herein may be constructed
from a number of layers. Accordingly, one aspect of the present
technology relates to a microfluidic cartridge that comprises a
first, second, third, fourth, and fifth layers wherein one or more
layers define a plurality of microfluidic networks, each network
having various components configured to carry out PCR on a sample
in which the presence or absence of one or more polynucleotides is
to be determined. In various embodiments, one or more such layers
are optional.
[0125] FIGS. 11A-C show various views of a layer structure of an
exemplary microfluidic cartridge comprising a number of layers, as
further described herein. FIG. 11A shows an exploded view; FIG. 11B
shows a perspective view; and FIG. 11C shows a cross-sectional view
of a sample lane in the exemplary cartridge. Referring to FIGS.
11A-C, an exemplary microfluidic cartridge 400 includes first 420,
second 422, third 424, fourth 426, and fifth layers in two
non-contiguous parts 428, 430 (as shown) that enclose a
microfluidic network having various components configured to
process multiple samples in parallel that include one or more
polynucleotides to be determined.
[0126] Microfluidic cartridge 400 can be fabricated as desired. The
cartridge can include a microfluidic substrate layer 424, typically
injection molded out of a plastic, such as a zeonor plastic (cyclic
olefin polymer), having a PCR channel and valve channels on a first
side and vent channels and various inlet holes, including wax
loading holes and liquid inlet holes, on a second side (disposed
toward hydrophobic vent membrane 426). It is advantageous that all
the microfluidic network defining structures, such as PCR reactors,
valves, inlet holes, and air vents, are defined on the same single
substrate 424. This attribute facilitates manufacture and assembly
of the cartridge. Additionally, the material from which this
substrate is formed is rigid or nondeformable, non-venting to air
and other gases, and has a low autofluorescence to facilitate
detection of polynucleotides during an amplification reaction
performed in the microfluidic circuitry defined therein. Rigidity
is advantageous because it facilitates effective and uniform
contact with a heat unit as further described herein. Use of a
non-venting material is also advantageous because it reduces the
likelihood that the concentration of various species in liquid form
will change during analysis. Use of a material having low
auto-fluorescence is also important so that background fluorescence
does not detract from measurement of fluorescence from the analyte
of interest.
[0127] The cartridge can further include, disposed on top of the
substrate 424, an oleophobic/hydrophobic vent membrane layer 426 of
a porous material, such as 0.2 to 1.0 micron pore-size membrane of
modified polytetrafluorethylene, the membrane being typically
between about 25 and about 100 microns thick, and configured to
cover the vent channels of microfluidic substrate 424, and attached
thereto using, for example, heat bonding.
[0128] Typically, the microfluidic cartridge further includes a
layer 428, 430 of polypropylene or other plastic label with
pressure sensitive adhesive (typically between about 50 and 150
microns thick) configured to seal the wax loading holes of the
valves in substrate 424, trap air used for valve actuation, and
serve as a location for operator markings. In FIG. 4A, this layer
is shown in two separate pieces, 428, 430, though it would be
understood by one of ordinary skill in the art that a single piece
layer would be appropriate.
[0129] In various embodiments, the label is a computer-readable
label. For example, the label can include a bar code, a radio
frequency tag or one or more computer-readable characters. The
label can be formed of a mechanically compliant material. For
example, the mechanically compliant material of the label can have
a thickness of between about 0.05 and about 2 millimeters and a
Shore hardness of between about 25 and about 100. The label can be
positioned such that it can be read by a sample identification
verifier as further described herein.
[0130] The cartridge can further include a heat sealable laminate
layer 422 (typically between about 100 and about 125 microns thick)
attached to the bottom surface of the microfluidic substrate 424
using, for example, heat bonding. This layer serves to seal the PCR
channels and vent channels in substrate 424. The cartridge can
further include a thermal interface material layer 420 (typically
about 125 microns thick), attached to the bottom of the heat
sealable laminate layer using, for example, pressure sensitive
adhesive. The layer 420 can be compressible and have a higher
thermal conductivity than common plastics, thereby serving to
transfer heat across the laminate more efficiently. Typically,
however, layer 420 is not present.
[0131] The application of pressure to contact the cartridge to the
heater of an instrument that receives the cartridge generally
assists in achieving better thermal contact between the heater and
the heat-receiveable parts of the cartridge, and also prevents the
bottom laminate structure from expanding, as would happen if the
PCR channel was only partially filled with liquid and the air
entrapped therein would be thermally expanded during
thermocycling.
[0132] In use, cartridge 400 is typically thermally associated with
an array of heat sources configured to operate the components
(e.g., valves, gates, actuators, and processing region 410) of the
device. Exemplary such heater arrays are further described herein.
Additional embodiments of heater arrays are described in U.S.
patent application Ser. No. 11/940,315, entitled "Heater Unit for
Microfluidic Diagnostic System" and filed on even date herewith,
the specification of which is incorporated herein by reference in
its entirety. In some embodiments, the heat sources are controlled
by a computer processor and actuated according to a desired
protocol. Processors configured to operate microfluidic devices are
described in, e.g., U.S. application Ser. No. 09/819,105, filed
Mar. 28, 2001, which application is incorporated herein by
reference.
[0133] In various embodiments, during transport and storage, the
microfluidic cartridge can be further surrounded by a sealed pouch.
The microfluidic cartridge can be sealed in the pouch with an inert
gas. The microfluidic cartridge can be disposable for example after
one or more of its sample lanes have been used.
Highly Multiplexed Embodiments
[0134] Embodiments of the cartridge described herein may be
constructed that have high-density microfluidic circuitry on a
single cartridge that thereby permit processing of multiple samples
in parallel, or in sequence, on a single cartridge. Preferred
numbers of such multiple samples include 20, 24, 36, 40, 48, 50,
60, 64, 72, 80, 84, 96, and 100, but it would be understood that
still other numbers are consistent with the apparatus and cartridge
herein, where deemed convenient and practical.
[0135] Accordingly, different configurations of lanes, sample
inlets, and associated heater networks than those explicitly
depicted in the FIGs and examples that can facilitate processing
such numbers of samples on a single cartridge are within the scope
of the instant disclosure. Similarly, alternative configurations of
detectors and heating elements for use in conjunction with such a
highly multiplexed cartridge are also within the scope of the
description herein.
[0136] It is also to be understood that the microfluidic cartridges
described herein are not to be limited to rectangular shapes, but
can include cartridges having circular, elliptical, triangular,
rhombohedral, square, and other shapes. Such shapes may also be
adapted to include some irregularity, such as a cut-out, to
facilitate placement in a complementary apparatus as further
described herein.
[0137] In an exemplary embodiment, a highly multiplexed cartridge
has 48 sample lanes, and permits independent control of each valve
in each lane by suitably configured heater circuitry, with 2 banks
of thermo cycling protocols per lane, as shown in FIG. 12. In the
embodiment in FIG. 12, the heaters (shown superimposed on the
lanes) are arranged in three arrays 502, 504, with 506, and 508.
The heaters are themselves disposed within one or more substrates.
Heater arrays 502, 508 in two separate glass regions only apply
heat to valves in the microfluidic networks in each lane. Because
of the low thermal conductivity of glass, the individual valves may
be heated separately from one another. This permits samples to be
loaded into the cartridge at different times, and passed to the PCR
reaction chambers independently of one another. The PCR heaters
504,506 are mounted on a silicon substrate--and are not readily
heated individually, but thereby permit batch processing of PCR
samples, where multiple samples from different lanes are amplified
by the same set of heating/cooling cycles. It is preferable for the
PCR heaters to be arranged in 2 banks (the heater arrays 506 on the
left and right 508 are not in electrical communication with one
another), thereby permitting a separate degree of sample
control.
[0138] FIG. 13 shows a representative 48-sample cartridge 600
compatible with the heater arrays of FIG. 12, and having a
configuration of inlets 602 different to that depicted o other
cartridges herein. The inlet configuration is exemplary and has
been designed to maximize efficiency of space usage on the
cartridge. The inlet configuration can be compatible with an
automatic pipetting machine that has dispensing heads situated at a
9 mm spacing. For example, such a machine having 4 heads can load 4
inlets at once, in 12 discrete steps, for the cartridge of FIG. 13.
Other configurations of inlets though not explicitly described or
depicted are compatible with the technology described herein.
[0139] FIG. 14 shows, in close up, an exemplary spacing of valves
702, channels 704, and vents 796, in adjacent lanes 708 of a
multi-sample microfluidic cartridge for example as shown in FIG.
13.
[0140] FIGS. 15 and 16 show close-ups of, respectively, heater
arrays 804 compatible with, and inlets 902 on, the exemplary
cartridge shown in FIG. 14.
[0141] FIGS. 17A and 17B show various views of an embodiment of a
radially-configured highly-multiplexed cartridge, having a number
of inlets 1002, microfluidic lanes 1004, valves 1005, and PCR
reaction chambers 1006. FIG. 17C shows an array of heater elements
1008 compatible with the cartridge layout of FIG. 17A.
[0142] The various embodiments shown in FIGS. 12-17C are compatible
with liquid dispensers, receiving bays, and detectors that are
configured differently from the other specific examples described
herein.
[0143] During the design and manufacture of highly multiplexed
cartridges, photolithographic processing steps such as etching,
hole drilling/photo-chemical drilling/sand-blasting/ion-milling
processes should be optimized to give well defined holes and
microchannel pattern. Proper distances between channels should be
identified and maintained to obtain good bonding between the
microchannel substrate and the heat conducting substrate layer. In
particular, it is desirable that minimal distances are maintained
between pairs of adjacent microchannels to promote, reliable
bonding of the laminate in between the channels.
[0144] The fabrication by injection molding of these complicated
microfluidic structures having multiple channels and multiple inlet
holes entails proper consideration of dimensional repeatability of
these structures over multiple shots from the injection molding
master pattern. Proper consideration is also attached to the
placement of ejector pins to push out the structure from the mold
without causing warp, bend or stretching of it. For example,
impression of the ejector pins on the microfluidic substrate should
not sink into the substrate thereby preventing planarity of the
surface of the cartridge. The accurate placement of various inlet
holes (such as sample inlet holes, valve inlet holes and vent
holes) relative to adjacent microfluidic channels is also important
because the presence of these holes can cause knit-lines to form
that might cause unintended leak from a hole to a microchannel.
Highly multiplexed microfluidic substrates may be fabricated in
other materials such as glass, silicon.
[0145] The size of the substrate relative to the number of holes is
also factor during fabrication because it is easy to make a
substrate having just a simple microfluidic network with a few
holes (maybe fewer than 10 holes) and a few microchannels, but
making a substrate having over 24, or over 48, or over 72 holes,
etc., is more difficult.
Microfluidic Networks
[0146] Particular components of exemplary microfluidic networks are
further described herein.
[0147] Channels of a microfluidic network in a lane of cartridge
typically have at least one sub-millimeter cross-sectional
dimension. For example, channels of such a network may have a width
and/or a depth of about 1 mm or less (e.g., about 750 microns or
less, about 500 microns, or less, about 250 microns or less).
[0148] FIG. 18 shows a plan view of a representative microfluidic
circuit found in one lane of a multi-lane cartridge such as shown
in FIGS. 10A and 10B. It would be understood by one skilled in the
art that other configurations of microfluidic network would be
consistent with the function of the cartridges and apparatus
described herein. In operation of the cartridge, in sequence,
sample is introduced through liquid inlet 202, optionally flows
into a bubble removal vent channel 208 (which permits adventitious
air bubbles introduced into the sample during entry, to escape),
and continues along a channel 216. Typically, when using a robotic
dispenser of liquid sample, the volume is dispensed accurately
enough that formation of bubbles is not a significant problem, and
the presence of vent channel 208 is not necessary. Thus, in certain
embodiments, the bubble removal vent channel 208 is not present and
sample flows directly into channel 216. Throughout the operation of
cartridge 200, the fluid is manipulated as a microdroplet (not
shown in the FIGs). Valves 204 and 206 are initially both open, so
that a microdroplet of sample-containing fluid can be pumped into
PCR reactor channel 210 from inlet hole 202 under influence of
force from the sample injection operation. Upon initiating of
processing, the detector present on top of the PCR reactor 210
checks for the presence of liquid in the PCR channel, and then
valves 204 and 206 are closed to isolate the PCR reaction mix from
the outside. In one embodiment, the checking of the presence of
liquid in the PCR channel is by measuring the heat ramp rate, such
as by one or more temperature sensors in the heating unit. A
channel with liquid absent will heat up faster than one in which,
e.g., a sample, is present.
