US007507575 B2

(12) United States Patent (10) Patent No.: US 7,507,575 B2

Bedingham et al. (45) Date of Patent: Mar. 24, 2009
(54) MULTIPLEX FLUORESCENCE DETECTION 5,296,958 A * 3/1994 Roddy et al. ................ 359,204
DEVICE HAVING REMOVABLE OPTICAL 5,414,600 A 5/1995 Stroblet al. .................. 362,32
MODULES 5,473,437 A 12/1995 Blumenfeld et al. ........ 356,417
5,585,069 A 12/1996 Zanzucchi et al. .......... 422/100
(75) Inventors: William Bedingham, Woodbury, MN 5,639,668 A 6/1997 Neel et al. .................. 436,172
(US); Peter D. Ludowise, Cottage 5,751,874. A * 5/1998 Chudoba et al. .............. 385/72
Grove, MN (US); Barry W. Robole, 5,766,889 A 6/1998 Atwood ..................... 435,912
Woodville, WI (US) 5,928,907 A 7/1999 Woudenberg et al. ...... 435,912
73) Assignee:
9. 3M Innovative Properties
p Company,
pany
St.Paul, MN (US) (Continued)
*) Notice: Subject to anyy disclaimer, the term of this FOREIGN PATENT DOCUMENTS
patent is extended or adjusted under 35 DE 2 O55944 5, 1972
U.S.C. 154(b) by 249 days.
(21) Appl. No.: 11/174,754
(Continued)
(22) Filed: Jul. 5, 2005 OTHER PUBLICATIONS
(65) Prior Publication Data Definition from m-w.com.
US 2006/022.3169 A1 Oct. 5, 2006
(Continued)
Related U.S. Application Data Primary Examiner BJ Forman
(60) Provisional application No. 60/667,461, filed on Apr. Assistant Examiner—Robert T. Crow
1, 2005.
(57) ABSTRACT
(51) Int. Cl.
CI2M I/34 (2006.01)
CI2M I/00 (2006.01) Techniques are described for the detection of multiple target
CI2O I/68 (2006.01) species in real-time PCR (polymerase chain reaction). For
(52) U.S. Cl. .............. 435/287.2:435/287.1:435/283.1; example, a system comprises a data acquisition device and a
435/6 detection device coupled to the data acquisition device. The
(58) Field of Classification Search ....................... None
detection device includes a rotating disk having a plurality of
See application file for complete search history. process chambers having a plurality of species that emit fluo
rescent light at different wavelengths. The device further
(56) References Cited includes a plurality of removable optical modules. Each of the
removable optical modules is optically configured to excite
U.S. PATENT DOCUMENTS the species and capture fluorescent light emitted by the spe
3,949,231 A 4, 1976 Blunck et al. ............... 250,493 cies at different wavelengths. A fiber optic bundle coupled to
4,343,991 A 8, 1982 Fujiwara et al. ............. 250,227 the plurality of removable optical modules conveys the fluo
4,726,676 A * 2/1988 Maslaney et al. .......... 356,731 rescent light from the optical modules to a single detector.
4.909,990 A 3, 1990 Block et al. .............. 422,82.11
4,927,766 A 5/1990 Auerbach et al. ............. 436/44 36 Claims, 23 Drawing Sheets

ik.gif 83.
ask
Asia
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is: is
is
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i.
 US 7,507,575 B2
Page 2

U.S. PATENT DOCUMENTS 2004/O126279 A1* 7/2004 Renzi et al. ................. 422/100
2004/0224317 A1 1 1/2004 Kordunsky et al. . ... 435/6
5,994,150 A 1 1/1999 Challener et al. ........... 436,518 2005/0012199 A1 1/2005 Rosenau et al. ............. 257,696
6,015,674. A 1/2000 Woudenberg et al. .......... 435/6 2005/0014249 A1 1/2005 Staimer et al. .............. 435/287
6,144,448 A 11, 2000 Mitoma - - - - - - - - - - - - - - - - - - - - - - 356,317 2005/0023765 A1 2/2005 Coombs 277 345
6,161,946 A 12/2000 Bishop et al. 2005/0048595 A1 3/2005 Yamatsu et al. ............... 435/18
6,232,075 B1* 5/2001 Williams ....................... 435/6 2005/0059062 A1 3/2005 Kaiser ........................... 435/6
6,339,473 B1 1/2002 Gordon ... 356,440 2005, OO64582 A1 3, 2005 Wittwer et al. 435/287
6,342,349 B1 1/2002 Virtanen .. ... 435/6 2005/0074784 A1 4/2005 Vo-Dinh ........................ 435/6
6,442,116 B2 * 8/2002 Asano ..... 369/4728 2005, 0109396 A1 5/2005 Zucchelli et al. .............. 137.67
6,537.211 B1* 3/2003 Wang et al. . ... 600,178 2005/0130177 A1 6/2005 Bedingham et al. ... 435/6
6,563,113 B1* 5/2003 Amann et al. ... ... 250,309 2005/0151972 A1 7/2005 Boege et al. ................ 356,417
6,563,581 B1 5/2003 Oldham et al. .............. 356/317 2006/0223169 A1 10/2006 Bedingham et al. 435/287
6,597.450 B1, 7/2003 Andrews et al. 356.317 2006/0223172 A1 10/2006 Bedingham et al. ......... 435/288
6,597,832 B2 * 7/2003 Cheng ........... ... 385.25 2007/00093.82 A1 1/2007 Bedingham et al. ........... 422/63
6,616,304 B2 9/2003 Li ................. 362/302 2007/0009383 A1 1/2007 Bedingham et al. ........... 422/63
6,627,159 B1 9/2003 Bedingham et al. ......... 422/100 2007/0010007 A1 1/2007 Aysta et al. ................. 435/287
6,734.401 B2 5/2004 Bedingham et al. ......... 219/388
6,803,999 B1 10/2004 Gordon ......... ... 356,73 FOREIGN PATENT DOCUMENTS
6,806,954 B2 10/2004 Sandstrom 356.317
6,821,771 B2 11/2004 Festoc ........ 435/287 JP 61-20839 1, 1986
6,833,536 B2 12/2004 Shigeura ..... 219/553 WO WO91,03915 3, 1991
6,992.278 B2 1/2006 Soberg et al. . 250,231 WO WO 98,38510 9, 1998
6,992,769 B2 1/2006 Gordon ............ ... 356/440 WO WOO1/O1112 1, 2001
7,088.650 B1* 8/2006 Worthington et al. ... 369/47.15 WO WO O2/O73605 9, 2002
7,238,269 B2 7/2007 Gason et al. WO WOO3,058253 T 2003
7,322.254 B2 1/2008 Bedingham et al. WO WOO3,O98278 11, 2003
2001/002903.6 A1 10, 2001 Landers et al. . 435/91.1 WO WOO3,O982.79 11, 2003
2001/0046712 A1* 1 1/2001 Hang et al. .... 436/172 WO WOO3,102226 12/2003
2001/0052927 A1* 12/2001 Takase et al. ..... 347257 WO WO 2004/079343 9, 2004
2002/0039333 A1* 4/2002 Tsukahara et al. 360,446 WO WO 2004/087950 10, 2004
2002/0043626 A1 4/2002 Booker et al. ............... 250/459
2002/0047003 A1* 4/2002 Bedingham et al. ......... 219/388 OTHER PUBLICATIONS
2002/0048533 Al 4/2002 Harms et al. ........, ... 422.99 Wenner et al.; “Biosensing on the CD Microfluidic Platform with
2002fOO64885 A1 5/2002 Bedingham et al. 436,174 Genetically Engineered Proteins'; Society of Automotive Engineers,
2002/0076354 A1 6/2002 Cohen ......................... 422,72 Inc.; Paper 2000-01-2513; pp. 1-6; 2000.
2002/0104884 A1* 8, 2002 Meier et al. ............ 235,462.25 Lee et al.; "A novel real-time PCR machine with a miniature spec
2002/0172980 A1* 11/2002 Phan et al. ................... 435.7.1 trometer for fluorescence sensing in a micro liter volume glass cap
2003.0054563 A1 3/2003 Ljungstrom et al. ......... 436,172 illary': Sensors and Actuators B 100 (2004) 401-410.
2003/0190.184 A1* 10, 2003 O'Brien et al. ............. 403,122 Lee et al., “Development of a CCD-based fluorimeter for real-time
2003/0219754 A1 1 1/2003 Oleksy et al. .................. 435/6 PCR machine'; Sensors and Actuators B 107 (2005) 872-881.
2004/0067051 A1 4/2004 Kylberg et al. .............. 392/.407
2004/0072335 A1 4/2004 Boege et al. ................ 435/287 * cited by examiner
U.S. Patent Mar. 24 9 2009 Sheet 1 of 23 US 7,507,575 B2

(81)
I
U.S. Patent Mar. 24, 2009 Sheet 2 of 23 US 7,507,575 B2
U.S. Patent Mar. 24, 2009 Sheet 3 of 23 US 7,507,575 B2

52 56

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U.S. Patent Mar. 24, 2009 Sheet 4 of 23 US 7,507,575 B2
U.S. Patent Mar. 24, 2009 Sheet 5 of 23 US 7,507,575 B2
U.S. Patent Mar. 24, 2009 Sheet 6 of 23 US 7,507,575 B2
U.S. Patent Mar. 24, 2009 Sheet 7 of 23
U.S. Patent Mar. 24, 2009 Sheet 8 of 23 US 7,507,575 B2

102
U.S. Patent Mar. 24, 2009 Sheet 9 of 23 US 7,507,575 B2
U.S. Patent Mar. 24, 2009 Sheet 10 of 23 US 7,507,575 B2

1O

LIGHT SOURCE
DISK MOTOR
126 3O

MEMORY FILTER
124 34
LENS
38
FILTER
a sm 40

PROCESSOR
COLLECTING
LENS
122 2
- -

-2 8 - a.
HEATING
ELEMENT DETECTOR
134 18
SLOT SENSOR
TRIGGER
27
LASER COMMUNICATION
INTERFACE
POWER SOURCE
136 130 132.