[0149] Both valves 204 and 206 are closed prior to thermocycling to
prevent or reduce any evaporation of liquid, bubble generation, or
movement of fluid from the PCR reactor. End vent 214 is configured
to prevent a user from introducing an excess amount of liquid into
the microfluidic cartridge, as well as playing a role of containing
any sample from spilling over to unintended parts of the cartridge.
A user may input sample volumes as small as an amount to fill the
region from the bubble removal vent (if present) to the middle of
the microreactor, or up to valve 204 or beyond valve 204. The use
of microvalves prevents both loss of liquid or vapor thereby
enabling even a partially filled reactor to successfully complete a
PCR thermocycling reaction.
[0150] The reactor 210 is a microfluidic channel that is heated
through a series of cycles to carry out amplification of
nucleotides in the sample, as further described herein, and
according to amplification protocols known to those of ordinary
skill in the art. The inside walls of the channel in the PCR
reactor are typically made very smooth and polished to a shiny
finish (for example, using a polish selected from SPI A1, SPI A2,
SPI A3, SPI B1, or SPI B2) during manufacture. This is in order to
minimize any microscopic quantities of air trapped in the surface
of the PCR channel, which would causing bubbling during the
thermocycling steps. The presence of bubbles especially in the
detection region of the PCR channel could also cause a false or
inaccurate reading while monitoring progress of the PCR.
Additionally, the PCR channel can be made shallow such that the
temperature gradient across the depth of the channel is
minimized.
[0151] The region of the cartridge 212 above PCR reactor 210 is a
thinned down section to reduce thermal mass and autofluorescence
from plastic in the cartridge. It permits a detector to more
reliably monitor progress of the reaction and also to detect
fluorescence from a probe that binds to a quantity of amplified
nucleotide. Exemplary probes are further described herein. The
region 212 can be made of thinner material than the rest of the
cartridge so as to permit the PCR channel to be more responsive to
a heating cycle (for example, to rapidly heat and cool between
temperatures appropriate for denaturing and annealing steps), and
so as to reduce glare, autofluorescence, and undue absorption of
fluorescence.
[0152] After PCR has been carried out on a sample, and presence or
absence of a polynucleotide of interest has been determined, it is
preferred that the amplified sample remains in the cartridge and
that the cartridge is either used again (if one or more lanes
remain unused), or disposed of. Should a user wish to run a post
amplification analysis, such as gel electrophoresis, the user may
pierce a hole through the laminate of the cartridge, and recover an
amount--typically about 1.5 microliter--of PCR product. The user
may also place the individual PCR lane on a special narrow heated
plate, maintained at a temperature to melt the wax in the valve,
and then aspirate the reacted sample from the inlet hole of that
PCR lane.
[0153] In various embodiments, the microfluidic network can
optionally include at least one reservoir configured to contain
waste.
[0154] Table 1 outlines typical volumes, pumping pressures, and
operation times associated with various components of a
microfluidic cartridge described herein.
TABLE-US-00001 TABLE 1 Pumping Displacement Time of Operation
Pressure Volume Operation Moving valve ~1-2 psi <1 .mu.l 5-15
seconds wax plugs Operation Pump Used Pump Design Pump Actuation
Moving valve Thermopneumatic 1 .mu.l of Heat trapped wax plugs pump
trapped air air to ~70-90 C.
Valves
[0155] A valve (sometimes referred to herein as a microvalve) is a
component in communication with a channel, such that the valve has
a normally open state allowing material to pass along a channel
from a position on one side of the valve (e.g., upstream of the
valve) to a position on the other side of the valve (e.g.,
downstream of the valve). Upon actuation of the valve, the valve
transitions to a closed state that prevents material from passing
along the channel from one side of the valve to the other. For
example, in one embodiment, a valve can include a mass of a
thermally responsive substance (TRS) that is relatively immobile at
a first temperature and more mobile at a second temperature. The
first and second temperatures are insufficiently high to damage
materials, such as polymer layers of a microfluidic cartridge in
which the valve is situated. A mass of TRS can be an essentially
solid mass or an agglomeration of smaller particles that cooperate
to obstruct the passage when the valve is closed. Examples of TRS's
include a eutectic alloy (e.g., a solder), wax (e.g., an olefin),
polymers, plastics, and combinations thereof. The TRS can also be a
blend of variety of materials, such as an emulsion of thermoelastic
polymer blended with air microbubbles (to enable higher thermal
expansion, as well as reversible expansion and contraction),
polymer blended with expancel material (offering higher thermal
expansion), polymer blended with heat conducting microspheres
(offering faster heat conduction and hence, faster melting
profiles), or a polymer blended with magnetic microspheres (to
permit magnetic actuation of the melted thermoresponsive
material).
[0156] Generally, for such a valve, the second temperature is less
than about 90.degree. C. and the first temperature is less than the
second temperature (e.g., about 70.degree. C. or less). Typically,
a chamber is in gaseous communication with the mass of TRS. The
valve is in communication with a source of heat that can be
selectively applied to the chamber of air and to the TRS. Upon
heating gas (e.g., air) in the chamber and heating the mass of TRS
to the second temperature, gas pressure within the chamber due to
expansion of the volume of gas, forces the mass to move into the
channel, thereby obstructing material from passing therealong.
[0157] An exemplary valve is shown in FIG. 19A. The valve of FIG.
19A has two chambers of air 1203, 1205 in contact with,
respectively, each of two channels 1207, 1208 containing TRS. The
air chambers also serve as loading ports for TRS during manufacture
of the valve, as further described herein. In order to make the
valve sealing very robust and reliable, the flow channel 1201
(along which, e.g., sample passes) at the valve junction is made
narrow (typically 150 .mu.m wide, and 150 .mu.m deep or narrower),
and the constricted portion of the flow channel is made at least
0.5 or 1 mm long such that the TRS seals up a long narrow channel
thereby reducing any leakage through the walls of the channel. In
the case of a bad seal, there may be leakage of fluid around walls
of channel, past the TRS, when the valve is in the closed state. In
order to minimize this, the flow channel is narrowed and elongated
as much as possible. In order to accommodate such a length of
channel on a cartridge where space may be at a premium, the flow
channel can incorporate one or more curves 1209 as shown in FIG.
19A. The valve operates by heating air in the TRS-loading port,
which forces the TRS forwards into the flow-channel in a manner so
that it does not come back to its original position. In this way,
both air and TRS are heated during operation.
[0158] In various other embodiments, a valve for use with a
microfluidic network in a microfluidic cartridge herein can be a
bent valve as shown in FIG. 19B. Such a configuration reduces the
footprint of the valve and hence reduces cost per part for highly
dense microfluidic cartridges. A single valve loading hole 1211 is
positioned in the center, that serves as an inlet for thermally
responsive substance. The leftmost vent 1213 can be configured to
be an inlet for, e.g., sample, and the rightmost vent 1215 acts as
an exit for, e.g., air. This configuration can be used as a
prototype for testing such attributes as valve and channel geometry
and materials.
[0159] In various other embodiments, a valve for use with a
microfluidic network can include a curved valve as shown in FIG.
19C, in order to reduce the effective cross-section of the valve,
thereby enabling manufacture of cheaper dense microfluidic devices.
Such a valve can function with a single valve loading hole and air
chamber 1221 instead of a pair as shown in FIG. 19A.
Gates
[0160] FIG. 19D shows an exemplary gate as may optionally be used
in a microfluidic network herein. A gate can be a component that
can have a closed state that does not allow material to pass along
a channel from a position on one side of the gate to another side
of the gate, and an open state that does allow material to pass
along a channel from a position on one side of the gate to another
side of the gate. Actuation of an open gate can transition the gate
to a closed state in which material is not permitted to pass from
one side of the gate (e.g., upstream of the gate) to the other side
of the gate (e.g., downstream of the gate). Upon actuation, a
closed gate can transition to an open state in which material is
permitted to pass from one side of the gate (e.g., upstream of the
gate) to the other side of the gate (e.g., downstream of the
gate).
[0161] In various embodiments, a microfluidic network can include a
narrow gate 380 as shown in FIG. 19D where a gate loading channel
382 used for loading wax from a wax loading hole 384 to a gate
junction 386 can be narrower (e.g., approximately 150 .mu.m wide
and 100 microns deep). An upstream channel 388 as well as a
downstream channel 390 of the gate junction 386 can be made wide
(e.g., .about.500 .mu.m) and deep (e.g., .about.500 .mu.m) to help
ensure the wax stops at the gate junction 386. The amount of gate
material melted and moved out of the gate junction 386 may be
minimized for optimal gate 380 opening. As an off-cartridge heater
may be used to melt the thermally responsive substance in gate 380,
a misalignment of the heater could cause the wax in the gate
loading channel 382 to be melted as well. Therefore, narrowing the
dimension of the loading channel may increase reliability of gate
opening. In the case of excessive amounts of wax melted at the gate
junction 386 and gate loading channel 382, the increased
cross-sectional area of the downstream channel 390 adjacent to the
gate junction 386 can prevent wax from clogging the downstream
channel 390 during gate 380 opening. The dimensions of the upstream
channel 388 at the gate junction 386 can be made similar to the
downstream channel 390 to ensure correct wax loading during gate
fabrication.
[0162] In various embodiments, the gate can be configured to
minimize the effective area or footprint of the gate within the
network and thus bent gate configurations, although not shown
herein are consistent with the foregoing description.
Vents
[0163] In various embodiments, the microfluidic network can include
at least one hydrophobic vent in addition to an end vent. A vent is
a general outlet (hole) that may or may not be covered with a
hydrophobic membrane. An exit hole is an example of a vent that
need not be covered by a membrane.
[0164] A hydrophobic vent (e.g., a vent in FIG. 20) is a structure
that permits gas to exit a channel while limiting (e.g.,
preventing) quantities of liquid from exiting the channel.
Typically, hydrophobic vents include a layer of porous hydrophobic
material (e.g., a porous filter such as a porous hydrophobic
membrane from GE Osmonics, Minnetonka, Minn.) that defines a wall
of the channel. As described elsewhere herein, hydrophobic vents
can be used to position a microdroplet of sample at a desired
location within a microfluidic network.
[0165] The hydrophobic vents of the present technology are
preferably constructed so that the amount of air that escapes
through them is maximized while minimizing the volume of the
channel below the vent surface. Accordingly, it is preferable that
the vent is constructed so as to have a hydrophobic membrane 1303
of large surface area and a shallow cross section of the
microchannel below the vent surface.
[0166] Hydrophobic vents are useful for bubble removal and
typically have a length of at least about 2.5 mm (e.g., at least
about 5 mm, at least about 7.5 mm) along a channel 1305 (see FIG.
13). The length of the hydrophobic vent is typically at least about
5 times (e.g., at least about 10 times, at least about 20 times)
larger than a depth of the channel within the hydrophobic vent. For
example, in some embodiments, the channel depth within the
hydrophobic vent is about 300 microns or less (e.g., about 250
microns or less, about 200 microns or less, about 150 microns or
less).
[0167] The depth of the channel within the hydrophobic vent is
typically about 75% or less (e.g., about 65% or less, about 60% or
less) of the depth of the channel upstream 1301 and downstream (not
shown) of the hydrophobic vent. For example, in some embodiments
the channel depth within the hydrophobic vent is about 150 microns
and the channel depth upstream and downstream of the hydrophobic
vent is about 250 microns. Other dimensions are consistent with the
description herein.
[0168] A width of the channel within the hydrophobic vent is
typically at least about 25% wider (e.g., at least about 50% wider)
than a width of the channel upstream from the vent and downstream
from the vent. For example, in an exemplary embodiment, the width
of the channel within the hydrophobic vent is about 400 microns,
and the width of the channel upstream and downstream from the vent
is about 250 microns. Other dimensions are consistent with the
description herein.
[0169] The vent in FIG. 20 is shown in a linear configuration
though it would be understood that it need not be so. A bent,
kinked, curved, S-shaped, V-shaped, or U-shaped (as in item 208
FIG. 11) vent is also consistent with the manner of construction
and operation described herein.
Use of Cutaways in Cartridge and Substrate to Improve Rate of
Cooling During PCR Cycling
[0170] During a PCR amplification of a nucleotide sample, a number
of thermal cycles are carried out. For improved efficiency, the
cooling between each application of heat is preferably as rapid as
possible. Improved rate of cooling can be achieved with various
modifications to the heating substrate and/or the cartridge, as
shown in FIG. 21.
[0171] One way to achieve rapid cooling is to cutaway portions of
the microfluidic cartridge substrate, as shown in FIG. 22A. The
upper panel of FIG. 22A is a cross-section of an exemplary
microfluidic cartridge taken along the dashed line A-A' as marked
on the lower panel of FIG. 22A. PCR reaction chamber 1601, and
representative heaters 1603 are shown. Also shown are two cutaway
portions, one of which labeled 1601, that are situated alongside
the heaters that are positioned along the long side of the PCR
reaction chamber. Cutaway portions such as 1601 reduce the thermal
mass of the cartridge, and also permit air to circulate within the
cutaway portions. Both of these aspects permit heat to be conducted
away quickly from the immediate vicinity of the PCR reaction
chamber. Other configurations of cutouts, such as in shape,
position, and number, are consistent with the present
technology.
[0172] Another way to achieve rapid cooling is to cutaway portions
of the heater substrate, as shown in FIG. 22B. The lower panel of
FIG. 22B is a cross-section of an exemplary microfluidic cartridge
and heater substrate taken along the dashed line A-A' as marked on
the upper panel of FIG. 22B. PCR reaction chamber 901, and
representative heaters 1003 are shown. Also shown are four cutaway
portions, one of which labeled 1205, that are situated alongside
the heaters that are situated along the long side of the PCR
reaction chamber. Cutaway portions such as 1205 reduce the thermal
mass of the heater substrate, and also permit air to circulate
within the cutaway portions. Both of these aspects permit heat to
be conducted away quickly from the immediate vicinity of the PCR
reaction chamber. Four separate cutaway portions are shown in FIG.
22A so that control circuitry to the various heaters is not
disrupted. Other configurations of cutouts, such as in shape,
position, and number, are consistent with the present technology.
These cutouts may be created by a method selected from: selective
etching using wet etching processes, deep reactive ion etching,
selective etching using CO.sub.2 laser or femtosecond laser (to
prevent surface cracks or stress near the surface), selective
mechanical drilling, selective ultrasonic drilling, or selective
abrasive particle blasting. Care has to be taken to maintain
mechanically intergrity of the heater while reducing as much
material as possible.
[0173] FIG. 22C shows a combination of cutouts and use of ambient
air cooling to increase the cooling rate during the cooling stage
of thermocycling. A substantial amount of cooling happens by
convective loss from the bottom surface of the heater surface to
ambient air. The driving force for this convective loss is the
differential in temperatures between the glass surface and the air
temperature. By decreasing the ambient air temperature by use of,
for example, a peltier cooler, the rate of cooling can be
increased. The convective heat loss may also be increased by
keeping the air at a velocity higher than zero.
[0174] An example of thermal cycling performance in a PCR reaction
chamber obtained with a configuration as described herein, is shown
in FIG. 23 for a protocol that is set to heat up the reaction
mixture to 92.degree. C., and maintain the temperature for 1
second, then cool to 62.degree. C., and stay for 10 seconds. The
cycle time shown is about 29 seconds, with 8 seconds required to
heat from 62.degree. C. and stabilize at 92.degree. C., and 10
seconds required to cool from 92.degree. C., and stabilize at
62.degree. C. To minimize the overall time required for a PCR
effective to produce detectable quantities of amplified material,
it is important to minimize the time required for each cycle. Cycle
times in the range 15-30 s, such as 18-25 s, and 20-22 s, are
desirable. In general, an average PCR cycle time of 25 seconds as
well as cycle times as low as 20 seconds are typical with the
technology described herein. Using reaction volumes less than a
microliter (such as a few hundred nanoliters or less) permits use
of an associated smaller PCR chamber, and enables cycle times as
low as 15 seconds. An average cycle time of 25 seconds and as low
as 20 seconds can be achieved by technology described herein, even
without any forced cooling or implementing any thermal mass
reductions described elsewhere herein.
Manufacturing Process for Cartridge
[0175] FIG. 24 shows a flow-chart 1800 for an embodiment of an
assembly process for an exemplary cartridge as shown in FIG. 11A
herein. It would be understood by one of ordinary skill in the art,
both that various steps may be performed in a different order from
the order set forth in FIG. 24, and additionally that any given
step may be carried out by alternative methods to those described
in the figure. It would also be understood that, where separate
serial steps are illustrated for carrying out two or more
functions, such functions may be performed synchronously and
combined into single steps and remain consistent with the overall
process described herein.
[0176] At 1802, a laminate layer is applied to a microfluidic
substrate that has previously been engineered, for example by
injection molding, to have a microfluidic network constructed in
it; edges are trimmed from the laminate where they spill over the
bounds of the substrate.
[0177] At 1804, wax is dispensed and loaded into the microvalves of
the microfluidic network in the microfluidic substrate. An
exemplary process for carrying this out is further described
herein.
[0178] At 1806, the substrate is inspected to ensure that wax from
step 1804 is loaded properly and that the laminate from step 1802
adheres properly to it. If a substrate does not satisfy either or
both of these tests, it is usually discarded. If substrates
repeatedly fail either or both of these tests, then the wax
dispensing, or laminate application steps, as applicable, are
reviewed.
[0179] At 1808, a hydrophobic vent membrane is applied to, and heat
bonded to, the top of the microfluidic substrate covering at least
the one or more vent holes, and on the opposite face of the
substrate from the laminate. Edges of the membrane that are in
excess of the boundary of the substrate are trimmed.
[0180] At 1810, the assembly is inspected to ensure that the
hydrophobic vent membrane is bonded well to the microfluidic
substrate without heat-clogging the microfluidic channels. If any
of the channels is blocked, or if the bond between the membrane and
the substrate is imperfect, the assembly is discarded, and, in the
case of repeated discard events, the foregoing process step 1808 is
reviewed.
[0181] At 1812, optionally, a thermally conductive pad layer is
applied to the bottom laminate of the cartridge.
[0182] At 1814, two label strips are applied to the top of the
microfluidic substrate, one to cover the valves, and a second to
protect the vent membranes. It would be understood that a single
label strip may be devised to fulfill both of these roles.
[0183] At 1816, additional labels are printed or applied to show
identifying characteristics, such as a barcode #, lot # and expiry
date on the cartridge. Preferably one or more of these labels has a
space and a writable surface that permits a user to make an
identifying annotation on the label, by hand.
[0184] Optionally, at 1818, to facilitate transport and delivery to
a customer, assembled and labeled cartridges are stacked, and
cartridges packed into groups, such as groups of 25, or groups of
10, or groups of 20, or groups of 48 or 50. Preferably the
packaging is via an inert and/or moisture-free medium.
Wax Loading in Valves
[0185] In general, a valve as shown in, e.g., FIGS. 25A-C, is
constructed by depositing a precisely controlled amount of a TRS
(such as wax) into a loading inlet machined in the microfluidic
substrate. FIGS. 25A and 25B show how a combination of controlled
hot drop dispensing into a heated microchannel device of the right
dimensions and geometry is used to accurately load wax into a
microchannel of a microfluidic cartridge to form a valve. The top
of FIG. 25A shows a plan view of a valve inlet 190 and loading
channel 1902, connecting to a flow channel 1904. The lower portions
of FIG. 25A show the progression of a dispensed wax droplet 1906
(having a volume of 75 nl.+-.15 nl) through the inlet 1901 and into
the loading channel 1902.
[0186] To accomplish those steps, a heated dispenser head can be
accurately positioned over the inlet hole of the micro channel in
the microfluidic device, and can dispense molten wax drops in
volumes as small as 75 nanoliters with an accuracy of 20%. A
suitable dispenser is also one that can deposit amounts smaller
than 100 nl with a precision of +/-20%. The dispenser should also
be capable of heating and maintaining the dispensing temperature of
the TRS to be dispensed. For example, it may have a reservoir to
hold the solution of TRS. It is also desirable that the dispense
head can have freedom of movement at least in a horizontal (x-y)
plane so that it can easily move to various locations of a
microfluidic substrate and dispense volumes of TRS into valve
inlets at such locations without having to be re-set, repositioned
manually, or recalibrated in between each dispense operation.
[0187] The inlet hole of the microfluidic cartridge, or other
microchannel device, is dimensioned in such a way that the droplet
of 75 nl can be accurately propelled to the bottom of the inlet
hole using, for example, compressed air, or in a manner similar to
an inkjet printing method. The microfluidic cartridge is maintained
at a temperature above the melting point of the wax thereby
permitting the wax to stay in a molten state immediately after it
is dispensed. After the drop falls to the bottom of the inlet hole
1901, the molten wax is drawn into the narrow channel by capillary
action, as shown in the sequence of views in FIG. 25B. A shoulder
between the inlet hole 1901 and the loading channel can facilitate
motion of the TRS. The volume of the narrow section can be designed
to be approximately equal to a maximum typical amount that is
dispensed into the inlet hole. The narrow section can also be
designed so that even though the wax dispensed may vary
considerably between a minimum and a maximum shot size, the wax
always fills up to, and stops at, the micro channel junction 1907
because the T-junction provides a higher cross section than that of
the narrow section and thus reduces the capillary forces.
PCR Reagent Mixtures
[0188] In various embodiments, the sample for introduction into a
lane of the microfluidic cartridge can include a PCR reagent
mixture comprising a polymerase enzyme and a plurality of
nucleotides.
[0189] In various embodiments, preparation of a PCR-ready sample
for use with an apparatus and cartridge as described herein can
include contacting a neutralized polynucleotide sample with a PCR
reagent mixture comprising a polymerase enzyme and a plurality of
nucleotides (in some embodiments, the PCR reagent mixture can
further include a positive control plasmid and a fluorogenic
hybridization probe selective for at least a portion of the
plasmid).
[0190] The PCR-ready sample can be prepared, for example, using the
following steps: (1) collect sample in sample collection buffer,
(2) transfer entire sample to lysis tube, mix, heat, and incubate
for seven minutes, (3) place on magnetic rack, allow beads to
separate, aspirate supernatant, (4) add 100 .mu.l of Buffer 1, mix,
place on magnetic rack, allow beads to separate, aspirate
supernatant, (5) add 10 .mu.l of Buffer 2, mix, place in high
temperature heat block for 3 minutes, place on magnetic rack, allow
beads to separate, transfer 5 .mu.l supernatant, and (6) Add 5
.mu.l of Buffer 3, transfer 1 to 10 .mu.l of supernatant for PCR
amplification and detection.
[0191] The PCR reagent mixture can be in the form of one or more
lyophilized pellets and the steps by which the PCR-ready sample is
prepared can involve reconstituting the PCR pellet by contacting it
with liquid to create a PCR reagent mixture solution. In yet
another embodiment, each of the PCR lanes may have dried down or
lyophilized ASR reagents preloaded such that the user only needs to
input prepared polynucleotide sample into the PCR. In another
embodiment, the PCR lanes may have only the application-specific
probes and primers pre-measured and pre-loaded, and the user inputs
a sample mixed with the PCR reagents.
[0192] In various embodiments, the PCR-ready sample can include at
least one probe that can be selective for a polynucleotide
sequence, wherein the steps by which the PCR-ready sample is
prepared involve contacting the neutralized polynucleotide sample
or a PCR amplicon thereof with the probe. The probe can be a
fluorogenic hybridization probe. The fluorogenic hybridization
probe can include a polynucleotide sequence coupled to a
fluorescent reporter dye and a fluorescence quencher dye.
[0193] In various embodiments, the PCR-ready sample further
includes a sample buffer.
[0194] In various embodiments, the PCR-ready sample includes at
least one probe that is selective for a polynucleotide sequence,
e.g., the polynucleotide sequence that is characteristic of a
pathogen selected from the group consisting of gram positive
bacteria, gram negative bacteria, yeast, fungi, protozoa, and
viruses.
[0195] In various embodiments, the PCR reagent mixture can further
include a polymerase enzyme, a positive control plasmid and a
fluorogenic hybridization probe selective for at least a portion of
the plasmid.