Fig. 8
U.S. Patent Mar. 24, 2009 Sheet 11 of 23 US 7,507,575 B2

OLHE/\CN ES
z?T
U.S. Patent Mar. 24, 2009 Sheet 12 of 23 US 7,507,575 B2

CREATE PROGRAM
PARAMETERS
15O
LOAD DISK

152
START PROGRAM

154
SPN DISK

156 166
DETECT CHECK DISK
FLUORESCENCE TEMPERATURE
158
168
SYNCHRONIZE MODULE DISK
DETECTION WITH DISK TEMPERATURE TO
TARGET
16O

SAVE DATA <G> 17O

162
172
HOLD
TEMPERATURE

164
STOP DISK
SPINNING END

Fig. 10
U.S. Patent Mar. 24, 2009 Sheet 13 of 23 US 7,507,575 B2

149
TURN ON
MODULE LED

151
SPIN DISK ONE
ROTATION

153
COLLECT LIGHT
FROM CHAMBERS

155
PLACE SAMPLED
DATA IN CORRECT BIN

157
ANOTHER
ROTATION?

161

TURN OFF LED EE

Fig. 11
U.S. Patent Mar. 24, 2009 Sheet 14 of 23 US 7,507,575 B2

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U.S. Patent Sheet 16 of 23 US 7,507,575 B2

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U.S. Patent Mar. 24, 2009 Sheet 18 of 23 US 7,507,575 B2

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U.S. Patent Mar. 24, 2009 Sheet 21 of 23 US 7,507,575 B2

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U.S. Patent Mar. 24, 2009 Sheet 22 of 23 US 7,507,575 B2