[0196] In various embodiments, the probe can be selective for a
polynucleotide sequence that is characteristic of an organism, for
example any organism that employs deoxyribonucleic acid or
ribonucleic acid polynucleotides. Thus, the probe can be selective
for any organism. Suitable organisms include mammals (including
humans), birds, reptiles, amphibians, fish, domesticated animals,
wild animals, extinct organisms, bacteria, fungi, viruses, plants,
and the like. The probe can also be selective for components of
organisms that employ their own polynucleotides, for example
mitochondria. In some embodiments, the probe is selective for
microorganisms, for example, organisms used in food production (for
example, yeasts employed in fermented products, molds or bacteria
employed in cheeses, and the like) or pathogens (e.g., of humans,
domesticated or wild mammals, domesticated or wild birds, and the
like). In some embodiments, the probe is selective for organisms
selected from the group consisting of gram positive bacteria, gram
negative bacteria, yeast, fungi, protozoa, and viruses.
[0197] In various embodiments, the probe can be selective for a
polynucleotide sequence that is characteristic of an organism
selected from the group consisting of Staphylococcus spp., e.g., S.
epidermidis, S. aureus, Methicillin-resistant Staphylococcus aureus
(MRSA), Vancomycin-resistant Staphylococcus; Streptococcus (e.g.,
.alpha., .beta. or .gamma.-hemolytic, Group A, B, C, D or G) such
as S. pyogenes, S. agalactiae; E. faecalis, E. durans, and E.
faecium (formerly S. faecalis, S. durans, S. faecium);
nonenterococcal group D streptococci, e.g., S. bovis and S.
equines; Streptococci viridans, e.g., S. mutans, S. sanguis, S.
salivarius, S. mitior, A. milleri, S. constellatus, S. intermedius,
and S. anginosus; S. iniae; S. pneumoniae; Neisseria, e.g., N.
meningitides, N. gonorrhoeae, saprophytic Neisseria sp;
Erysipelothrix, e.g., E. rhusiopathiae; Listeria spp., e.g., L.
monocytogenes, rarely L. ivanovii and L. seeligeri; Bacillus, e.g.,
B. anthracis, B. cereus, B. subtilis, B. subtilus niger, B.
thuringiensis; Nocardia asteroids; Legionella, e.g., L.
pneumonophilia, Pneumocystis, e.g., P. carinii; Enterobacteriaceae
such as Salmonella, Shigella, Escherichia (e.g., E. coli, E. coli
O157:H7); Klebsiella, Enterobacter, Serratia, Proteus, Morganella,
Providencia, Yersinia, and the like, e.g., Salmonella, e.g., S.
typhi S. paratyphi A, B (S. schottmuelleri), and C (S.
hirschfeldii), S. dublin S. choleraesuis, S. enteritidis, S.
typhimurium, S. heidelberg, S. newport, S. infantis, S. agona, S.
montevideo, and S. saint-paul; Shigella e.g., subgroups: A, B, C,
and D, such as S. flexneri, S. sonnei, S. boydii, S. dysenteriae;
Proteus (P. mirabilis, P. vulgaris, and P. myxofaciens), Morganella
(M. morganii); Providencia (P. rettgeri, P. alcalifaciens, and P.
stuartii); Yersinia, e.g., Y. pestis, Y. enterocolitica;
Haemophilus, e.g., H. influenzae, H. parainfluenzae H. aphrophilus,
H. ducreyi; Brucella, e.g., B. abortus, B. melitensis, B. suis, B.
canis; Francisella, e.g., F. tularensis; Pseudomonas, e.g., P.
aeruginosa, P. paucimobilis, P. putida, P. fluorescens, P.
acidovorans, Burkholderia (Pseudomonas) pseudomallei, Burkholderia
mallei, Burkholderia cepacia and Stenotrophomonas maltophilia;
Campylobacter, e.g., C. fetus fetus, C. jejuni, C. pylori
(Helicobacter pylori); Vibrio, e.g., V. cholerae, V.
parahaemolyticus, V. mimicus, V. alginolyticus, V. hollisae, V.
vulnificus, and the nonagglutinable vibrios; Clostridia, e.g., C.
perfringens, C. tetani, C. difficile, C. botulinum; Actinomyces,
e.g., A. israelii; Bacteroides, e.g., B. fragilis, B.
thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. caccae,
and B. merdae; Prevotella, e.g., P. melaninogenica; genus
Fusobacterium; Treponema, e.g. T. pallidum subspecies endemicum, T.
pallidum subspecies pertenue, T. carateum, and T. pallidum
subspecies pallidum; genus Borrelia, e.g., B burgdorferi; genus
Leptospira; Streptobacillus, e.g., S. moniliformis; Spirillum,
e.g., S. minus; Mycobacterium, e.g., M. tuberculosis, M. bovis, M.
africanum, M. avium M. intracellulare, M. kansasii, M. xenopi, M.
marinum, M. ulcerans, the M. fortuitum complex (M. fortuitum and M.
chelonei), M. leprae, M. asiaticum, M. chelonei subsp. abscessus,
M. fallax, M. fortuitum, M. malmoense, M. shimoidei, M. simiae, M.
szulgai, M. xenopi; Mycoplasma, e.g., M. hominis, M. orale, M.
salivarium, M. fermentans, M. pneumoniae, M. bovis, M.
tuberculosis, M. avium, M. leprae; Mycoplasma, e.g., M. genitalium;
Ureaplasma, e.g., U. urealyticum; Trichomonas, e.g., T. vaginalis;
Cryptococcus, e.g., C. neoformans; Histoplasma, e.g., H.
capsulatum; Candida, e.g., C. albicans; Aspergillus sp;
Coccidioides, e.g., C. immitis; Blastomyces, e.g. B. dermatitidis;
Paracoccidioides, e.g., P. brasiliensis; Penicillium, e.g., P.
marneffei; Sporothrix, e.g., S. schenckii; Rhizopus, Rhizomucor,
Absidia, and Basidiobolus; diseases caused by Bipolaris,
Cladophialophora, Cladosporium, Drechslera, Exophiala, Fonsecaea,
Phialophora, Xylohypha, Ochroconis, Rhinocladiella,
Scolecobasidium, and Wangiella; Trichosporon, e.g., T. beigelii;
Blastoschizomyces, e.g., B. capitatus; Plasmodium, e.g., P.
falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp;
protozoa of the genus Trypanosoma, e.g., T. cruzi; Leishmania,
e.g., L. donovani, L. major L. tropica, L. mexicana, L.
braziliensis, L. viannia braziliensis; Toxoplasma, e.g., T. gondii;
Amoebas of the genera Naegleria or Acanthamoeba; Entamoeba
histolytica; Giardia lamblia; genus Cryptosporidium, e.g., C.
parvum; Isospora belli; Cyclospora cayetanensis; Ascaris
lumbricoides; Trichuris trichiura; Ancylostoma duodenale or Necator
americanus; Strongyloides stercoralis Toxocara, e.g., T. canis, T.
cati; Baylisascaris, e.g., B. procyonis; Trichinella, e.g., T.
spiralis; Dracunculus, e.g., D. medinensis; genus Filarioidea;
Wuchereria bancrofti; Brugia, e.g., B. malayi, or B. timori;
Onchocerca volvulus; Loa loa; Dirofilaria immitis; genus
Schistosoma, e.g., S. japonicum, S. mansoni, S. mekongi, S.
intercalatum, S. haematobium; Paragonimus, e.g., P. Westermani, P.
Skriabini; Clonorchis sinensis; Fasciola hepatica; Opisthorchis sp;
Fasciolopsis buski; Diphyllobothrium latum; Taenia, e.g., T.
saginata, T. solium; Echinococcus, e.g., E. granulosus, E.
multilocularis; Picornaviruses, rhinoviruses echoviruses,
coxsackieviruses, influenza virus; paramyxoviruses, e.g., types 1,
2, 3, and 4; adenoviruses; Herpesviruses, e.g., HSV-1 and HSV-2;
varicella-zoster virus; human T-lymphotrophic virus (type I and
type II); Arboviruses and Arenaviruses; Togaviridae, Flaviviridae,
Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; Viral
encephalitis (alphaviruses [e.g., Venezuelan equine encephalitis,
eastern equine encephalitis, western equine encephalitis]); Viral
hemorrhagic fevers (filoviruses [e.g., Ebola, Marburg] and
arenaviruses [e.g., Lassa, Machupo]); Smallpox (variola);
retroviruses e.g., human immunodeficiency viruses 1 and 2; human
papillomavirus [HPV] types 6, 11, 16, 18, 31, 33, and 35.
[0198] In various embodiments, the probe can be selective for a
polynucleotide sequence that is characteristic of an organisms
selected from the group consisting of Pseudomonas aeruginosa,
Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumoniae,
Escherichia coli, Acinetobacter Baumannii, Serratia marcescens,
Enterobacter aerogenes, Enterococcus faecium, vancomycin-resistant
enterococcus (VRE), Staphylococcus aureus, methecillin-resistant
Staphylococcus aureus (MRSA), Streptococcus viridans, Listeria
monocytogenes, Enterococcus spp., Streptococcus Group B,
Streptococcus Group C, Streptococcus Group G, Streptococcus Group
F, Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus
epidermidis, Gardenerella vaginalis, Micrococcus sps., Haemophilus
influenzae, Neisseria gonorrhoeee, Moraxella catarrahlis,
Salmonella sps., Chlamydia trachomatis, Peptostreptococcus
productus, Peptostreptococcus anaerobius, Lactobacillus fermentum,
Eubacterium lentum, Candida glabrata, Candida albicans, Chlamydia
spp., Camplobacter spp., Salmonella spp., smallpox (variola major),
Yersina Pestis, Herpes Simplex Virus I (HSV I), and Herpes Simplex
Virus II (HSV II).
[0199] In various embodiments, the probe can be selective for a
polynucleotide sequence that is characteristic of Group B
Streptococcus.
[0200] In various embodiments, a method of carrying out PCR on a
sample can further include one or more of the following steps:
heating the biological sample in the microfluidic cartridge;
pressurizing the biological sample in the microfluidic cartridge at
a pressure differential compared to ambient pressure of between
about 20 kilopascals and 200 kilopascals, or in some embodiments,
between about 70 kilopascals and 110 kilopascals.
[0201] In some embodiments, the method for sampling a
polynucleotide can include the steps of: placing a microfluidic
cartridge containing a PCR-ready sample in a receiving bay of a
suitably configured apparatus; carrying out PCR on the sample under
thermal cycling conditions suitable for creating PCR amplicons from
the neutralized polynucleotide in the sample, the PCR-ready sample
comprising a polymerase enzyme, a positive control plasmid, a
fluorogenic hybridization probe selective for at least a portion of
the plasmid, and a plurality of nucleotides; contacting the
neutralized polynucleotide sample or a PCR amplicon thereof with
the at least one fluorogenic probe that is selective for a
polynucleotide sequence, wherein the probe is selective for a
polynucleotide sequence that is characteristic of an organism
selected from the group consisting of gram positive bacteria, gram
negative bacteria, yeast, fungi, protozoa, and viruses; and
detecting the fluorogenic probe, the presence of the organism for
which the one fluorogenic probe is selective is determined.
[0202] Carrying out PCR on a PCR-ready sample can additionally
include: independently contacting each of the neutralized
polynucleotide sample and a negative control polynucleotide with
the PCR reagent mixture under thermal cycling conditions suitable
for independently creating PCR amplicons of the neutralized
polynucleotide sample and PCR amplicons of the negative control
polynucleotide; and/or contacting the neutralized polynucleotide
sample or a PCR amplicon thereof and the negative control
polynucleotide or a PCR amplicon thereof with at least one probe
that is selective for a polynucleotide sequence.
[0203] In various embodiments, a method of using the apparatus and
cartridge described herein can further include one or more of the
following steps: determining the presence of a polynucleotide
sequence in the biological sample, the polynucleotide sequence
corresponding to the probe, if the probe is detected in the
neutralized polynucleotide sample or a PCR amplicon thereof;
determining that the sample was contaminated if the probe is
detected in the negative control polynucleotide or a PCR amplicon
thereof; and/or in some embodiments, wherein the PCR reagent
mixture further comprises a positive control plasmid and a plasmid
probe selective for at least a portion of the plasmid, the method
further including determining that a PCR amplification has occurred
if the plasmid probe is detected.
Kit
[0204] In various embodiments, the microfluidic cartridge as
described herein can be provided in the form of a kit, wherein the
kit can include a microfluidic cartridge, and a liquid transfer
member (such as a syringe or a pipette). In various embodiments,
the kit can further include instructions to employ the liquid
transfer member to transfer a sample containing extracted nucleic
acid from a sample container via a sample inlet to the microfluidic
network on the microfluidic cartridge. In some embodiments, the
microfluidic cartridge and the liquid transfer member can be sealed
in a pouch with an inert gas.