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U.S. Patent Mar. 24, 2009 Sheet 23 of 23 US 7,507,575 B2
 US 7,507,575 B2
1. 2
MULTIPLEX FLUORESCIENCE DETECTION tiple, parallel reactions at different wavelengths. The reaction
DEVICE HAVING REMOVABLE OPTICAL may, for example, occur within a single process chamber
MODULES (e.g., well) of a rotating disk. Additionally, each optical mod
ule may be removable to quickly change the detection capa
CROSS REFERENCE TO RELATED bilities of the device.
APPLICATIONS The plurality of optical modules may be optically coupled
to a single detector by a multi-legged optical fiberbundle. In
This application claims the benefit of U.S. Provisional this manner, multiplexing can beachieved by using a plurality
Application No. 60/667,461, filed Apr. 1, 2005, the entire of optical modules and a single detector, e.g., a photomulti
content of which is incorporated herein by reference. 10 plier tube. The optical components in each optical module
may be selected to maximize sensitivity and minimize the
TECHNICAL FIELD amount of spectral crosstalk, i.e., signals from one dye on
another optical module.
The invention relates to assaying systems and, more par In one embodiment, a device comprises a motor to rotate a
ticularly, techniques for the detection of multiple target spe 15 disk having a plurality of process chambers each holding a
cies using fluorescent dyes. respective sample and a plurality of fluorescent dyes, a plu
BACKGROUND
rality of optical modules, and a housing having a plurality of
locations adapted to receive the optical modules wherein each
Optical disc systems are often used to perform various of the optical modules includes an optical channel having a
biological, chemical or bio-chemical assays. In a typical sys light source selected for a different one of the dyes and a lens
to capture fluorescent light emitted from the disk.
tem, a rotatable disc is used as a medium for storing and In another embodiment, a system comprises a data acqui
processing fluid specimens, such as blood, plasma, serum, sition device. The system further comprises a detection
urine or other fluid.
One type of analysis is polymerase chain reaction (PCR), 25
device coupled to the data acquisition device, wherein the
which is often used for nucleic acid sequence analysis. In detection device comprises a motor to rotate a disk having a
particular, PCR is often used for DNA sequencing, cloning, plurality of process chambers each holding a respective
genetic mapping, and other forms of nucleic acid sequence sample and a plurality of fluorescent dyes, a plurality of
analysis. optical modules, and a housing having a plurality of locations
In general, PCR relies on the ability of DNA-copying 30
adapted to receive the optical modules wherein each of the
enzymes to remain stable at high temperatures. There are optical modules includes an optical channel having a light
three major steps in PCR: denaturation, annealing, and exten source selected for a different one of the dyes and a lens to
Sion. During the denaturation, a liquid sample is heated at capture fluorescent light emitted from the disk.
approximately 94° C. During this process, double DNA In an additional embodiment, a method comprises rotating
strands "melt” open into single stranded DNA and all enzy 35
a disk having a plurality of process chambers each having a
matic reactions stop. During annealing, the single stranded plurality of species that emit fluorescent light at different
DNA is cooled to 54°C. At this temperature, primers bind or wavelengths, exciting the disk with a plurality of light beams
“anneal to the ends of the DNA strands. During extension, to produce a plurality of emitted fluorescent light beams,
the sample is heated to 75°C. At this temperature, nucleotides capturing the fluorescent light beams with a plurality of dif
add to the primers and eventually a complementary copy of 40
ferent optical modules, wherein the modules are optically
the DNA template is formed. configured for the different wavelengths, containing the plu
There are a number of existing PCR instruments designed rality of different modules within a housing.
to determine levels of specific DNA and RNA sequences in The invention may provide one or more advantages. For
the sample during the PCR in real-time. Many of the instru example, the modular design may allow a technician to
ments are based on the use of fluorescent dyes. In particular, 45
quickly and efficiently interchange detection modules
many conventional real-time PCR instruments detect a fluo depending on the particular reactions being performed.
rescent signal produced proportionally during amplification Moreover, the technician may select detection modules that
of a PCR product. are optically optimized for different reactions. Further, dif
Conventional real-time PCR instruments use different ferent combinations of detection modules may be installed
methods for detection of different fluorescent dyes. For 50 and utilized within the real-time, multiplex PCR device.
example, some conventional PCR instruments incorporate While the device may be capable of conducting real-time
white light sources with filter wheels for spectrally resolving PCR, the device may be capable of analyzing any type of
each dye. The white light Sources are tungsten halogen bulbs, biological reaction while it occurs. The device may be able to
which have a lifetime maxima of a few thousand hours. The modulate the temperature of each reaction independently or
filter wheels are typically complicated electromechanical 55 as a selected group, and the device may be able to Support
parts that are Susceptible to wear. multiple stages of reactions by including a valve between two
chambers. This valve may be opened during reactions
SUMMARY through the use of a laser which delivers a burst of energy to
the valve.
In general, the invention relates to techniques for the detec 60 In some embodiments, the device may be portable and
tion of multiple target species in real-time PCR (polymerase robust to allow operation in remote areas or temporary labo
chain reaction), referred to herein as multiplex PCR. In par ratories. The device may include a data acquisition computer
ticular, a multiplex fluorescence detection device is described for analyzing the reactions in real-time, or the device may
that incorporates a plurality of optical modules. Each of the communicate the data to another device through wired or
optical modules may be optimized for detection of a respec 65 wireless communication interfaces.
tive fluorescent dye at a discrete wavelength band. In other The details of one or more embodiments of the invention
words, the optical modules may be used to interrogate mul are set forth in the accompanying drawings and the descrip
 US 7,507,575 B2
3 4
tion below. Other features, objects, and advantages of the rescent light energy at different wavelengths as the disk 13
invention will be apparent from the description and drawings, rotates. For example, excitation sources within modules 16
and from the claims. may be sequentially activated for periods sufficient to collect
data at the corresponding wavelengths. That is, an optical
BRIEF DESCRIPTION OF DRAWINGS module 16A may be activated for a period of time to collect
data at a first range of wavelengths selected for a first dye
FIG. 1 is a block diagram illustrating an exemplary corresponding to a first reaction. The excitation Source may
embodiment of a multiplex fluorescence detection device. then be deactivated, and an excitation source within module
FIG. 2 is a schematic diagram illustrating an exemplary 16B may be activated to interrogate sample 22 at a second
detection module, which may correspond to any of a plurality 10 range of wavelengths selected for a second dye corresponding
of detection modules of the fluorescence detection device of to a second reaction. This process continues until data has
FIG 1. been captured from all optical modules 16. In one embodi
FIG. 3 is a perspective diagram illustrating a front view of ment, each of the excitation Sources within optical modules
an exemplary set of removable optical modules within the 16 is activated for an initial period of approximately two
device housing. 15 seconds to reach steady state followed by an interrogation
FIG. 4 is a perspective diagram illustrating the exemplary period which lasts for 10-50 rotations of disk 13. In other
set of removable optical modules within the device housing. embodiments, the excitation sources may be sequenced for
FIG. 5 is a perspective diagram illustrating a front side shorter (e.g., 1 or 2 milliseconds) or longer periods. In some
view of an exemplary set of removable optical modules hav embodiments, more than one optical module may be acti
ing one module removed to expose a module connector. vated simultaneously for concurrent interrogation of sample
FIGS. 6A and 6B are perspective diagrams illustrating the 22 without stopping the rotation of disk 13.
components within exemplary main removable optical mod Although a single sample 22 is illustrated, disk 13 may
ules. contain a plurality of chambers holding samples. Optical
FIGS. 7A and 7B are perspective diagrams illustrating the modules 16 may interrogate some or all of the different cham
components within exemplary Supplemental removable opti 25 bers at different wavelengths. In one embodiment, disk 13
cal modules. includes 96 chambers space around a circumference of disk
FIG. 8 is a block diagram illustrating an example embodi 13. With a 96 chamber disk and four optical modules 16,
ment of the multiplex fluorescence detection device in further device 10 may be capable of acquiring data from 384 different
detail. species.
FIG.9 is a block diagram of theasingle detector coupled to 30 In one embodiment, optical modules 16 include excitation
four optical fibers of the optical fiber bundle. Sources that are inexpensive high power light emitting diodes
FIG. 10 is a flow diagram illustrating exemplary operation (LEDs), which are commercially available in a variety of
of the multiplex fluorescence detection device. wavelengths and have long lifetimes (e.g., 100,000 hours or
FIG. 11 is a flow diagram illustrating an exemplary method more). In another embodiment, conventional halogen bulbs
if detecting light and sampling data from the disk. 35 or mercury lamps may be used as excitation Sources.
FIGS. 12 and 13 show the absorption and emission spectra As illustrated in FIG. 1, each of optical modules 16 may be
of commonly used fluorescent dyes that may be utilized for coupled to one leg of a fiber optic bundle 14. Fiber optic
multiplex PCR. bundle 14 provides a flexible mechanism for collection of
FIGS. 14A and 14B illustrate raw data acquired from two fluorescent signals from optical modules 16 without loss of
exemplary detection modules with a single detector during a 40 sensitivity. In general, a fiber optic bundle comprises multiple
PCR analysis. optical fibers laid side by side and bonded together at the ends
FIG. 15 is a graph that shows the data once adjusted for a and encased in a flexible protective jacket. Alternatively, fiber
time offset. optic bundle 14 may comprise a smaller number of discrete,
FIGS. 16A and 16B show a limit of detection (LOD) for the large diameter multimode fibers, either glass or plastic, hav
data received from two exemplary detection modules. 45 ing a common end. For example, for a four-optical module
FIG. 17 is an exemplary screen shot of a temperature con device, fiber optic bundle 16 may comprise four discrete
trol user interface. multimode fibers, each having a 1 mm core diameter. The
FIG. 18 is an exemplary screen shot of an optical control common end of the bundle contains the four fibers bound
user interface. together. In this example, the aperture of detector 18 may be
FIG. 19 is an exemplary screenshot of a real-time PCR user 50 8 mm, which is more than sufficient for coupling to the four
interface. fibers.
In this example, fiber optic bundle 14 couples optical mod
DETAILED DESCRIPTION ules 16 to a single detector 18. The optical fibers carry the
fluorescent light collected by optical modules 16 and effec
FIG. 1 is a block diagram illustrating an exemplary 55 tively deliver the captured light to detector 18. In one embodi
embodiment of a multiplex fluorescence detection device 10. ment, detector 18 is a photomultiplier tube. In another
In the illustrated example, device 10 has four optical modules embodiment, the detector may include multiple photomulti
16 that provide four “channels' for optical detection of four plier elements, one for each optical fiber, within the single
different dyes. In particular, device 10 has four optical mod detector. In other embodiments, one or more solid-state
ules 16 that excite different regions of rotating disk 13 at any 60 detectors may be used.
given time, and collect emitted fluorescent light energy at The use of a single detector 18 may be advantageous in that
different wavelengths from the dyes. As a result, modules 16 it allows use of a highly sensitive and possibly expensive
may be used to interrogate multiple, parallel reactions occur detector (e.g., a photomultiplier), while maintaining a mini
ring within sample 22. mal cost in that only a single detector need be used. A single
The multiple reactions may, for example, occur simulta 65 detector is discussed herein; however, one or more detectors
neously within a single chamber of a rotating disk 13. Each of may be included for detecting a greater number of dyes. For
optical modules 16 interrogates sample 22 and collects fluo example, four additional optical modules 16 and a second
 US 7,507,575 B2
5 6
detector may be added to the system to allow for the detection ing platform 25. As a result, optical modules 16 are over
of eight different wavelengths emitted from one disk. An lapped with different chambers at any one time.
exemplary fiber optic bundle coupled to a single detector for Detection device 10 also includes a heating element (not
use with rotating disk 13 is described in U.S. patent applica shown) for modulating the temperature of the sample 22 on
tion Ser. No. 1 1/174,755, entitled “MULTIPLEX FLUO disk 13. The heating element may comprise a cylindrical
RESCENCE DETECTION DEVICE HAVING FIBER halogen bulb contained within a reflective enclosure. The
BUNDLE COUPLING MULTIPLE OPTICAL MODULES reflective chamber is shaped to focus radiation from the bulb
TO A COMMON DETECTOR, filed on Jul. 5, 2005, the onto a radial section of disk 13. Generally, the heated area of
entire content of which is hereby incorporated by reference. disk 13 would resemble a ring as disk 13 spins. In this
Optical modules 16 are removable from the device and 10 embodiment, the shape of the reflective enclosure may be a
easily interchangeable with other optical modules that are combination of elliptical and spherical geometries that allow
optimized for interrogation at different wavelengths. For precise focusing. In other embodiments, the reflective enclo
example, optical modules 16 may be physically mounted sure may be of a different shape or the bulb may broadly
within locations of a module housing. Each of optical mod irradiate a larger area. In other embodiments, the reflective
ules 16 may be easily inserted within a respective location of 15 enclosure may be shaped to focus the radiation from the bulb
the housing along guides (e.g., recessed grooves) that mate onto a single area of the disk 13, Such as a single process
with one or more marking (e.g., guide pins) of the optical chamber containing a sample 22.
module. Each optical module includes an optical output port In some embodiments, the heating element may heat air
(shown in FIGS. 6A and 7A) for coupling to one leg of fiber and force the hot air over one or more samples to modulate the
optic bundle 14. The optical output port may have a threaded temperature. Additionally, the samples may be heated
end coupled to a threaded connector of the leg. Alternatively, directly by the disk. In this case, the heating element may be
a form of “quick-connect may be used (e.g., a slidable con located in platform 25 and thermally couple to disk 13. Elec
nection having an o-ring and a catch pin) that allows fiber trical resistance within the heating element may heat a
optic bundle 14 to be slidably engaged and disengaged from selected region of the disk as controlled by control unit 23.
the optical output port. Moreover, each of optical modules 16 25 For example, a region may contain one or more chambers,
may have one or more electrical contacts for electronically possibly the entire disk. An exemplary heating element for
coupling to control unit 23 when fully inserted. use with rotating disk 13 is described in U.S. patent applica
The modular architecture of device 10 allows the device to tion Ser. No. 1 1/174,691, entitled “HEATING ELEMENT
FOR A ROTATING MULTIPLEX FLUORESCENCE
be easily adapted for all of the fluorescent dyes used in a given 30 DETECTION DEVICE, filed on Jul. 5, 2005, the entire
analysis environment, such as multiplex PCR. Other chemis content of which is hereby incorporated by reference.
tries that may be used in device 10 include Invader (Third Alternatively, or in addition, device 10 may also includes a
Wave, Madison, Wis.), Transcripted-mediated Amplification cooling component (not shown). A fan is included in device
(GenProbe, San Diego, Calif.), fluorescence labeled enzyme 10 to supply cold air, i.e., room temperature air, to disk 13.
linked immunosorbent assay (ELISA) or fluorescence in situ 35 Cooling may be needed to modulate the temperature of the
hybridization (FISH). The modular architecture of device 10 sample appropriately and store samples after an experiment
may provide another advantage in that the sensitivity of each has completed. In other embodiments, the cooling component
optical module 16 can be optimized by choice of the corre may include thermal coupling between platform 25 and disk
sponding excitation source (not shown) and excitation and 13, as platform 25 may reduce its temperature when needed.
detection filters for a small specific target range of wave 40 For example, Some biological samples may be stored at 4
lengths in order to selectively excite and detect a correspond degrees Celsius to reduce enzyme activity or protein denatur
ing dye in the multiplex reaction. 1ng.
For purpose of example, device 10 is illustrated in a 4-color Detection device 10 may also be capable of controlling
multiplex arrangement, but more or less channels can be used reaction species contained within a process chamber. For
with the appropriate fiber optic bundle 14. This modular 45 example, it may be beneficial to load some species in a pro
design allows a user to easily upgrade device 10 in the field by cess chamber to generate one reaction and later adding
simply adding another optical module 16 to base 20 and another species to the sample once the first reaction has ter
inserting one leg of fiber optic bundle 14 into the new optical minated. A laser homing valve may be added to control a
module. Optical modules 16 may have integrated electronics valve position separating an inner holding chamber from the
that identify the optical modules and download calibration 50 process chamber, thereby controlling the addition of species
data into an internal control module or other internal electron to the chamber during rotation of disk 13. This laser device
ics (e.g., control unit 23) of device 10. may be located within one of optical modules 16 or separate
In the example of FIG. 1, samples 22 are contained in from the optical modules. Directly below the laser, under disk
chambers of disk 13, which is mounted on a rotating platform 13, may be a laser sensor for positioning the laser relative to
under the control of control unit 23. A slot sensor trigger 27 55 disk 13.
provides an output signal utilized by control unit 23 and data In one embodiment, the laser is a near infrared (NIR) laser
acquisition device 21 for synchronizing data acquisition with with at least two power settings. Under a low power setting,
chamber position during disk rotation. Slot sensor trigger 27 the laser positioning sensor may indicate that the laser is in
may be a mechanical or optical sensor. For example, the position over the chamber valve by recognizing the NIR light
sensor may be a laser which sends a beam of light to disk 13 60 though a slot in disk 13. Once the laser is in position, control
and control unit 23 uses a sensor detecting light passing unit 23 directs the laser to output a short burst of high power
through a slot in disk 13 to locate the chambers on the disk. In energy to heat the valve and open it. The open valve may then
other embodiments, disk 13 may include a tab, protrusion or allow the inner fluid specimen to flow toward from the inside
reflective surface in addition to or in place of the slot. Slot chamber to the outside process chamber and conduct a second
sensor trigger 27 may use any physical structure or mecha 65 reaction. In some embodiments, disk 13 may contain a plu
nism to locate the radial position of disk 13 as it rotates. rality of valves to generate a plurality of reactions in
Optical modules 16 may be physically mounted above rotat sequence. More than one set of laser and laser sensor may also
 US 7,507,575 B2
7 8
be used when utilizing multiple chamber valves. An exem tion Ser. No. 60/260,063 filed on Jan. 6, 2001 and titled
plary laser homing valve control system for use with rotating “SAMPLE PROCESSING DEVICES, SYSTEMS AND
disk 13 is described in U.S. patent application Ser. No. METHODS”; U.S. Provisional Patent Application Ser. No.
11/174,957, entitled “VALVE CONTROL SYSTEM FORA 60/284,637 filed on Apr. 18, 2001 and titled “ENHANCED
ROTATING MULTIPLEX FLUORESCENCE DETEC 5 SAMPLE PROCESSING DEVICES, SYSTEMS AND
TION DEVICE, filed on Jul. 5, 2005, the entire content of METHODS”; and U.S. Patent Application Publication No.
which is hereby incorporated by reference. US 20O2/OO48533 titled SAMPLE PROCESSING
Data acquisition device 21 may collect data from device 10 DEVICES AND CARRIERS.” Other potential device con
for each dye either sequentially or in parallel. In one embodi structions may be found in, e.g., U.S. Pat. No. 6,627,159 titled
ment, data acquisition system 21 collects the data from opti 10 “CENTRIFUGAL FILLING OF SAMPLE PROCESSING
cal modules 16 in sequence, and corrects the spatial overlap DEVICES' (Bedingham et al.). The entire content of these
by a trigger delay for each one of the optical modules mea disclosures are incorporated herein by reference.
Sured from slot sensor trigger 27. FIG. 2 is a schematic diagram illustrating an exemplary
One application for device 10 is real-time PCR, but the optical module 16A, which may correspond to any of optical
techniques described herein may be extended to other plat 15 modules 16 of FIG. 1. In this example, optical module 16A
forms that utilize fluorescence detection at multiple wave contains a high-power excitation Source, LED 30, a collimat
lengths. Device 10 may combine rapid thermal cycling, ulti ing lens 32, an excitation filter 34, a dichrotic filter 36, a
lizing the heating element, and centrifugally driven focusing lens 38, a detection filter 40, and a lens 42 to focus
microfluidics for isolation, amplification, and detection of the fluorescence into one leg of fiber optic bundle 14.
nucleic acids. By making use of multiplex fluorescence detec Consequently, the excitation light from LED 30 is colli
tion, multiple target species may be detected and analyzed in mated by collimating lens 32, filtered by excitation filter 34,
parallel. transmitted through dichrotic filter 36, and focused into the
For real-time PCR, fluorescence is used to measure the sample 22 by focusing lens 38. The resulting fluorescence
amount of amplification in one of three general techniques. emitted by the sample is collected by the same focusing lens
The first technique is the use of a dye, such as Sybr Green 25 38, reflected off ofdichrotic filter36, and filtered by detection
(Molecular Probes, Eugene, Oreg.), whose fluorescence filter 40 before focused into one leg of fiber optic bundle 14.
increases upon binding to double-stranded DNA. The second The optic bundle 14 then transfers the light to detector 18.
technique uses fluorescently labeled probes whose fluores LED 30, collimating lens 32, excitation filter 34, dichrotic
cence changes when bound to the amplified target sequence filter 36, focusing lens 38, detection filter 40, and lens 42 are
(hybridization probes, hairpin probes, etc.). This technique is 30 selected based on the specific absorption and emission bands
similar to using a double-stranded DNA binding dye, but is of the multiplex dye with which optical module 16A is to be
more specific because the probe will bind only to a certain used. In this manner, multiple optical modules 16 may be
section of the target sequence. The third technique is the use configured and loaded within device 10 to target different
ofhydrolysis probes (TaqmanTM, Applied BioSystems, Foster dyes.
City Calif.), in which the exonuclease activity of the poly 35 Table 1 lists exemplary components that may be used in a
merase enzyme cleaves a quencher molecule from the probe 4-channel multiplex fluorescence detection device 10 for a
during the extension phase of PCR, making it fluorescently variety of fluorescent dyes. FAM, HEX, JOE, VIC, TET, ROX
active. are trademarks of Applera, Norwalk, Calif. Tamra is a trade
In each of the approaches, fluorescence is linearly propor mark of AnaSpec, San Jose, Calif. Texas Red is a trademark of
tional to the amplified target concentration. Data acquisition 40 Molecular Probes. Cy 5 is a trademark of Amersham, Buck
system 21 measures an output signal from detector 18 (or inghamshire, United Kingdom.
alternatively optionally sampled and communicated by con
trol unit 23) during the PCR reaction to observe the amplifi TABLE 1
cation in near real-time. In multiplex PCR, the multiple tar
gets are labeled with different dyes that are measured 45 Optical Excitation Detection
Module LED Filter Filter Dye
independently. Generally speaking, each dye will have dif
ferent absorbance and emission spectra. For this reason, opti 1 blue 475 nm. 520 nm FAM, Sybr Green
cal modules 16 may have excitation Sources, lenses and 2
3
green
Orange
530 mm
580 mm
555 mm
610 nm
HEX, JOE, VIC, TET
TAMRA, ROX, Texas Red
related filters that are optically selected for interrogation of 4 red 630 mm 670 nm Cy 5
sample 22 at different wavelengths. 50
Some examples of Suitable construction techniques or
materials that may be adapted for use in connection with the One advantage of the described modular, multiplex detec
present invention may be described in, e.g., commonly-as tion architecture is the flexibility in optimizing detection for a
signed U.S. Pat. No. 6,734.401 titled “ENHANCED wide variety of dyes. Conceivably a user may have a bank of
SAMPLE PROCESSING DEVICES SYSTEMS AND 55 several different optical modules that can be plugged into
METHODS” (Bedingham et al.) and U.S. Patent Application device 10 as needed, of which N can used at any one time,
Publication No. US 2002/0064885 titled “SAMPLE PRO where N is the maximum number of channels supported by
CESSING DEVICES.” Other useable device constructions the device. Therefore, device 10 and optical modules 16 may
may be found in, e.g., U.S. Provisional Patent Application be used with any fluorescent dye and PCR detection method.
Ser. No. 60/214,508 filed on Jun. 28, 2000 and entitled 60 A larger fiber optic bundle may be used to Support a larger
“THERMAL PROCESSING DEVICES AND METHODS: number of detection channels. Moreover, multiple fiber optic
U.S. Provisional Patent Application Ser. No. 60/214,642 filed bundles may be used with multiple detectors. For example,
on Jun. 28, 2000 and entitled “SAMPLE PROCESSING two 4-legged fiber optic bundles may be used with eight
DEVICES, SYSTEMS AND METHODS”; U.S. Provisional optical modules 16 and two detectors 18.
Patent Application Ser. No. 60/237,072 filed on Oct. 2, 2000 65 FIG.3 is a perspective diagram illustrating a front view of
and entitled “SAMPLE PROCESSING DEVICES, SYS an exemplary set of removable optical modules within the
TEMS AND METHODS”; U.S. Provisional Patent Applica device housing. In the example of FIG. 3, device 10 includes
 US 7,507,575 B2
10
base arm 44 and module housing 46. Main optical module 48, different wavelength of fluorescence from the samples in disk
Supplemental optical module 52 and Supplemental optical 13. Therefore, main optical module 48 may connect to two
module 56 are contained within module housing 46. Optical legs of fiber optic bundle 14. In this manner, main optical
modules 48, 52 and 56 produce optical output beams 49, 53 module 48 may be viewed as a dual-channeled optical module
and 57, respectively, that sequentially excite different process having two independent optical excitation and collection
chambers of disk 13. In other words, output beams 49.53 and channels. In some embodiments, main optical module 48 may
57 follow the curvature of disk 13 to each excite the same contain optical components for more than two optical mod
radial position of the disk which contains the process cham ules. In other cases, module housing 46 contains a plurality
bers. Slot sensor trigger 27 includes infrared light source 31 (e.g., two or more) of single-channeled optical modules, such
which produces light 35 that is detected by detector 33. 10 as supplemental optical modules 52 and 56.
Each of optical modules 48.52 and 56 includes a respective As illustrated in FIG. 3, main optical module 48 may also
release lever 50, 54 or 58, respectively, for engaging module contain components for a laser valve control system 51 (lo
housing 46. Each release lever may provide an upward bias to cated within optical module 48). Laser valve control system
engage a respective latch formed within module housing 46. 51 detects disk 13 location by a small slot located near the