[0205] Typically when transferring a sample from liquid dispenser,
such as a pipette tip, to an inlet on the microfluidic cartridge, a
volume of air is simultaneously introduced into the microfluidic
network, the volume of air being between about 0.5 mL and about 5
mL. Presence of air in the microfluidic network, however, is not
essential to operation of the cartridge described herein.
[0206] In various embodiments, the kit can further include at least
one computer-readable label on the cartridge. The label can
include, for example, a bar code, a radio frequency tag or one or
more computer-readable characters. When used in conjunction with a
similar computer-readable label on a sample container, such as a
vial or a pouch, matching of diagnostic results with sample is
thereby facilitated.
[0207] In some embodiments, a sample identifier of the apparatus
described elsewhere herein is employed to read a label on the
microfluidic cartridge and/or a label on the biological sample.
Heater Unit
[0208] An exemplary heater unit 2020 is shown in FIG. 26. The unit
is configured to deliver localized heat to various selected regions
of a cartridge received in a receiving bay 2014. Heater unit 2020
is configured to be disposed within a diagnostic apparatus during
operation, as further described herein, and in certain embodiments
is removable from that apparatus, for example to facilitate
cleaning, or to permit reconfiguration of the heater circuitry. In
various embodiments, heater unit 2020 can be specific to particular
designs of microfluidic networks and microfluidic substrate
layouts.
[0209] Shown in FIG. 26 is a heater unit having a recessed surface
2044 that provides a platform for supporting a microfluidic
cartridge when in receiving bay 2014. In one embodiment, the
cartridge rests directly on surface 2044. Surface 2044 is shown as
recessed, in FIG. 2, but need not be so and, for example, may be
raised or may be flush with the surrounding area of the heater
unit. Surface 2044 is typically a layer of material that overlies a
heater chip or board, or a heater substrate, that contains heater
micro-circuitry configured to selectively and specifically heat
regions of a microfluidic substrate, such as in a cartridge, in the
receiving bay 2014.
[0210] Area 2044 is configured to accept a microfluidic cartridge
in a single orientation. Therefore area 2044 can be equipped with a
registration member such as a mechanical key that prevents a user
from placing a cartridge into receiving bay 2014 in the wrong
configuration. Shown in FIG. 26 as an exemplary mechanical key 2045
is a diagonally cutout corner of area 2044 into which a
complementarily cutoff corner of a microfluidic cartridge fits.
Other registration members are consistent with the heater unit
described herein, for example, a feature engineered on one or more
edges of a cartridge including but not limited to: several, such as
two or more, cut-out corners, one or more notches cut into one or
more edges of the cartridge; or one or more protrusions fabricated
into one or more edges of the cartridge. Alternative registration
members include one or more lugs or bumps engineered into an
underside of a cartridge, complementary to one or more recessed
sockets or holes in surface 2044 (not shown in the embodiment of
FIG. 26). Alternative registration members include one or more
recessed sockets or holes engineered into an underside of a
cartridge, complementary to one or more lugs or bumps on surface
2044. In general, the pattern of features is such that the
cartridge possesses at least one element of asymmetry so that it
can only be inserted in a single orientation into the receiving
bay.
[0211] Also shown in FIG. 26 is a hand-grasp 2042 that facilitates
removal and insertion of the heater unit into an apparatus by a
user. Cutaway 2048 permits a user to easily remove a cartridge from
receiving bay 2014 after a processing run where, e.g., a user's
thumb or finger when grabbing the top of the cartridge, is afforded
comfort space by cutaway 2048. Both cutaways 2042 and 2048 are
shown as semicircular recesses in the embodiment of FIG. 26, but it
would be understood that they are not so limited in shape. Thus,
rectangular, square, triangular, half-oval, contoured, and other
shaped recesses are also consistent with a heater unit as described
herein.
[0212] In the embodiment of FIG. 26, which is designed to be
compatible with an exemplary apparatus as further described herein,
the front of the heater unit is at the left of the figure. At the
rear of heater unit 2020 is an electrical connection 2050, such as
an RS-232 connection, that permits electrical signals to be
directed to heaters located at specific regions of area 2044 during
sample processing and analysis, as further described herein. Thus,
underneath area 2044 and not shown in FIG. 2 can be an array of
heat sources, such as resistive heaters, that are configured to
align with specified locations of a microfluidic cartridge properly
inserted into the receiving bay. Surface 2044 is able to be cleaned
periodically, for example with common cleaning agents (e.g., a 10%
bleach solution), to ensure that any liquid spills that may occur
during sample handling do not cause any short circuiting. Such
cleaning can be carried out frequently when the heater unit is
disposed in a diagnostic apparatus, and less frequently but more
thoroughly when the unit is removed.
[0213] Other non-essential features of heater unit 2020 are as
follows. One or more air vents 2052 can be situated on one or more
sides (such as front, rear, or flanking) or faces (such as top or
bottom) of heater unit 2020, to permit excess heat to escape, when
heaters underneath receiving bay 2014, are in operation. The
configuration of air vents in FIG. 26, as a linear array of square
vents, is exemplary and it would be understood that other numbers
and shapes thereof are consistent with routine fabrication and use
of a heater unit. For example, although 5 square air vents are
shown, other numbers such as 1, 2, 3, 4, 6, 8, or 10 air vents are
possible, arranged on one side, or spread over two or more sides
and/or faces of the heater unit. In further embodiments, air vents
may be circular, rectangular, oval, triangular, polygonal, and
having curved or squared vertices, or still other shapes, including
irregular shapes. In further embodiments two or more vents need not
be disposed in a line, parallel with one another and with an edge
of the heater unit but may be disposed offset from one another.
[0214] Heater unit 2020 may further comprise one or more guiding
members 2047 that facilitate inserting the heater unit into an
apparatus as further described herein, for an embodiment in which
heater unit 2020 is removable by a user. Heater unit is
advantageously removable because it permits system 2000 to be
easily reconfigured for a different type of analysis, such as
employing a different cartridge with a different registration
member and/or microfluidic network, in conjunction with the same or
a different sequence of processing operations. In other
embodiments, heater unit 2020 is designed to be fixed and only
removable, e.g., for cleaning, replacement, or maintenance, by the
manufacturer or an authorized maintenance agent, and not routinely
by the user. Guiding members 2047 may perform one or more roles of
ensuring that the heater unit is aligned correctly in the
apparatus, and ensuring that the heater unit makes a tight fit and
does not significantly move during processing and analysis of a
sample, or during transport of the apparatus.
[0215] Guiding members shown in the embodiment of FIG. 26 are on
either side of receiving bay 2044 and stretch along a substantial
fraction of the length of unit 2020, but such an arrangement of
guiding members is exemplary. Other guiding members are consistent
with use herein, and include but are not limited to other numbers
of guiding members such as 1, 3, 4, 5, 6, or 8, and other positions
thereof, including positioned in area 2051 of unit 2020, and need
not stretch along as much of the length of unit 2020 as shown in
FIG. 26, or may stretch along its entire length. Guiding members
2047 are shown having a non-constant thickness along their lengths.
It is consistent herein that other guiding members may have
essentially constant thickness along their lengths. At the end of
the heater unit that is inserted into an apparatus, in the
embodiment shown, the edges are beveled to facilitate proper
placement.
[0216] Also shown in FIG. 26 is an optional region of fluorescent
material, such as optically fluorescent material 2069, on area 2051
of heater unit 2020. The region of fluorescent material is
configured to be detected by a detection system further described
herein. The region 2069 is used for verifying the state of optics
in the detection system prior to sample processing and analysis and
therefore acts as a control, or a standard. For example, in one
embodiment a lid of the apparatus in which the heater unit is
disposed, when in an open position, permits ambient light to reach
region 2069 and thereby cause the fluorescent material to emit a
characteristic frequency or spectrum of light that can be measured
by the detector for, e.g., standardization or calibration purposes.
In another embodiment, instead of relying on ambient light to cause
the fluorescent material to fluoresce, light source from the
detection system itself, such as one or more LED's, is used to
shine on region 2069. The region 2069 is therefore positioned to
align with a position of a detector. Region 2069 is shown as
rectangular, but may be configured in other shapes such as square,
circular, elliptical, triangular, polygonal, and having curved or
squared vertices. It is also to be understood that the region 2069
may be situated at other places on the heater unit 2020, according
to convenience and in order to be complementary to the detection
system deployed.
[0217] In particular and not shown in FIG. 26, heater/sensor unit
2020 can include, for example, a multiplexing function in a
discrete multiplexing circuit board (MUX board), one or more
heaters (e.g., a microheater), one or more temperature sensors
(optionally combined together as a single heater/sensor unit with
one or more respective microheaters, e.g., as photolithographically
fabricated on fused silica substrates). The micro-heaters can
provide thermal energy that can actuate various microfluidic
components on a suitably positioned microfluidic cartridge. A
sensor (e.g., as a resistive temperature detector (RTD)) can enable
real time monitoring of the micro-heaters, for example through a
feedback based mechanism to allow for rapid and accurate control of
the temperature. One or more microheaters can be aligned with
corresponding microfluidic components (e.g., valves, pumps, gates,
reaction chambers) to be heated on a suitably positioned
microfluidic cartridge. A microheater can be designed to be
slightly bigger than the corresponding microfluidic component(s) on
the microfluidic cartridge so that even though the cartridge may be
slightly misaligned, such as off-centered, from the heater, the
individual components can be heated effectively.
Heater Configurations to Ensure Uniform Heating of a Region
[0218] The microfluidic substrates described herein are configured
to accept heat from a contact heat source, such as found in a
heater unit described herein. The heater unit typically comprises a
heater board or heater chip that is configured to deliver heat to
specific regions of the microfluidic substrate, including but not
limited to one or more microfluidic components, at specific times.
For example, the heat source is configured so that particular
heating elements are situated adjacent to specific components of
the microfluidic network on the substrate. In certain embodiments,
the apparatus uniformly controls the heating of a region of a
microfluidic network. In an exemplary embodiment, multiple heaters
can be configured to simultaneously and uniformly heat a region,
such as the PCR reaction chamber, of the microfluidic substrate.
The term heater unit, as used herein, may be used interchangeably
to describe either the heater board or an item such as shown in
FIG. 26.
[0219] Referring to FIGS. 27A and 27B, an exemplary set of heaters
configured to heat, cyclically, PCR reaction chamber 1001 is shown.
It is to be understood that heater configurations to actuate other
regions of a microfluidic cartridge such as other gates, valves,
and actuators, may be designed and deployed according to similar
principles to those governing the heaters shown in FIGS. 27A and
27B.
[0220] Referring to FIGS. 27A and 27B, an exemplary PCR reaction
chamber 1001 in a microfluidic substrate, typically a chamber or
channel having a volume .about.1.6 .mu.l, is configured with a long
side and a short side, each with an associated heating element. A
PCR reaction chamber may also be referred to as a PCR reactor,
herein, and the region of a cartridge in which the reaction chamber
is situated may be called a zone. The heater substrate therefore
includes four heaters disposed along the sides of, and configured
to heat, a given PCR reaction chamber, as shown in the exemplary
embodiment of FIG. 27A: long top heater 1005, long bottom heater
1003, short left heater 1007, and short right heater 1009. The
small gap between long top heater 1005 and long bottom heater 1003
results in a negligible temperature gradient (less than 1.degree.
C. difference across the width of the PCR channel at any point
along the length of the PCR reaction chamber) and therefore an
effectively uniform temperature throughout the PCR reaction
chamber. The heaters on the short edges of the PCR reactor provide
heat to counteract the gradient created by the two long heaters
from the center of the reactor to the edge of the reactor.
[0221] It would be understood by one of ordinary skill in the art
that still other configurations of one or more heater(s) situated
about a PCR reaction chamber are consistent with the methods and
apparatus described herein. For example, a `long` side of the
reaction chamber can be configured to be heated by two or more
heaters. Specific orientations and configurations of heaters are
used to create uniform zones of heating even on substrates having
poor thermal conductivity because the poor thermal conductivity of
glass, or quartz, polyimide, FR4, ceramic, or fused silica
substrates is utilized to help in the independent operation of
various microfluidic components such as valves and independent
operation of the various PCR lanes. It would be further understood
by one of ordinary skill in the art, that the principles underlying
the configuration of heaters around a PCR reaction chamber are
similarly applicable to the arrangement of heaters adjacent to
other components of the microfluidic cartridge, such as actuators,
valves, and gates.
[0222] Generally, the heating of microfluidic components, such as a
PCR reaction chamber, is controlled by passing currents through
suitably configured microfabricated heaters. Under control of
suitable circuitry, the lanes of a multi-lane cartridge can then be
controlled independently of one another. This can lead to a greater
energy efficiency of the apparatus, because not all heaters are
heating at the same time, and a given heater is receiving current
for only that fraction of the time when it is required to heat.
Control systems and methods of controllably heating various heating
elements are further described in U.S. patent application Ser. No.
11/940,315, filed Nov. 14, 2007 and entitled "Heater Unit for
Microfluidic Diagnostic System".
[0223] In certain embodiments, each heater has an associated
temperature sensor. In the embodiment of FIG. 27A, a single
temperature sensor 1011 is used for both long heaters. A
temperature sensor 1013 for short left heater, and a temperature
sensor 1015 for short right heater are also shown. The temperature
sensor in the middle of the reactor is used to provide feedback and
control the amount of power supplied to the two long heaters,
whereas each of the short heaters has a dedicated temperature
sensor placed adjacent to it in order to control it. As further
described herein, temperature sensors are preferably configured to
transmit information about temperature in their vicinity to a
processor in the apparatus at such times as the heaters are not
receiving current that causes them to heat. This can be achieved
with appropriate control of current cycles.
[0224] In order to reduce the number of sensor or heater elements
required to control a PCR heater, the heaters may be used to sense
as well as heat, and thereby obviate the need to have a separate
dedicated sensor for each heater. In another embodiment, each of
the four heaters may be designed to have an appropriate wattage,
and connect the four heaters in series or in parallel to reduce the
number of electronically-controllable elements from four to just
one, thereby reducing the burden on the associated electronic
circuitry.
[0225] FIG. 27B shows expanded views of heaters and temperature
sensors used in conjunction with a PCR reaction chamber of FIG.
27A. Temperature sensors 1001 and 1013 are designed to have a room
temperature resistance of approximately 200-300 ohms. This value of
resistance is determined by controlling the thickness of the metal
layer deposited (e.g., a sandwich of 400 .ANG. TiW/3,000 .ANG.
Au/400 .ANG. TiW), and etching the winding metal line to have a
width of approximately 10-25 .mu.m and 20-40 mm length. The use of
metal in this layer gives it a temperature coefficient of
resistivity of the order of 0.5-20.degree. C./ohms, preferably in
the range of 1.5-3.degree. C./ohms. Measuring the resistance at
higher temperatures enables determination of the exact temperature
of the location of these sensors.
[0226] The configuration for uniform heating, shown in FIG. 27A for
a single PCR reaction chamber, can also be applied to a multi-lane
PCR cartridge in which multiple independent PCR reactions
occur.
[0227] Each heater can be independently controlled by a processor
and/or control circuitry used in conjunction with the apparatus
described herein. FIG. 27C shows thermal images, from the top
surface of a microfluidic cartridge when heated by heaters
configured as in FIGS. 27A and 27B, when each heater in turn is
activated, as follows: (A): Long Top only; (B) Long Bottom only;
(C) Short Left only; (D) Short Right only; and (E) All Four Heaters
on. Panel (F) shows a view of the reaction chamber and heaters on
the same scale as the other image panels in FIG. 27C. Also shown in
the figure is a temperature bar.
[0228] The configuration for uniform heating, shown in FIG. 27A for
a single PCR reaction chamber, can be applied to a multi-lane PCR
cartridge in which multiple independent PCR reactions occur. See,
e.g., FIG. 28, which shows an array of heater elements suitable to
heat a cartridge herein.
Heater Multiplexing (Under Software Control)
[0229] Another aspect of the heater unit described herein, relates
to a control of heat within the system and its components. The
method leads to a greater energy efficiency of the apparatus
described herein, because not all heaters are heating at the same
time, and a given heater is receiving current for only part of the
time.
[0230] Generally, the heating of microfluidic components, such as a
PCR reaction chamber, is controlled by passing currents through
suitably configured microfabricated heaters. The heating can be
further controlled by periodically turning the current on and off
with varying pulse width modulation (PWM), wherein pulse width
modulation refers to the on-time/off-time ratio for the current.
The current can be supplied by connecting a microfabricated heater
to a high voltage source (for example, 30 V), which can be gated by
the PWM signal. In some embodiments, the device includes 48 PWM
signal generators. Operation of a PWM generator includes generating
a signal with a chosen, programmable, period (the end count) and a
particular granularity. For instance, the signal can be 4000
(micro-seconds) with a granularity of 1 .mu.s, in which case the
PWM generator can maintain a counter beginning at zero and
advancing in increments of 1 .mu.s until it reaches 4000 .mu.s,
when it returns to zero. Thus, the amount of heat produced can be
adjusted by adjusting the end count. A high end count corresponds
to a greater length of time during which the microfabricated heater
receives current and therefore a greater amount of heat produced.
It would be understood that the granularity and signal width can
take values other than those provided here without departing from
the principles described herein.
Fluorescence Detection System, Including Lenses and Filters, and
Multiple Parallel Detection for a Multi-Lane Cartridge
[0231] The detection system herein is configured to monitor
fluorescence coming from one or more species involved in a
biochemical reaction. The system can be, for example, an optical
detector having a light source that selectively emits light in an
absorption band of a fluorescent dye, and a light detector that
selectively detects light in an emission band of the fluorescent
dye, wherein the fluorescent dye corresponds to a fluorescent
polynucleotide probe or a fragment thereof, as further described
elsewhere herein. Alternatively, the optical detector can include a
bandpass-filtered diode that selectively emits light in the
absorption band of the fluorescent dye and a bandpass filtered
photodiode that selectively detects light in the emission band of
the fluorescent dye. For example, the optical detector can be
configured to independently detect a plurality of fluorescent dyes
having different fluorescent emission spectra, wherein each
fluorescent dye corresponds to a fluorescent polynucleotide probe
or a fragment thereof. For example, the optical detector can be
configured to independently detect a plurality of fluorescent dyes
at a plurality of different locations of, for example, a
microfluidic substrate, wherein each fluorescent dye corresponds to
a fluorescent polynucleotide probe or a fragment thereof. The
detector further has potential for 2, 3 or 4 color detection and is
controlled by software, preferably custom software, configured to
sample information from the detector.
[0232] The detection system described herein is capable of
detecting a fluorescence signal from nanoliter scale PCR reactions.
Advantageously, the detector is formed from inexpensive components,
having no moving parts. The detector can be configured to couple to
a microfluidic cartridge as further described herein, and can also
be part of a pressure application system, such as a sliding lid on
an apparatus in which the detector is situated, that keeps the
cartridge in place.
[0233] FIGS. 29-31B depict an embodiment of a highly sensitive
fluorescence detection system that includes light emitting diodes
(LED's), photodiodes, and filters/lenses for monitoring, in
real-time, one or more fluorescent signals emanating from the
microfluidic channel. The embodiment in FIGS. 29-31B displays a
two-color detection system having a modular design that couples
with a single microfluidic channel found, for example, in a
microfluidic cartridge. It would be understood by one skilled in
the art that the description herein could also be adapted to create
a detector that just detects a single color of light. FIGS. 31A and
31B show elements of optical detector elements 1220 including light
sources 1232 (for example, light emitting diodes), lenses 1234,
light detectors 1236 (for example, photodiodes) and filters 1238.
The detector comprises two LED's (blue and red, respectively) and
two photodiodes. The two LED's are configured to transmit a beam of
focused light on to a particular region of the cartridge. The two
photo diodes are configured to receive light that is emitted from
the region of the cartridge. One photodiode is configured to detect
emitted red light, and the other photodiode is configured to detect
emitted blue light. Thus, in this embodiment, two colors can be
detected simultaneously from a single location. Such a detection
system can be configured to receive light from multiple
microfluidic channels by being mounted on an assembly that permits
it to slide over and across the multiple channels. The filters can
be, for example, bandpass filters, the filters at the light sources
corresponding to the absorption band of one or more fluorogenic
probes and the filters at the detectors corresponding to the
emission band of the fluorogenic probes.
[0234] FIGS. 32 and 33 show an exemplary read-head comprising a
multiplexed 2 color detection system that is configured to mate
with a multi-lane microfluidic cartridge. FIG. 32 shows a view of
the exterior of a multiplexed read-head. FIG. 33 is an exploded
view that shows how various detectors are configured within an
exemplary multiplexed read head, and in communication with an
electronic circuit board.
[0235] Each of the detection systems multiplexed in the assembly of
FIGS. 32 and 33 is similar in construction to the embodiment of
FIGS. 29-31B. The module in FIGS. 32 and 33 is configured to detect
fluorescence from each of 12 microfluidic channels, as found in,
for example, the respective lanes of a 12-lane microfluidic
cartridge. Such a module therefore comprises 24 independently
controllable detectors, arranged as 12 pairs of identical detection
elements. Each pair of elements is then capable of dual-color
detection of a pre-determined set of fluorescent probes. It would
be understood by one of ordinary skill in the art that other
numbers of pairs of detectors are consistent with the apparatus
described herein. For example, 4, 6, 8, 10, 16, 20, 24, 25, 30, 32,
36, 40, and 48 pairs are also consistent and can be configured
according to methods and criteria understood by one of ordinary
skill in the art.
Detection Sensitivity, Time Constant and Gain
[0236] A typical circuit that includes a detector as described
herein includes, in series, a preamplifier, a buffer/inverter, a
filter, and a digitizer. Sensitivity is important so that high gain
is very desirable. In one embodiment of the preamplifier, a very
large, for example 100 G.OMEGA., resistor is placed in parallel
with the diode. Other values of a resistor would be consistent with
the technology herein: such values typically fall in the range
0.5-100 G.OMEGA., such as 1-50 G.OMEGA., or 2-10 G.OMEGA.. An
exemplary pre-amplifier circuit configured in this way is shown in
FIG. 7. Symbols in the figure have their standard meanings in
electronic circuit diagrams.
[0237] The FIG. 34 shows a current-to-voltage
converter/pre-amplifier circuit suitable for use with the detection
system. D11 is the photodetector that collects the fluorescent
light coming from the microfluidic channel and converts it into an
electric current. The accompanying circuitry is used to convert
these fluorescent currents into voltages suitable for measurement
and output as a final measure of the fluorescence.
[0238] A resistor-capacitor circuit in FIG. 34 contains capacitor
C45 and resistor R25. Together, the values of capacitance of C45
and resistance of R25 are chosen so as to impact the time constant
.tau..sub.c, (equal to the product of R25 and C45) of the circuit
as well as gain of the detection circuit. The higher the time
constant, the more sluggish is the response of the system to
incident light. It typically takes the duration of a few time
constants for the photodetector to completely charge to its maximum
current or to discharge to zero from its saturation value. It is
important for the photo current to decay to zero between
measurements, however. As the PCR systems described herein are
intended to afford rapid detection measurements, the product
R.sub.25C.sub.45 should therefore be made as low as possible.
However, the gain of the pre-amplifier whose circuitry is shown is
directly proportional to the (fluorescent-activated) current
generated in the photodetector and the resistance R.sub.25. As the
fluorescence signal from the microfluidic channel device is very
faint (due to low liquid volume as well as small path lengths of
excitation), it is thus important to maximize the value of
R.sub.25. In some embodiments, R.sub.25 is as high as 100 Giga-Ohms
(for example, in the range 10-100 G.OMEGA.), effectively behaving
as an open-circuit. With such values, the time-constant can take on
a value of approximately 50-100 ms by using a low-value capacitor
for C45. For example, the lowest possible available standard
off-the-shelf capacitor has a value of 1 pF (1 picoFarad). A
time-constant in the range 50-100 ms ensures that the photocurrent
decays to zero in approximately 0.5 s (approx. 6 cycles) and
therefore that approximately 2 samplings can be made per second.
Other time constants are consistent with effective use of the
technology herein, such as in the range 10 ms-1 s, or in the range
50 ms-500 ms, or in the range 100-200 ms. The actual time constant
suitable for a given application will vary according to
circumstance and choice of capacitor and resistor values.
Additionally, the gain achieved by the pre-amplifier circuit herein
may be in the range of 10.sup.7-5.times.10.sup.9, for example may
be 1.times.10.sup.9.
[0239] As the resistance value for R25 is very high (.about.100
G.OMEGA.), the manner of assembly of this resistor on the optics
board is important for the overall efficiency of the circuit.
Effective cleaning of the circuit during assembly and before use is
important to achieve an optimal time-constant and gain for the
optics circuit.