A technician or other user depresses release levers 50, 54 or 15 outer edge of disk 13. A detector (not shown) detects low
58, respectively, in order to unlatch and remove optical mod power laser light 55 to map the location of disk 13 with
ule 48, 52 or 56 from module housing 46. Barcode reader 29 respect to the motor which spins the disk. The control unit 23
includes laser 62 for identifying disk 13. uses the map to locate valves (not shown) on disk 13.
Base arm 44 extends from detection device 10 and provides Laser valve control system 51 focuses laser light 55 on the
support for module housing 46 and optical modules 48, 52 valves that separate holding chambers towards the center of
and 56. Module housing 46 may be securely mounted atop disk 13 from process chambers near the outer edge of disk 13.
base arm 44. Module housing 46 may contain a location When the contents of the holding chambers are to be moved
adapted to receive a respective one of optical modules 48, 52 to the associated process chambers, laser valve control sys
and 56. Although described for exemplary purposes with tem 51 applies laser light 55 to heat a valve separating the
respect to module housing 46, module housing 46 of detec 25 chambers, causing the value open and providing fluid com
tion device 10 may have a plurality of locations for receiving munication between the two chambers. In particular, once the
optical modules 48, 52 and 56. In other words, a separate valve is open, the contents from the inner holding chamber
housing need not be used for optical modules 48, 52 and 56. may then flow towards the outer process chamber as disk 13
Each location of module housing 46 may contain one or is spinning. Detection device 10 may then monitor the sub
more tracks or guides which help to correctly position the 30 sequent reaction in the process chamber. Contents within a
associated optical module within the location when a techni chamber may include Substances in a fluid or Solid state.
cian or other user inserts the optical module. These guides In some embodiments, laser valve control system 51 may
may be located along the top, bottom, or sides of each loca be contained within a single-channeled optical module, e.g.,
tions. Each of optical modules 48, 52 and 56 may include Supplemental optical module 54 or Supplemental optical
guides or tracks that mate with the guides or tracks of the 35 module 56. In other embodiments, laser valve control system
locations of module housing 46. For example, module hous 51 may be mounted to detection device 10 separately from
ing 46 may have protruding guides which mate with recessed any of optical modules 48, 52 or 56. In this case, laser valve
guides in optical modules 48, 52 and 56. control system 51 may be removable and adapted to engage a
In some embodiments, module housing 46 may not com location within module housing 46 or a different housing of
pletely enclose each of optical modules 48, 52 and 56. For 40 detection device 10.
example, module housing 46 may provide mounting points to In the example of FIG. 3, slot sensor trigger 27 is located
secure each of optical modules 48, 52 and 56 to base arm 44, near the removable modules, on either side of disk 13. In one
but portions or all of each optical module may be exposed. In embodiment, slot sensor trigger 27 contains a light source 31
other embodiments, module housing 46 may completely to emit infrared (IR) light 35. Detector 33 detects IR light 35
enclose each of optical modules 48.52 and 56. For example, 45 when the slot in disk 13 allows the light to pass through the
module housing 46 may include a single door that closes over disk to detector 33. Control unit 23 may use this information
optical modules 48.52 and 56, or a respective door for each of to synchronize disk 13 location as it is spinning with data
the modules. This embodiment may be appropriate for appli from optical modules 48,54 and 56. In some embodiments,
cations where the modules are seldom removed or detection slot sensor trigger 27 may extend from base arm 44 to reach
device 10 is subjected to extreme environmental conditions. 50 the outer edge of disk 13 during device 10 operation. In other
A technician may easily remove any of optical modules 48, embodiments, a mechanical detector may be used to detect
52 or 56, and may be completed by using only one hand. For the position of disk 13.
example, the technician may rest his or her forefinger under a Barcode reader 29 uses laser 62 to read a barcode located
molded lip located beneath release lever 54 of optical module on the side edge of disk 13. The barcode identifies the type of
52. The technicians thumb may then press down release lever 55 disk 13 to allow proper operation of device 10. In some
54 to release optical module 52 from module housing 46. embodiments, the barcode may identify the actual disk to
While grasping optical module 52 between the thumb and assist a technician in tracking data to specific samples from
forefinger, the technician may pullback on the optical module multiple disks 13.
to remove the optical module from detection device 10. Other All surface components of optical modules 48, 52 and 56
methods may be used to remove any of optical module 48, 52 60 may be constructed of a polymer, composite, or metal alloy.
or 56, including methods utilizing two-handed removal. For example, high molecular weight polyurethane may be
Inserting any of optical module 48, 52 or 56 may be accom used in forming the Surface components. In other cases, an
plished in a reversed manner with one or two hands. aluminum alloy or carbon fiber structure may be created. In
In the example of FIG. 3, the components of two optical any case, the material may be resistant to heat, fatigue, stress,
modules are combined to form main optical module 48. Main 65 and corrosion. As detection device 10 may come into contract
optical module 48 may contain light sources that produce two with biological materials, the structures may be sterilizable in
different wavelengths of light and detectors for detecting each the event chamber contents leak out of disk 13.
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11 12
FIG. 4 is an perspective diagram illustrating the exemplary urable to detect samples on different sized disks or samples
set of removable optical modules 48.52 and 56 within module located within an interior of disk 13. For example, a larger
housing 46 of detection device 10. In the example of FIG. 4, disk containing more process chambers or larger process
base arm 44 supports barcode reader 29 as well as the remov chambers may be used by moving the base arm 44 further
able optical modules 48, 52 and 56 attached within module away from the center of disk 13. Module housing 46 may also
housing 46. Disk 13 is located beneath optical modules 48,52 have a configurable position for each of optical module 48,52
and 56 with the process chambers located under a respective or 56 so that each module may be movable to one or more
optical path of each of the modules at different moments in circular paths of process chambers around disk 13.
time. FIG. 5 is perspective diagram illustrating a front side view
Within module housing 46, the fronts of supplementary 10 of an exemplary set of removable optical modules having one
module 56 and main optical module 48 can be seen. Supple module removed to expose a module connector. In particular,
mentary module 56 contains molded lip 59 and release lever module housing 46 is not shown in FIG. 5, and optical module
58. As previously described, molded lip 59 may be used to 56 has been removed to expose optical modules 52 and 48
grasp module 56 when removing or inserting the module into along with the connections for removed module 56.
module housing 46. All of optical modules 48.52 and 56 may 15 Release lever 58 (FIG. 3) of optical module 56 securely
have a respective molded lip and release lever, or a single attaches to attachment post 69 mounted to base arm 44. In this
release lever may be used to remove all of the optical mod example, attachment post 69 extends into optical module 56
ules. In some embodiments, optical modules 48, 52 and 56 and couples to release lever 58. In other embodiments, other
may contain a different component for grasping the module. attachment mechanisms may be used to fix optical module 56
For example, each of optical modules 48, 52 and 56 may to base arm 44. Such as a screw or Snap fixation device.
contain a handle for removing the respective module in a Base arm 44 provides two different operational connec
vertical or horizontal direction from module housing 46. tions within module housing 46 for receiving and engaging
The location of optical modules 48, 52 and 56 within optical module 56, once inserted. In particular, base arm 44
module housing 46 may be fixed in order to separately excite provides electrical contact board 66, which includes electrical
different samples within disk 13 at any particular moment in 25 connections 70 for coupling to the electrical contacts (not
time. For example, main optical module 48 may be located shown) contained within optical module 56. Electrical con
slightly further toward base arm 44 than Supplemental optical nections 70 allow control unit 23 to communicate with elec
modules 52 and 56, which are offset to a location at either side trical components within module 56. For example, module 56
of the main module. Moreover, optical modules 48.52 and 56 may include electrical circuits, hardware, firmware, or any
may be offset in a horizontal direction (indicated by the arrow 30 combination thereof. In one example, the internal electrical
in FIG. 4, where X is the distance the outside light beams are components may store and output to control unit 23 unique
offset from the inside light beams) so that the excitation light identification information, such as a serial number. Alterna
beams produced by the modules follows the curvature of disk tively, or in addition, the electrical components may provide
13. In this arrangement, the light beams produced by optical information describing the specific characteristics of the opti
modules 48, 52 and 56 traverse the same path as disk 13 35 cal components contained within the removable module 56.
rotates, thereby exciting and collecting light from process For example, the electrical components may include pro
chambers located along the path. In other embodiments, opti grammable read-only memory (PROM), flash memory, or
cal modules 48.52 and 56 are aligned such that the excitation other internal or removable storage media. Other embodi
light beams traverse different paths around rotating disk 13. ments may include a set of resistors, a circuit or an imbedded
In this example, base arm 44 contains electrical contact 40 processor for outputting a unique signature of optical mod
board 66 which extends into module housing 46. Inside mod ules 48, 52 or 56 to control unit 23. In another example,
ule housing 46, electrical contact board 66 may contain elec optical module 56 may include a laser Source and other com
trical contacts for each of optical modules 48, 52 and 56. ponents that form part of a laser valve control system, i.e.
Electrical contact board 66 may be electrically coupled to laser valve control system 51.
control unit 23. In some embodiments, each of optical mod 45 Electrical contact board 66 may be removed and replaced
ules 48, 52 and 56 may have a separate associated electrical with another version associated with a different removable
contact board which is connected to control unit 23. optical module. This option may support upgrades in device
Fiber optic coupler 68 couples one leg of the fiber optic capability. In other embodiments, connections 70 may con
bundle 14 to an optical output port of optical module 56. tain more or less connection pins.
Although not shown, each of optical modules 48, 52 and 56 50 In addition, base arm 44 and module housing 46 provide
include an optical output port adapted to engage a respective optical channel 72 within the location for receiving optical
fiber optic coupler mounted to module housing 46. The con module 56. Optical channel 72 is connected to fiber optic
nection between fiber optic coupler 68 and the leg of fiber coupler 68 (FIG. 4) that interfaces with a leg of fiber optic
optic bundle 14 may be a threaded screw lock, Snap closure or bundle 14. Optical channel 72 inserts into a location within
friction fit. 55 optical module 56. The light captured by optical module 56
Barcode reader 29 produces laser light 64 for reading the may be directed through optical channel 72, fiber optic cou
barcode of disk 13. The laser light 64 follows a direct path pler 68 and fiber optic bundle 15 to the detector. Fittings
where it interacts with the outer edge of disk 13. The light 64 between these connections may be tight to ensure that light
may spread out to cover a large area of disk 13 at one time. does not escape or enter the optical path.
Barcode reader 29 reads the barcode on disk 13 when the disk 60 In some embodiments, the connections to optical module
is rotating at slow speeds. In other embodiments, barcode 56 may be arranged in a different configuration. For example,
reader 29 may read the barcode periodically during operation the connections may be located in another position for accept
to make sure a new disk has not been loaded in device 10. The ing optical module 56 from another direction. In other
barcode reader 29 may detect more than one barcode on disk embodiments, electrical connections may be located on one
13 in other embodiments. 65 side of optical module 56 while an optical connection is
In some embodiments, base arm 44 may be movable with located on a second Surface of module 56. In any case, the
respect to disk 13. In this case, base arm 44 could be config electrical and optical connections located within the location
 US 7,507,575 B2
13 14
of module housing 46 accommodate a removable optical light 49 leaves LED 82, the light is expanded by collimating
module, i.e., optical module 56 in this example. lens 84 before the light enters excitation filter 86. The light 49
The optical and electrical connections of module 56 of one wavelength band is passed by dichrotic filter 88 and is
described in FIG.5 may be used with any module, including focused on a sample by focusing lens 90. The light 49 excites
optical modules 48 and 52. In addition, the connections for the sample and fluorescence is collected by focusing lens 90
each optical module may not be identical. Since connections and delivered to detection filter 92 by dichrotic filter 88. The
may be modified for coupling with a desired removable opti resulting wavelength band of light is collected by lens 94 and
cal module, the connections utilized by any particular optical delivered to optical output port 17 where the collected fluo
module inserted within a particular location of module hous rescent light enters a leg of fiber optic bundle 14 for convey
ing 46 may vary at any time. 10 ance to detector 18.
FIG. 6A is perspective diagram illustrating the components Internal housing 78 may support all components included
within an exemplary main removable optical module 48A. In in the excitation of the sample and detection of fluorescent
the example of FIG. 6A, main optical module 48A includes light emitted by the sample for a selected wavelength. On the
release lever 50, pivot pin 51 and latch 74. Internal housing 78 other side of internal housing 78, a similar configuration of
separates each side of module 48A and contains electrical 15 optical components may be included to produce light of a
contacts pad 80 connected to ribbon 81. Optical components different wavelength and detect the corresponding different
include LED 82, collimating lens 84, excitation filter 86, fluorescent wavelength. Separation of each side may elimi
dichrotic filter 88, focusing lens 90, detection filter 92 and nate light contamination from one side entering the optical
lens 94. Optical output port 17 couples to a leg of fiber optic channel of the other side.
bundle 14. A separate set of optical components for a second Housed partially between each side of module 48A may be
optical channel (not shown) are located on the other side of the components of the laser valve control system 51, includ
internal housing 78. In addition, main module 48A includes ing connector 96, laser diode 98 and focusing lens 100. Inter
connector 96, laser diode 98 and focusing lens 100 as part of nal housing 78 may provide physical Support for these com
a laser valve control system 51 controlled by control unit 23. ponents. Ribbon 81 is connected to connector 96 for
Release lever 50 is attached to optical module 48A by a 25 communicating drive signals and power to the laser source.
pivot pin 61. Pivot pin 61 allows release lever 50 to rotate Laser diode 98 is connected to connector 96 and produces the
about the axis of the pin. When release lever 50 is depressed, laser energy 55 used to open valves on disk 13. Laser diode 98
arm 63 rotates counter-clockwise to raise latch 74. Once latch delivers this near-infrared (NIR) light to focusing lens 100 for
74 is raised, optical module 48A may be free for removal from directing the laser energy 55 to specific valves on disk 13. An
module housing 46. There may be a spring or other mecha 30 NIR sensor may be located below disk 13 for locating par
nism maintaining a bias force against release lever 50 to ticular valves that need to be opened. In other embodiments,
maintain latch 74 in a down position. In some embodiments, these components may be housed separately from the optical
a spring may be included around pivot pin 61 to provide a components.
moment arm that keeps latch 74 in the down, or latched, In some embodiments, emission lens 98 and focusing lens
position. In other embodiments, other mounting mechanisms 35 100 of laser valve control system 51 may be contained within
may be added to or used in place of the described lever. For a single-channeled optical module. Such as Supplemental
example, optical module 48A may be attached to module optical module 52 and 56 (FIG. 3).
housing 46 by one or more screws or pins. FIG. 6B is a perspective diagram illustrating the compo
Mounting board 76 may be installed within optical module nents within a different optical module substantially similar
48A for attaching communication ribbon 81 and LED 82. 40 to FIG. 6A. Optical module 48B includes many of the same
Ribbon 81 is connected to electrical contacts pad 80 and components as optical module 48A. Differences include nut
provides a connection between the pad and electrical compo 85, flex circuit 87 and flex circuit connector 89.
nents within optical module 48A. Contacts pad 80 and ribbon Optical module 48B does not require a latch mechanism
81 may carry the information required for both sides of main for attaching to module housing 46. Alternatively, nut 85 is
optical module 48A, including the laser valve control system 45 threaded and is engaged by a matching threaded bolt attached
51 and any internal memory or other storage medium. Ribbon through module housing 46. Once tightened, optical module
81 may be flexible for weaving within optical module 48A. 48B is securely attached to detection device 10. In other
Ribbon 81 may contain a plurality of electrically conductive embodiments, a different fastening device may be used. For
wires to communicate signals between the electrical compo example, a pin or track may lock optical module 48B into
nents and control unit 23 and/or to deliverpower to the elec 50 place.
trical components. In some embodiments, each electrical Flex circuit 87 provides the electrical connection between
component may have a separate cable connecting the com components of optical module 48B with control unit 23. Flex
ponent with control unit 23. A technician may need to dis circuit 87 is flexible to move between multiple locations. Flex
connect a cable or flex circuit from module housing 46 when circuit connector 89 is coupled to flex circuit 87 and provides
removing optical module 48A from the housing. 55 a secure connection between optical module 48B. Flex circuit
In some embodiments, optical module 48A may contain a connector 89 must be disengaged to completely remove opti
detector for detecting light from disk 13 and electronics for cal module 48B from module housing 46.
processing and storing the data. The electronics may contain FIG. 7A is a perspective diagram illustrating the compo
a telemetry circuit for wirelessly transmitting data represent nents within an exemplary Supplemental optical module that
ing the detected light to control unit 23. Wireless communi 60 may be easily removed from or inserted into detection device
cation may be performed by infrared light, radio frequency, 10. In the example of FIG. 7A, optical module 56A includes
Bluetooth, or other telemetry technique. Optical module 48A release lever 58, pivot pin 59 and latch 102, similar to main
may also include a battery to power the electronics, which optical module 48A. Optical module 56A also includes elec
may be rechargeable by control unit 23. trical contacts pad 106 connected to ribbon 107. Ribbon 107
LED 82 is affixed to mounting board 76 and electrically 65 may also be connected to mounting board 104. Similar to
coupled to ribbon 81. LED 82 produces excitation light 49 of main optical module 48A, optical components include LED
a predetermined wavelength to excite the sample 22. After 108, collimating lens 110, excitation filter 112, dichrotic filter
 US 7,507,575 B2
15 16
114, focusing lens 116, detection filter 118 and lens 120. cations. For example, any optical modules may be modified to
Optical output port 19 couples to a leg of fiber optic bundle be inserted from a different direction or to be placed within
14. the device at a different position with respect to disk 13. In any
Release lever 58 is attached to optical module 56A by a case, the optical modules may be removable to provide modi
pivot pin 65. Pivot pin 65 allows the release lever to rotate fication flexibility to device 10.
about the axis of the pin. When release lever 58 is depressed, FIG. 7B is a perspective diagram illustrating the compo
arm 67 rotates counter-clockwise to raise latch 102. Once nents withina different Supplemental optical module Substan
latch 102 is raised, optical module 56A may be free for tially similar to FIG. 7A. Optical module 56B includes many
removal from module housing 46. There may be a spring or of the same components as optical module 56A. Differences
other mechanism maintaining a bias force against release 10 include nut 91, flex circuit 93 and flex circuit connector 95.
lever 58 to maintain latch 102 in a down position. Alterna Optical module 56B does not require a latch mechanism
tively, a spring may be located above latch 102. In some for attaching to module housing 46. Alternatively, nut 91 is
embodiments, a spring may be included around pivot pin 65 threaded and is engaged by a matching threaded bolt attached
to provide a moment arm that keeps latch 102 in the down, or through module housing 46. Once tightened, optical module
latched, position. In other embodiments, other mounting 15 56B is securely attached to detection device 10. In other
mechanisms may be added to or used in place of the described embodiments, a different fastening device may be used. For
lever. For example, optical module 56A may be attached to example, a pin or track may lock optical module 56B into
module housing 46 by one or more screws or pins. place.
Mounting board 104 may be installed within optical mod Flex circuit 93 provides the electrical connection between
ule 56A for attaching communication ribbon 107 and LED components of optical module 56B with control unit 23. Flex
108. Ribbon 107 is connected to electrical contacts pad 106 circuit 93 is flexible to move between multiple locations. Flex
and provides a connection between the pad and electrical circuit connector 95 is coupled to flex circuit 93 and provides
components within optical module 56A. Contacts pad 106 a secure connection between optical module 56B. Flex circuit
and ribbon 107 may carry the information required for oper connector 95 must be disengaged to completely remove opti
ating the optical components. Ribbon 107 may be flexible for 25 cal module 56B from module housing 46.
weaving within optical module 56A. Ribbon 107 may contain FIG. 8 is a functional block diagram of the multiplex fluo
a plurality of electrically conductive wires to communicate rescence detection device 10. In particular, FIG. 8 indicates
signals between the components and control unit 23 and/or the electrical connections between device components and
deliverpower to the electrical components. In some embodi the general paths of light through the components. In the
ments, each electrical component may have a separate cable 30 example of FIG. 8, device 10 includes at least one processor
connecting the component with control unit 23. A technician 122 or other control logic, memory 124, disk motor 126, light
may need to disconnect a cable or flex circuit from module source 30, excitation filter 34, lens 38, detection filter 40,
housing 46 when removing optical module 56A from the collecting lens 42, detector 18, slot sensor trigger 27, com
housing. munication interface 130, heating element 134, laser 136 and
In some embodiments, optical module 56A may contain a 35 power source 132. As shown in FIG. 3, lens 38 and collecting
detector for detecting light from disk 13 and electronics for lens 42 need not be electrically connected to another compo
processing and storing the data. The electronics may contain nent. Further, light source 30, filters 34 and 40, lens 38 and
a telemetry circuit for wirelessly transmitting data represent collecting lens 42 are representative of one optical module 16.
ing the detected light to control unit 23. Wireless communi Although not illustrated in FIG. 8, device 10 may contain
cation may be performed by infrared light, radio frequency, 40 additional optical modules 16, as described previously. In that
Bluetooth, or other telemetry technique. Optical module 56A case, each additional optical module may include compo
may also include a battery to power the electronics, which nents arranged substantially similarly as to those shown in
may be rechargeable by control unit 23. FIG 8.
LED 108 is affixed to mounting board 104 and electrically Light follows a certain path through several components in
coupled to ribbon 107. LED 108 produces excitation light 101 45 FIG. 8. Once light is emitted by light source 30, it enters
of a predetermined wavelength to excite the sample 22. After excitation filter 34 and leaves as light of a discrete wave
light 101 leaves LED 108, the light is expanded by collimat length. It then passes through lens 38 where it leaves detection
ing lens 110 before the light enters excitation filter 112. The device 10 and excites sample 22 within a process chamber
light 101 of one wavelength band is passed by dichrotic filter (not shown). Sample 22 responds by fluorescing at a different
114 and is focused on a sample by focusinglens 116. The light 50 wavelength, at which time this fluorescent light enters lens 38
101 excites the sample and fluorescence is collected by focus and is filtered by detection filter 40. Filter 40 removes back
ing lens 116 and delivered to detection filter 118 by dichrotic ground light of wavelengths outside of the desired fluores
filter 114. The resulting wavelength band of light is collected cence from Sample 22. The remaining light is sent through
by lens 120 and delivered to optical output port 19 where the collecting lens 42 and enters a leg of fiber optic bundle 14
collected fluorescent light enters a leg offiber optic bundle 14 55 before being detected by detector 18. Detector 18 subse
for conveyance to detector 18. quently amplifies the received light signal.
Supplemental optical module 56A may also contain the Processor 122, memory 124 and communication interface
components of the laser valve control system 51. Laser valve 130 may be part of control unit 23. Processor 122 controls
control system 51 may be the only system used within device disk motor 126 to rotate or spin disk 13 as needed to collect
10 or one of a plurality of laser valve control systems. The 60 fluorescence information or move fluid through disk 13. Pro
components used for this system may be similar to the com cessor 122 may use disk position information received from
ponents described in optical module 48A of FIG. 6A. slot sensor trigger 27 to identify the location of chambers on
The components of supplemental optical module 56A may disk 13 during rotation and synchronize the acquisition of
be similar to any Supplemental optical module or any optical florescence data received from the disk.
module used to emit and detect one wavelength band of light. 65 Processor 122 may also control when the light source 30
In some embodiments, the components may be altered in within optical module 16 is powered on and off. In some
configuration to accommodate different experimental appli embodiments, processor 122 controls excitation filter 34 and
 US 7,507,575 B2
17 18
detection filter 40. Depending on the sample being illumi other embodiments may include opening times of shorter or
nated, processor 122 may change the filter to allow a different greater duration. A laser energy and pulse duration may be
wavelength of excitation light to reach the sample or a differ controlled by processor 122 through communication with
ent wavelength of fluorescence to reach collecting lens 42. In laser 136.
some embodiments, one or both filters may be optimized for Processor 122 utilizes communication interface 130 to
the light source 30 of the particular optical module 16 and not communicate with data acquisition system 21. The commu
changeable by processor 122. nication interface 130 may include a single method or com
Collecting lens 42 is coupled to one leg of fiber bundle 14 bination of methods to transfer data. Some methods may