[0240] It is also important to test each photo-diode that is used,
because many do not perform according to specification.
Sensitivity and Aperturing
[0241] The LED light passes through a filter before passing through
the sample in the microfluidic channel (as described herein,
typically 300.mu. deep). This is a very small optical path-length
for the light in the sample. The generated fluorescence then also
goes through a second filter, and into a photo-detector.
Ultimately, then, the detector must be capable of detecting very
little fluorescence. Various aspects of the detector configuration
can improve sensitivity, however.
[0242] The illumination optics can be designed so that the
excitation light falling on the PCR reactor is incident along an
area that is similar to the shape of the reactor. As the reactor is
typically long and narrow, the illumination spot should be long and
narrow, i.e., extended, as well. The length of the spot can be
adjusted by altering a number of factors, including: the diameter
of the bore where the LED is placed (the tube that holds the filter
and lens has an aperturing effect); the distance of the LED from
the PCR reactor; and the use of proper lens at the right distance
in between. As the width of the beam incident on the reactor is
determined by the bore of the optical element (approximately 6 mm
in diameter), it is typical to use an aperture (a slit having a
width approximately equal to the width of the reactor, and a length
equal to the length of the detection volume) to make an optimal
illumination spot. A typical spot, then, is commensurate with the
dimensions of a PCR reaction chamber, for example 1.5 mm wide by 7
mm long. FIG. 35A shows the illumination spot across 12 PCR
reactors for the two different colors used. A typical aperture is
made of anodized aluminum and has very low autofluoresence in the
wavelengths of interest. FIG. 35B illustrates a cross-section of a
detector, showing an exemplary location for an aperture 802.
[0243] The optimal spot size and intensity is importantly dependent
on the ability to maintain the correct position of the LED's with
respect to the center of the optical axis. Special alignment
procedures and checks can be utilized to optimize this. The
different illuminations can also be normalized with respect to each
other by adjusting the power current through each of the LED's or
adjusting the fluorescence collection time (the duration for which
the photodetector is on before measuring the voltage) for each
detection spot. It is also important to align the detectors with
the axis of the micro-channels.
[0244] The aperturing is also important for successful fluorescence
detection because as the cross-sectional area of the incident beam
increases in size, so the background fluorescence increases, and
the fluorescence attributable only to the molecules of interest
(PCR probes) gets masked. Thus, as the beam area increases, the use
of an aperture increases the proportion of collected fluorescence
that originates only from the PCR reactor. Note that the aperture
used in the detector herein not only helps collect fluorescence
only from the reaction volume but it correspondingly adjusts the
excitation light to mostly excite the reaction volume. The
excitation and emission aperture is, of course, the same.
[0245] Based on a typical geometry of the optical excitation and
emission system and aperturing, show spot sizes as small as 0.5 mm
by 0.5 mm and as long as 8 mm.times.1.5 mm have been found to be
effective. By using a long detector (having an active area 6 mm by
1 mm) and proper lensing, the aperture design can extend the
detection spot to as long as 15-20 mm, while maintaining a width of
1-2 mm using an aperture. Correspondingly, the background
fluorescence decreases as the spot size is decreased, thereby
increasing the detection sensitivity.
Use of Detection System to Measure/Detect Fluid in PCR Chamber
[0246] The fluorescence detector is sensitive enough to be able to
collect fluorescence light from a PCR chamber of a microfluidic
substrate. The detector can also be used to detect the presence of
liquid in the chamber, a measurement that provides a determination
of whether or not to carry out a PCR cycle for that chamber. For
example, in a multi-sample cartridge, not all chambers will have
been loaded with sample; for those that are not, it would be
unnecessary to apply a heating protocol thereto. One way to
determine presence or absence of a liquid is as follows. A
background reading is taken prior to filling the chamber with
liquid. Another reading is taken after microfluidic operations have
been performed that should result in filling the PCR chamber with
liquid. The presence of liquid alters the fluorescence reading from
the chamber. A programmable threshold value can be used to tune an
algorithm programmed into a processor that controls operation of
the apparatus as further described herein (for example, the second
reading has to exceed the first reading by 20%). If the two
readings do not differ beyond the programmed margin, the liquid is
deemed to not have entered the chamber, and a PCR cycle is not
initiated for that chamber. Instead, a warning is issued to a
user.
Exemplary Electronics and Software
[0247] The heater unit described herein can be controlled by
various electronics circuitry, itself operating on receipt of
computer-controlled instructions. FIG. 36 illustrates exemplary
electronics architecture modules for operating a heater unit and
diagnostic apparatus. It would be understood by one of ordinary
skill in the art that other configurations of electronics
components are consistent with operation of the apparatus as
described herein. In the exemplary embodiment, the electronics
architecture is distributed across two components of the apparatus:
the Analyzer 2100 and a Heater unit 2102. The Analyzer apparatus as
further described herein contains, for example, an Optical
Detection Unit 2108, a Control Board 2114, a Backplane 2112, and a
LCD Touchscreen 2110. The Control Board includes a Card Engine 2116
further described herein, and Compact Flash memory 2118, as well as
other components. The Heater Assembly includes a Heater Board 2104
and a Heater Mux Board 2106, both further described herein.
[0248] In one embodiment, the Card Engine electronics module 2116
is a commercial, off the shelf "single board computer" containing a
processor, memory, and flash memory for operating system
storage.
[0249] The optional LCD+Touchscreen electronics module 2110 is a
user interface, for example, driven through a touchscreen, such as
a 640 pixel by 480 pixel 8 inch LCD and 5-wire touchscreen.
[0250] The Compact Flash electronics module 2118 is, for example, a
256 megabyte commercial, off the shelf, compact flash module for
application and data storage. Other media are alternatively usable,
such as USB-drive, smart media card, memory stick, and smart
data-card having the same or other storage capacities.
[0251] The Backplane electronics module 2112 is a point of
connection for the removable heater assembly 2102. Bare PC boards
with two connectors are sufficient to provide the necessary level
of connectivity.
[0252] The Control Board electronics module 2114 supports
peripherals to the Card Engine electronics module 2116. In one
embodiment, the peripherals include such devices as a USB
host+slave or hub, a USB CDROM interface, serial ports, and
ethernet ports. The Control Board 2114 can include a power monitor
with a dedicated processor. The Control Board may also include a
real time clock. The Control Board may further include a speaker.
The Control Board 2114 also includes a CPLD to provide SPI access
to all other modules and programming access to all other modules.
The Control Board includes a programmable high voltage power
supply. The Control Board includes a Serial-Deserializer interface
to the LCD+Touchscreen electronics module 2110 and to the Optical
Detection Unit electronics module 2108. The Control Board also
includes module connectors.
[0253] In the exemplary embodiment, the optical detection unit
electronics module 2108 contains a dedicated processor. The optical
detection unit 2108 contains a serializer-deserializer interface.
The optical detection unit 2108 contains LED drivers. The optical
detection unit also contains high gain-low noise photodiode
amplifiers. The optical detection unit can have power monitoring
capability. The optical detection unit can also be remotely
reprogrammable.
[0254] The Heater Board electronics module 2104 is preferably a
glass heater board. The Heater Board has PCB with bonding pads for
glass heater board and high density connectors.
[0255] In one embodiment, the heater mux (`multiplex`) board
electronics module 2106 has 24 high-speed ADC, 24 precision current
sources, and 96 optically isolated current drivers for heating. The
heater mux board has the ability to time-multiplex
heating/measurement. The heater mux board has multiplexer banks to
multiplex inputs to ADC, and to multiplex current source outputs.
The heater mux board has a FPGA with a soft processor core and
SDRAM. The heater mux board has a Power Monitor with a dedicated
processor. The Heater Mux Board can be remotely reprogrammable.
[0256] In another embodiment, control electronics can be spread
over four different circuit board assemblies. These include the
MAIN board: Can serve as the hub of the Analyzer control
electronics and manages communication and control of the other
various electronic subassemblies. The main board can also serve as
the electrical and communications interface with the external
world. An external power supply (12V DC/10 A; UL certified) can be
used to power the system. The unit can communicate via 5 USB ports,
a serial port and an Ethernet port. Finally, the main board can
incorporate several diagnostic/safety features to ensure safe and
robust operation of the Analyzer.
[0257] MUX Board: Upon instruction from the main board, the MUX
board can perform all the functions typically used for accurate
temperature control of the heaters and can coordinate the
collection of fluorescence data from the detector board.
[0258] LCD Board: Can contain the typical control elements to light
up the LCD panel and interpret the signals from the touch sensitive
screen. The LCD/touch screen combination can serve as a mode of
interaction with the user via a Graphical User Interface.
[0259] Detector Board: Can house typical control and processing
circuitry that can be employed to collect, digitize, filter, and
transmit the data from the fluorescence detection modules.
[0260] Certain software can be executed in each electronics module.
The Control Board Electronics Module executes, for example, Control
Board Power Monitor software. The Card Engine electronics module
executes an operating system, graphical user interface (GUI)
software, an analyzer module, and an application program interface
(api). The Optical Detection Unit electronics module executes an
optics software module. The Heater Mux Board electronics module
executes dedicated Heater Mux software, and Heater Mux Power
Monitor software. Each of the separate instances of software can be
modular and under a unified control of, for example, driver
software.
[0261] The exemplary electronics can use Linux, UNIX, Windows, or
MacOS, including any version thereof, as the operating system. The
operating system is preferably loaded with drivers for USB,
Ethernet, LCD, touchscreen, and removable media devices such as
compact flash. Miscellaneous programs for configuring the Ethernet
interface, managing USB connections, and updating via CD-ROM can
also be included.
[0262] In the embodiment of FIG. 36, the analyzer module is the
driver for specific hardware. The analyzer module provides access
to the Heater Mux Module, the Optical Detection Unit, the Control
Board Power Monitor, the Real Time Clock, the High Voltage Power
Supply, and the LCD backlight. The analyzer module provides
firmware programming access to the Control Board power monitor, the
Optical Detection Unit, and the Heater Mux Module.
[0263] The API provides uniform access to the analyzer module
driver. The API is responsible for error trapping, and interrupt
handling. The API is typically programmed to be thread safe.
[0264] The GUI software can be based on a commercial, off-the-shelf
PEG graphics library. The GUI can use the API to coordinate the
self-test of optical detection unit and heater assembly. The GUI
starts, stops, and monitors test progress. The GUI can also
implement an algorithm to arrive on diagnosis from fluorescence
data. The GUI provides access control to unit and in some
embodiments has an HIS/LIS interface.
[0265] The Control Board Power Monitor software monitors power
supplies, current and voltage, and signals error in case of a
fault.
[0266] The Optics Software performs fluorescence detection which is
precisely timed to turn on/off of LED with synchronous digitization
of the photodetector outputs. The Optics Software can also monitor
power supply voltages. The Optics Software can also have self test
ability.
[0267] The Heater Mux Module software implements a "protocol
player" which executes series of defined "steps" where each "step"
can turn on sets of heaters to implement a desired microfluidic
action. The Heater Mux Module software also has self test ability.
The Heater Mux Module software contains a fuzzy logic temperature
control algorithm.
[0268] The Heater Mux Power Monitor software monitors voltage and
current levels. The Heater Mux Power Monitor software can
participate in self-test, synchronous, monitoring of the current
levels while turning on different heaters.
EXAMPLES
[0269] The following are exemplary aspects of various parts and
functions of the system described herein.
[0270] Additional embodiments of a cartridge are found in U.S.
patent application Ser. No. 11/940,310, entitled "Microfluidic
Cartridge and Method of Making Same", and filed on even date
herewith, the specification of which is incorporated herein by
reference.
[0271] Additional embodiments of heater units and arrays are
described in U.S. patent application Ser. No. 11/940,315, entitled
"Heater Unit for Microfluidic Diagnostic System" and filed on even
date herewith, the specification of which is incorporated herein by
reference in its entirety.
[0272] Further description of suitably configured detectors are
described in U.S. patent application Ser. No. 11/940,321, filed on
Nov. 14, 2007 and entitled "Fluorescence Detector for Microfluidic
Diagnostic System", incorporated herein by reference.
Example 1
Analyzer Having Removable Heater Unit
[0273] This non-limiting example describes pictorially, various
embodiments of an apparatus, showing incorporation of a heater unit
and a microfluidic cartridge operated on by the heater unit.