that provides an optical path for the light from the collecting include a universal serial bus (USB) port or IEEE 1394 port
lens to detector 18. Processor 122 may control the operation 10 for hardwire connectivity with high data transfer rates. In
of detector 18. While detector 18 may constantly be detecting Some embodiments, a storage device may be directly attached
all light, Some embodiments many utilize other acquisition to one of these ports for data storage for post processing. The
modes. Processor 122 may determine when detector 18 col data may be pre-processed by processor 122 and ready for
lects data and may programmatically set other configuration viewing, or the raw data may need to be completely processed
parameters of detector 18. In one embodiment, detector 18 is 15 before analyzing can begin.
a photomultiplier tube that capture fluorescence information Communications with detection device 10 may also be
from light provided by collecting lens 42. In response, detec accomplished by radio frequency (RF) communication or a
tor 18 produces an output signal 128 (e.g., an analog output local area network (LAN) connection. Moreover, connectiv
signal) representative of the received light. Although not ity may be achieved by direct connection or through a net
shown in FIG. 8, detector 18 may concurrently receive light work access point, such as a hub or router, which may support
from other optical modules 16 of device 10. In that case, wired or wireless communications. For example detection
output signal 128 electrically represents a combination of the device 10 may transmit data on a certain RF frequency for
optical input received by detector 18 from the various optical reception by the target data acquisition device 21. Data acqui
modules 16. sition device 21 may be a general purpose computer, a note
Processor 122 may also control data flow from device 10. 25 book computer, a handheld computing device, or an applica
Data such as sampled fluorescence from detector 18, tem tion-specific device. Further, multiple data acquisition
perature of the samples from heating element 134 and related devices may receive the data simultaneously. In other
sensors, and disk rotation information may be stored into embodiments, the data acquisition device 21 may be included
memory 124 for analysis. Processor 122 may comprise any with detection device 10 as one integrated detection and
one or more of a microprocessor, digital signal processor 30 acquisition system.
(DSP), application specific integrated circuit (ASIC), field In addition, detection device 10 may be able to download
programmable gate array (FPGA), or other digital logic cir updated software, firmware, and calibration data from a
cuitry. Moreover, processor 122 provides an operating envi remote device over a network, Such as the internet. Commu
ronment for firmware, software, or combinations thereof, nication interface 130 may also enable processor 122 to moni
stored on a computer-readable medium, Such as memory 124. 35 tor inventory report any failures. If operational problems
Memory 124 may include one or more memories for stor occur, processor 122 may be able to output error information
ing a variety of information. For example, one memory may to assista user in trouble shooting the problems by providing
contain specific configuration parameters, executable operational data. For example, processor 122 may provide
instructions, and one may contain collected data. Therefore, information to help the user diagnose a failing heating ele
processor 122 may use data stored in memory 124 for con 40 ment or a synchronization problem.
trolling device operation and calibration. Memory 124 may Power source 132 delivers operating power to the compo
include any one or more of a random access memory (RAM), nents of device 10. Power source 132 may utilize electricity
read-only memory (ROM), electronically-erasable program from a standard 115 Volt electrical outlet or include a battery
mable ROM (EEPROM), flash memory, or the like. and a power generation circuit to produce the operating
Processor 122 may additionally control heating element 45 power. In some embodiments, the battery may be recharge
134. Based upon the instructions contained within memory able to allow extended operation. For example, device 10 may
124, the heating element 134 may be selectively driven to be portable to detection of biological samples in an emer
control the temperature of one or more chambers according to gency, Such as a disaster area. Recharging may be accom
desired heating profiles. Generally, heating element heats one plished through the 115 Volt electrical outlet. In other
radial section of disk 13 as the disk spins. Heating element 50 embodiments, traditional batteries may be used.
134 may comprise a halogen bulb and reflector for focusing FIG. 9 is a functional block diagram of the single detector
heating energy on a specific area of disk 13. In other embodi 18 coupled to four optical fibers of the optical fiberbundle. In
ments, heating element 134 may heat one or more chambers this embodiment, detector 18 is a photomultiplier tube. Each
sequentially. This embodiment would require disk 13 to be leg of fiber optic bundle 14, optical fiber 14A, optical fiber
stationary while a chamber is heated. In any embodiment, 55 14B, optical fiber 14C and optical fiber 14D, couples to an
heating element 134 may be capable of turning on and off optical input interface 138 of detector 18. In this manner, light
extremely quickly as needed. carried by any of optical fibers 14 is provided to a single
Laser 136 is used to control valve opening which allows optical input interface 138 of detector 18. The optical input
contents of a holding chamber to flow to another chamber on interface 138 provides the aggregate light to electron multi
disk 13, e.g., a reaction well or process chamber. Processor 60 plier 140. Anode 142 collects the electrons and produces a
122 and supporting hardware drives laser 136 to selectively corresponding analog signal as output signal.
open specific valves contained with disk 13. Processor 122 In other words, as shown, the optical fibers 14 fit within the
may interact with a laser sensor underneath disk 13 for deter input optical aperture for detector 18. Consequently, detector
mining the position of the laser relative to the desired valve. 18 may be used to detect light from each leg of optic bundle
When in position, processor 122 outputs signals to direct laser 65 14 simultaneously. Optical input interface 138 provides the
136 to produce a burst of energy targeted at the valve. In some light to electron multiplier 140. For a photomultiplier tube,
cases, the burst may last for approximately 0.5 seconds, while the photons from the optical fibers first hit a photoemissive
 US 7,507,575 B2
19 20
cathode, which in turn releases photoelectrons. The photo and save data until the program is complete (162). Once the
electrons then cascade by hitting a series of dynodes, more run is complete, control unit 23 stops the disk from spinning
photoelectrons being emitted upon contact with each dynode. (164).
The resulting group of electrons have essentially multiplied During this process, control unit 23 monitors the disk tem
the Small light signals originally transmitted by the optical 5 perature (166) and modulates the disk, or each sample, tem
fibers 14. The increased number of electrons finally are col perature to attain the target temperature for that time (168).
lected by anode 142. This current from anode 142 is trans The control unit 23 continues to monitor and control the
ferred by a current to Voltage amplifier 144 as an analog temperatures until the program is complete (170). Once the
output signal which is representative of the optical florescent run is complete, control unit 23 holds the temperature of the
signals from the sample provided by the plurality of optical 10 samples to a target storage temperature, usually 4 degrees
modules 16. Celsius (172).
Control unit 23 includes an analog to digital (A/D) con The operation of device 10 may vary from the example of
Verter 146 converts the analog signal to a stream of sampled FIG. 10. For example, the disk revolutions per minute may be
digital data, i.e., a digital signal. Processor 122 receives the modified throughout the program, and laser 136 may be uti
digital signal and stores the sampled data in memory 124 for 15 lized to open valves between chambers on the disk to allow
communication to data acquisition device 21, as described in for multiple reactions. These steps may occur in any order
above. In some embodiments, A/D converter 146 may be within the operation, depending on the program the user
contained within detector 18 instead of control unit 23. defines.
In this manner, a single detector 18 may be utilized to FIG.11 is a flow diagram illustrating an exemplary method
collect all light from the optic bundle 14 and produce a signal 20 if detecting light and sampling data from the disk. Initially, a
representative thereof. Once the signal is amplified by ampli user specifies which modules will detect fluorescence from
fier 144 and converted to a digital signal, it may be digitally disk 13, and control unit 23 turns on the LED of a module
separated into data corresponding to the light collected by (149). Once the LED has warmed to steady state, control unit
each individual optical modules 16. The entire (i.e., aggre 23 spins disk 13 one rotation at the rate of approximately 1470
gate) signal may be separated by frequency range into each 25 revolutions per minute (151). During that rotation, the mod
detected signal representative of each fluorescence. These ule collects light fluoresced from the process chambers of
frequencies may be separated by a digital filter applied by disk 13 (153), and control unit 23 places 16 samples from
data acquisition device 21 or within device 10. each process chamber in the memory BIN associated with
In other embodiments, the amplified signal may be sepa each process chamber (155).
rated by frequency using analog filters and sent to separate 30 If disk 13 must be spun another rotation (157), control unit
channels before A/D converter 146. Each channel may then 23 executes another revolution of disk 13 (151). If 16 revo
be separately digitized and sent to the data acquisition device. lutions have been sampled, the module has completed detec
In either case, the single detector is able to capture all flores tion with the LED. Therefore, each process chamber was
cence information from each optical module 16. Data acqui sampled a total of 256 times and data acquisition device 21
sition device 21 may then plot and analyze the signal acquired 35 integrates the samples to create a histogram of each process
from each chamber of disk 13 in real-time without the need chamber. Control unit 23 turns the LED off (159). If another
for multiple detectors. module must to used to continue detection (161), control unit
In some embodiments, detector 18 may not be a photomul 23 turns on the next module LED (149). If no other modules
tiplier tube. In general, detector 18 may be any type of analog are needed to collect data, control unit 23 discontinues the
or digital detection device capable of capturing light from 40 collection of data from disk 13.
multiple legs of an optical delivery mechanism, i.e., fiber In some embodiments, each process chamber may be
bundle 14, and producing a transmittable representation of sampled more or less times. Control unit 23 may spin disk 13
the captured light. at a faster rate to provide quicker results or spin disk 13 slower
FIG. 10 is a flow diagram illustrating the operation of the to acquire more samples. In other embodiments, LEDs from
multiplex fluorescence detection device 10. Initially, a user 45 two or more modules may be turned on to detect fluorescence
specifies program parameters on the data acquisition device simultaneously in multiple wavelengths.
21 or via an interface with control unit 23 (148). For example,
these parameters may include a Velocity and time period for EXAMPLE
rotating disk 13, define temperature profiles for the reaction,
and sample locations on disk 13. 50 FIGS. 12 and 13 show the absorption and emission spectra
Next, the user loads disk 13 into the detection device 10 of commonly used fluorescent dyes that may be utilized with
(150). Upon securing the device 10, the user starts the pro device 10 for multiplex PCR. In these examples, the absorp
gram (152), causing control unit 23 to begin spinning the disk tion maxima of the dyes vary from 480-620 nm, and the
(154) at the specified rate. After the disk has begun to spin, resulting emission maxima vary from 520-670 nm. The sig
two concurrent processes may occur. 55 nals for each dye in FIG. 12 are numbered as FAM174, Sybr
First, the detection device 10 starts to detect fluorescence 176, JOE 178, TET 180, HEX 182, ROX 184, Tx Red 186,
from the excitation light (156) produced by one or more and Cy5 188. The signals in FIG. 13 are FAM190, Sybr 192,
reactions within one or more samples. The detector 18 ampli TET 194, JOE 196, HEX 198, ROX 200, Tx Red 202, and
fies the fluorescence signals from each sample, which are Cy5 204. FAM, HEX, JOE, VIC, TET, ROX are trademarks of
synchronized to each respective sample and time at which the 60 Applera, Norwalk, Calif. Tamra is a trademark of AnaSpec,
fluorescence was emitted (158). During this process, proces San Jose, Calif. Texas Red is a trademark of Molecular
Sor 122 saves the captured data to memory 124 and may Probes. Cy 5 is a trademark of Amersham, Buckinghamshire,
communicate the data to data acquisition device 10 in real United Kingdom.
time to monitor the progress of the run and for additional In one example, a 96 chamber disk was filled with different
processing (160). Alternatively, processor 122 may save the 65 concentrations of FAM and ROX dye diluted instandard PCR
data within device 10 until the program is complete. The reaction buffer. Four replicates of each dye were added in a 2x
processor 122 continues to detect florescence of the samples dilution series, starting from 200 nM FAM and 2000 nM
 US 7,507,575 B2
21 22
ROX. Each sample volume was 10 uL. Chamber 82 had a perature as a function of time. In some embodiments, the
mixture of 5uL of 200 nM FAM and 5 uL of 2000 nM ROX. technician may manually change the values located within
Device 10 was constructed as a two-channel multiplex PCR temperature controls 252.
detection device having two optical modules 16 for detection Status indicator 256 is always visible to the technician.
of the dyes. 5 Status indicator 256 displays relevant operational times,
The first optical module (the FAM module) contained a cycle number, temperature and other important information.
blue LED, 475 nm excitation filter and a 520 nm detection Message window 258 displays current commands to control
filter. The second optical module (the ROX module) con unit 23. Window 258 includes a scroll bar for locating any
tained a green LED with a 560 nm excitation filter and a 610 command delivered to control unit 23 during device 10 opera