[0274] FIG. 37 shows an apparatus 1100 that includes a housing
having a display output 1102, an openable lid 1104, and a bar code
reader 1106. The cartridge is positioned in a single orientation in
a receiving bay under the lid, FIG. 38. The lid of the apparatus
can be closed to apply pressure to the cartridge, as shown in FIG.
39. The unit currently weighs about 20 lbs. and is approximately
10'' wide by 16'' deep by 13'' high.
[0275] FIGS. 40 and 41: The heating stage of the apparatus can be
removable for cleaning, maintenance, or to replace a custom heating
stage for a particular microfluidic cartridge. FIGS. 40 and 41 also
show how a heater unit is insertable and removable from a front
access door to an analyzer apparatus.
Example 2
Assembly of an Exemplary Heater Unit
[0276] FIG. 42A shows an exploded view of an exemplary heater unit.
The unit has a top cover and a bottom cover that together enclose a
Mux board (control board), a pressure support layer, and insulator
film, and a microthermal circuit on a PCB. The last of these is the
heat source that selectively heats regions of a microfluidic
substrate placed in contact therewith through the top cover.
[0277] An exemplary heater substrate, FIG. 42B, consists of a
photo-lithographically processed glass wafer bonded to a standard
0.100'' standard FR4 printed circuit board. The glass wafer is 0.5
mm thick and is cut into a rectangle the size of
.about.3.5.times.4.25 inches. The glass substrate has numerous
metal heaters and resistive temperature sensors
photo-lithographically etched on the surface of the glass wafer.
The substrate is aligned and bonded to the PCBoard using a
compliant epoxy, ensuring flatness to within 2-3 mils over the
surface of the wafer. The cured epoxy should withstand up to
120.degree. C. for two hours minimum. Approximately 300-400 bond
pads of the size of approximately 1 mm.times.0.25 mm, with exposed
gold surfaces, are located along the two long edges of the wafer.
These pads are wirebonded (ball-bonding) to corresponding pads on
the PCB using 1.5 mil gold wires. Wire bonding is athreading
process, standard in semiconductor FAB. Alternatively, a flip-chip
method may be used, though such methods are more complicated and
may warp the wafer because of thermal mismatch. Wire bonds should
have good integrity and pass defined pull strength. The substrate
is baked at 120.degree. C. for two hours and then the wire bonds
are encapsulated by a compliant epoxy that will protect the
wirebonds but not damage the bonds even at 120.degree. C.
Encapsulant should not spill over predefined area around the
wirebonds and should not be taller than a defined height. For
example, instead of laying epoxy all over the substrate, lines
(e.g., a hash pattern) of it are made so that epoxy cures and air
escapes through side. Alternatively, a laminate fill (adhesive on
both sides) can be used. Standard connectors are soldered to the
PCB and then the unit is tested using a test set-up to ensure all
heaters and sensors read the right resistance values.
[0278] Pictures of an exemplary Mux board and assembled heater unit
are shown in FIGS. 27-29.
Example 3
Pulse Width Modulation for Heater Circuitry
[0279] In various embodiments, the operation of a PWM generator can
also include a programmable start count in addition to the
aforementioned end count and granularity. In such embodiments,
multiple PWM generators can produce signals that can be selectively
non-overlapping (e.g., by multiplexing the on-time of the various
heaters) such that the current capacity of the high voltage power
is not exceeded. Multiple heaters can be controlled by different
PWM signal generators with varying start and end counts. The
heaters can be divided into banks, whereby a bank defines a group
of heaters of the same start count. For example, 36 PWM generators
can be grouped into six different banks, each corresponding to a
certain portion of the PWM cycle (500 ms for this example). The end
count for each PWM generator can be selectively programmed such
that not more than six heaters will be on at any given time. A
portion of a PWM cycle can be selected as dead time (count 3000 to
4000 for this example) during which no heating takes place and
sensitive temperature sensing circuits can use this time to sense
the temperature. The table below represents a PWM cycle for the
foregoing example:
TABLE-US-00002 Start Count End Count Max End count Bank 1 PWM
generator#1 0 150 500 PWM generator#2 0 220 500 . . . . . . . . .
PWM generator#6 0 376 500 Bank 2 PWM generator#7 500 704 1000 PWM
generator#8 500 676 1000 . . . . . . . . . . . . PWM generator#12
500 780 1000 Bank 3 PWM generator#13 1000 1240 1500 PWM
generator#14 1000 1101 1500 . . . . . . . . . . . . PWM
generator#18 1000 1409 1500 Bank 4 PWM generator#19 1500 1679 2000
PWM generator#20 1500 1989 2000 . . . . . . . . . . . . PWM
generator#24 1500 1502 2000 Bank 5 PWM generator#25 2000 2090 2500
PWM generator#26 2000 2499 2500 . . . . . . . . . . . . PWM
generator#30 2000 2301 2500 Bank 6 PWM generator#31 2500 2569 3000
PWM generator#32 2500 2790 3000 . . . . . . . . . . . . PWM
generator#36 2500 2678 3000
Example 4
Detector Integrated in Force Member
[0280] This non-limiting example describes pictorially, various
embodiments of a detection system integrated into a force member,
in an apparatus for carrying out diagnostics on microfluidic
samples.
[0281] FIG. 43A: The lid of the apparatus can be closed, which can
block ambient light from the sample bay, and place an optical
detector contained in the lid into position with respect to the
microfluidic cartridge.
[0282] FIG. 43B: The lid of the apparatus can be closed to apply
pressure to the cartridge. Application of minimal pressure on the
cartridge: after the slider compresses the cartridge, the slider
can compress the compliant label of the cartridge. This can cause
the bottom of the cartridge to be pressed down against the surface
of the heater unit present in the heater module. Springs present in
the slider can deliver, for example approximately 50 lb of pressure
to generate a minimum pressure, for example 2 psi over the entire
cartridge bottom.
[0283] Thermal interface: the cartridge bottom can have a layer of
mechanically compliant heat transfer laminate that can enable
thermal contact between the microfluidic substrate and the
microheater substrate of the heater module. A minimal pressure of 1
psi can be employed for reliable operation of the thermal valves,
gate and pumps present in the microfluidic cartridge.
[0284] Mechanicals and assembly: the Analyzer can have a simple
mechanical frame to hold the various modules in alignment. The
optics module can be placed in rails for easy opening and placement
of cartridges in the Analyzer and error-free alignment of the
optics upon closing. The heater/sensor module can be also placed on
rails or similar guiding members for easy removal and insertion of
the assembly.
[0285] Slider: the slider of the Analyzer can house the optical
detection system as well as the mechanical assembly that can
enables the optics jig to press down on the cartridge when the
handle of the slider is turned down onto the analyzer. The optics
jig can be suspended from the case of the slider at 4 points. Upon
closing the slider and turning the handle of the analyzer down, 4
cams can turn to push down a plate that presses on 4 springs. On
compression, the springs can deliver approximately 50 lb on the
optical block. See FIGS. 44A-44C.
[0286] The bottom surface of the optics block can be made flat to
within 100 microns, typically within 25 microns, and this flat
surface can press upon the compliant (shore hardness approximately
50-70) label (approximately 1.5 mm thick under no compression) of
the cartridge making the pressure more or less uniform over the
cartridge. An optional lock-in mechanism can also be incorporated
to prevent the slider from being accidentally knocked-off while in
use.
[0287] FIG. 45A shows a side view of a lever assembly 1200, with
lever 1210, gear unit 1212, and force member 1214. Assembly 1200
can be used to close the lid of the apparatus and (through force
members 1214) apply force to a microfluidic cartridge 1216 in the
receiving chamber 1217. One force member is visible in this cut
away view, but any number, for example 4, can be used. The force
members can be, for example, a manual spring loaded actuator as
shown, an automatic mechanical actuator, a material with sufficient
mechanical compliance and stiffness (e.g., a hard elastomeric
plug), and the like. The force applied to the microfluidic
cartridge 1216 can result in a pressure at the surface of the
microfluidic cartridge 1216 of at least about 0.7 psi to about 7
psi (between about 5 and about 50 kilopascals), or in some
embodiments about 2 psi (about 14 kilopascals.
[0288] FIG. 45B shows a side view of lever assembly 1200, with
microfluidic cartridge 1216 in the receiving chamber 1217. A heat
source 1219 (for example, a xenon bulb as shown) can function as a
radiant heat source directed at a sample inlet reservoir 1218,
where the heat can lyse cells in reservoir 1218. A thermally
conductive, mechanically compliant layer 1222 can lie at an
interface between microfluidic cartridge 1216 and thermal stage
1224. Typically, microfluidic cartridge 1216 and thermal stage 1224
can be planar at their respective interface surfaces, e.g., planar
within about 100 microns, or more typically within about 25
microns. Layer 1222 can improve thermal coupling between
microfluidic cartridge 1216 and thermal stage 1224. Optical
detector elements 1220 can be directed at the top surface of
microfluidic cartridge 1216.
[0289] FIGS. 45C and 45D show further cross-sectional views.
Example 6
Exemplary Optics Board
[0290] An exemplary optics board is shown schematically in FIG. 46,
and is used to collect and amplify the fluorescent signature of a
successful chemical reaction on a micro-fluidic cartridge, and
control the intensity of LED's using pulse-width modulation (PWM)
to illuminate the cartridge sample over up to four channels, each
with two color options. Additionally, it receives instructions and
sends results data back over an LVDS (low-voltage differential
signaling) SPI (serial peripheral interface). In some embodiments
there is a separate instance of this circuitry for each PCR channel
that is monitored.
[0291] The power board systems include: a +12V input; and +3.3V,
+3.6V, +5V, and -5V outputs, configured as follows: the +3.3V
output contains a linear regulator, is used to power the LVDS
interface, should maintain a +/-5% accuracy, and supply an output
current of 0.35 A; the +3.6V output contains a linear regulator, is
used to power the MSP430, should maintain a +/-5% accuracy, and
supply an output current of 0.35 A; the +5V output contains a
linear regulator, is used to power the plus rail for op-amps,
should maintain a +/-5% accuracy, and supply an output current of
0.35 A; the -5V output receives its power from the +5V supply, has
a mV reference, is used to power the minus rail for op-amps and for
the photo-detector bias, should maintain a +/-1% voltage accuracy,
and supply an output current of 6.25 mA+/-10%. Additionally, the
power board has an 80 ohm source resistance, and the main board
software can enable/disable the regulator outputs.
[0292] The main board interface uses a single channel of the LVDS
standard to communicate between boards. This takes place using SPI
signaling over the LVDS interface which is connected to the main
SPI port of the control processor. The interface also contains a
serial port for in-system programming.
[0293] The optical detection system of FIG. 46 comprises a control
processor, LED drivers, and a photo-detection system. In the
exemplary embodiment, the control processor is a TI MSP430F1611
consisting of a dual SPI (one for main board interface, and one for
ADC interface) and extended SRAM for data storage. It has the
functions of power monitoring, PWM LED control, and SPI linking to
the ADC and main board. The LED drivers contain NPN transistor
switches, are connected to the PWM outputs of the control
processor, can sink 10 mA @ 12V per LED (80 mA total), and are
single channel with 2 LEDs (one of each color) connected to each.
The photo-detection system has two channels and consists of a
photo-detector, high-sensitivity photo-diode detector, high gain
current to voltage converter, unity gain voltage inverting
amplifier, and an ADC. Additionally it contains a 16 channel
Sigma-delta (only utilizing the first 8 channels) which is
connected to the second SPI port of the control processor.
[0294] During assembly of the various components on to the PC
board, such as may occur on a production line, there are the
following considerations. The extremely high impedance of the
photo-detection circuit means that a rigorous cleaning procedure
must be employed. Such a procedure may include, for example: After
surface mount components are installed, the boards are washed on a
Weskleen and blow dried upon exiting conveyor. The belt speed can
be set at 20-30. The boards are soaked in an alcohol bath for
approximately 3 minutes, then their entire top and bottom surfaces
are scrubbed using a clean, soft bristle brush. The boards are
baked in a 105.degree. F. (40.degree. C.) oven for 30 minutes to
dry out all components.
[0295] After all the components are installed: the soldered areas
of the boards can be hand wash using deionized water and a soft
bristle brush. The same soldered areas can be hand washed using
alcohol and a soft bristle brush. The boards are allowed to air
dry. Once the board is cleaned, the optical circuitry must be
conformal coated to keep contaminates out.
[0296] The foregoing description is intended to illustrate various
aspects of the present technology. It is not intended that the
examples presented herein limit the scope of the present
technology. The technology now being fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
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