nm detection filter. Another option would be to incorporate an 10 tion. In some embodiments, message window 258 may dis
orange LED and an excitation filter at 580 nm to optimize for play error information or other important information to the
ROX detection. technician.
A PCR analysis was conducted, and fluorescent signals FIG. 18 is an exemplary screen shot of an optical control
from the samples were multiplexed into a bifurcated fiber user interface. Optical control screen 260 is highlighted and
optic bundle. The fiber bundle was interfaced with a single 15 shows signal graph 262. Histogram 264 shows the integrated
detector, specifically a photomultiplier tube (PMT). Data was signal of each process chamber. Screen 260 also includes
collected by a National Instruments data acquisition (DAQ) message window 266 and offset control 268.
board interfaced with a Visual Basic data acquisition program Signal graph 262 displays the raw optical data detected by
executing on a general-purpose computer. Data was acquired detection device 10. The signal displayed on graph 262 is the
while the disk was spinning at 1000 revolutions per minute raw signal from optical modules 48, 52 and 56 and includes
(nominally). The FAM module and the ROX module were cycles that correspond to the signal change between process
sequentially used to interrogate the samples. Each scan con chambers. The technician may change offset control 268 to
sisted of an average of 50 rotations. The raw data from the two match the binning of signal into appropriate bins representing
optical modules is shown in FIGS. 14A and 14B. each process chamber with the signal waveform. The loss of
The graph in FIG. 14A was acquired by powering the LED 25 signal between each peak represents detection of light from
in the FAM module, and the graph in FIG. 14B was acquired disk 13 between each process chamber. The corresponding
by powering the LED in the ROX module. signal is integrated to produce histogram 264 which displays
During the analysis, the collected data clearly showed that the detected signal from each of 96 process chambers. Con
there was a time offset associated with optical modules being trol unit 23 integrates 16 samples from a process chamber in
physically located over different chambers at any one time. 30 each of 16 rotations of disk 13. Histogram 264 therefore
An offset value was calculated by determining the time offset contains 256 samples of the contents in each samples process
between optical modules 1 and 2 for a particular chamber, i.e., chamber. In some embodiments, software may automatically
chamber 82 in this case. In other words, the time offset indi adjust offset control 268 by recognizing elements of the raw
cates the amount of time delay between data captured by the signal waveform. Message window 266 displays command
FAM module and data captured by the ROX module for the 35 information and error messages relating to optical control and
same chamber. light detection.
FIG. 15 is a graph that shows the offset-subtracted inte FIG. 19 is an exemplary screenshot of a real-time PCR user
grated data for each chamber. FAM is indicated by dotted line interface. Data screen 270 is highlighted and shows histo
bars, ROX is indicated by solid line bars, and the ROX data is gram 272 and product graph 274. Screen 270 shows the
placed over the FAM data. The data showed that there was no 40 real-time data being collected from the process chambers of
signal from the ROX dye on optical module 1 and no signal disk 13. Histogram 272 displays the integrated signal for each
from the FAM dye on optical module 2. There was a higher process chamber while product graph 274 displays the
background on optical module 1, which may be rectified by amount of amplified product as a function of cycle number. In
using an optimized set of filters. The data was analyzed to other embodiments, results for the process chambers may
determine the limit of detection (LOD), described as the 45 vary under different applications.
signal equivalent to the baseline noise level. The baseline Various embodiments of the invention have been
noise level was defined as the average often scans of a blank described. These and other embodiments are within the scope
chamber plus 3 times the standard deviation. of the following claims.
The LOD was determined by a linear least squares fit of the The invention claimed is:
integrated signal plotted against the concentration of the FAM 50 1. A detection device comprising:
and ROX standards. The LOD of the FAM and ROX modules a disk having a plurality of process chambers each holding
were calculated to be 1 and 4 nM, respectively, as shown in a respective sample and at least one of two or more
FIGS. 16A and 16B. fluorescent dyes, wherein at least one of the process
FIG. 17 is an exemplary screen shot of a temperature con chambers holds two or more of the fluorescent dyes;
trol user interface. Temperature control screen 250 is high 55 a motor to rotate the disk;
lighted and shows temperature controls 252. Temperature a plurality of optical modules; and
graph 254 outputs temperature readings while status indicator a housing having a plurality of locations adapted to receive
256 displays general information. Message window 258 dis the optical modules, each of the plurality of locations
plays commands when running detection device 10. having a respective fiber optic coupler mounted therein,
The technician may select temperature control screen 250 60 wherein each of the plurality of optical modules are
to view temperature information from device 10. Tempera removable from the locations of the housing:
ture control screen 250 is one of several screens which may be wherein each of the optical modules includes an optical
selected to display information associated with the operation channel having a light source selected for a different one
of control unit 23 or data acquisition device 21. Screen 250 of the dyes, a lens to capture fluorescent light emitted
includes temperature controls 252 which display numerical 65 from the disk, and an optical output port adapted to
information to the technician. Temperature graph 254 dis engage any of the fiber optic couplers mounted within
plays graphical temperature information as a graph of tem the plurality of locations of the module housing, wherein
 US 7,507,575 B2
23 24
in each of the optical modules the lens is optically 19. The detection device of claim 1, wherein the light
coupled to the output port to deliver the fluorescent light sources are selected for detection of different species of a
collected by the lens to the optical output port of the polymerase chain reaction (PCR) utilizing fluorescence
optical module. detection at multiple wavelengths.
2. The detection device of claim 1, further comprising: 5 20. The detection device of claim 1, wherein the device
a detector, and includes at least four optical modules.
a fiber optic bundle coupled to the optical output port of 21. The detection device of claim 1, wherein the plurality
each of the plurality of optical modules by the fiber optic of optical modules are aligned around the disk to sequentially
couplers to convey the fluorescent light from the mul interrogate one or more process chambers.
tiple optical modules to the detector. 10
22. The detection device of claim 1, wherein two or more
3. The detection device of claim 1, further comprising a optical modules interrogate one or more process chambers
plurality of attachment mechanisms to attach each of the without stopping the rotation of the disk.
plurality of optical modules to the housing. 23. The detection device of claim 1, wherein the plurality
4. The detection device of claim 1, wherein each of the of optical modules are arranged so that each optical module
15
attachment mechanisms includes a lever, a pivot pin, a spring, detects samples at its associated radial position on the disk.
a latch or a screw. 24. The detection device of claim 23, wherein at least two
5. The detection device of claim 1, wherein each of the of the plurality of optical modules are positioned over differ
locations includes a set of one or more electrical contacts, and ent radial positions of the disk.
wherein each optical module has a set of one or more electri 25. A detection system comprising:
cal contacts for electronically coupling to the electrical con a disk having a plurality of process chambers each holding
tacts of the locations when inserted into the housing. a respective sample and at least one of two or more
6. The detection device of claim 5, further comprising a fluorescent dyes, wherein at least one of the process
control unit, wherein the control unit controls the light Source chambers of the disk holds two or more of the fluores
within each of the optical modules via the electrical contacts. 25 cent dyes;
7. The detection device of claim 6, wherein each of the a data acquisition device; and
optical modules includes an electrical component that com a detection device coupled to the data acquisition device,
municates with the control unit via the electrical contacts.
8. The detection device of claim 6, wherein the electrical wherein the detection device comprises:
contacts include a flex circuit connection. a motor to rotate the disk;
9. The detection device of claim 7, wherein the electrical
30 a plurality of optical modules, and
component of each of the optical modules outputs unique a housing having a plurality of locations adapted to receive
identification information for the respective optical module. the optical modules, each of the plurality of locations
10. The detection device of claim 7, wherein the electrical having a respective fiber optic coupler mounted therein,
component of each of the optical modules outputs informa wherein each of the plurality of optical modules are
35
tion describing at least one optical characteristic of an optical removable from the locations of the housing:
component contained within the optical module. wherein each of the optical modules includes an optical
11. The detection device of claim 7, wherein the electrical channel having a light source selected for a different
component includes programmable read-only memory one of the dyes, a lens to capture fluorescent light
(PROM), Flash, an internal storage medium or a removable 40
emitted from the disk, and an optical output port
storage medium. adapted to engage any of the fiber optic couplers
12. The detection device of claim 7, wherein the electrical mounted within the plurality of locations of the mod
components include a laser source that emits a laser to open a ule housing, wherein in each of the optical modules
valve separating a process chamber from a holding chamber the lens is optically coupled to the output port to
on the disk. 45
deliver the fluorescent light collected by the lens to the
13. The detection device of claim 7, wherein one or more of optical output port of the optical module.
the optical modules includes a microprocessor. 26. The system of claim 25, further comprising:
14. The detection device of claim 1, wherein each of the a detector; and
plurality of optical modules further comprises an excitation a fiber optic bundle coupled to the plurality of optical
filter and a detection filter. 50 modules to convey the fluorescent light from the mul
15. The detection device of claim 1, wherein at least two of tiple optical modules to the detector.
the optical modules are combined to form a removable multi 27. The system of claim 26, wherein each of the locations
channel optical module having at least two separate optical includes a set of one or more electrical contacts, and wherein
channels having light sources selected for at least two differ each optical module has a set of one or more electrical con
ent ones of the dyes, and wherein the removable multi-chan 55 tacts for electronically coupling to the electrical contacts of
nel optical module occupies at least two of the locations the locations when inserted into the housing.
within the housing. 28. The system of claim 26, wherein the optical output port
16. The detection device of claim 1, further comprising a optically couples the optical module to a leg of the fiber optic
laser valve control system electrically coupled to one of the bundle by engaging the fiber optical coupler of the location
plurality of optical modules. 60 within which the optical module is positioned.
17. The detection device of claim 16, wherein the laser 29. The system of claim 28, wherein the optical output port
valve control system controls a laser within one of the optical has a slidable connection that allows the leg of the fiber optic
modules to selectively open a valve separating a process bundle to be slidably engaged and disengaged from the opti
chamber from a holding chamber on the disk. cal output port of the optical module.
18. The detection device of claim 1, wherein the light 65 30. The system of claim 29, further comprising a bias
Sources of the optical modules comprise light emitting diodes member associated with the slidable connection to force the
or laser diodes. leg of the fiber optic bundle against the optical output port.
 US 7,507,575 B2
25 26
31. The system of claim 28, wherein the optical output port 36. A detection device comprising:
has a threaded end for engaging a threaded connector of the a disk having a process chamber holding a sample and at
leg of the fiber optic bundle. least two or more fluorescent dyes;
32. The system of claim 25, further comprising a slot a motor to rotate the disk;
sensor trigger that provides an output signal for synchroniza one or more optical modules, and
tion of rotation of the disk with the captured fluorescent light a housing having a location adapted to receive one of the
emitted from the disk.
33. The system of claim 32, further comprising a control optical modules, the location having a fiber optic coupler
unit that uses the slot sensor trigger output signal to compute mounted therein,
a time offset between the modules, controls the light source 10 wherein each of the optical modules includes an optical
within each of the optical modules and processes data from channel having a light source selected for a different one
the captured fluorescent light emitted from the disk based on of the dyes, a lens to capture fluorescent light emitted
the time offset. from the disk, and an optical output port adapted to
34. The system of claim 25, further comprising a laser engage the fiber optic coupler mounted within the loca
valve control system electrically coupled to one of the plu 15 tion of the module housing, wherein the lens is optically
rality of optical modules. coupled to the output port to deliver the fluorescent light
35. The system of claim 34, wherein the laser valve control collected by the lens to the optical output port of the
system controls a laser within one of the optical modules to optical module.
selectively open a valve separating a process chamber from a
holding chamber on the disk.