U.S. patent application number 14/219905 was filed with the patent office on 2014-11-27 for portable high gain fluorescence detection system.
This patent application is currently assigned to Micronics, Inc.. The applicant listed for this patent is Micronics, Inc.. Invention is credited to C. Frederick Battrell, Troy D. Daiber, William Samuel Hunter.
Application Number | 20140349381 14/219905 |
Document ID | / |
Family ID | 42060531 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140349381 |
Kind Code |
A1 |
Battrell; C. Frederick ; et
al. |
November 27, 2014 |
PORTABLE HIGH GAIN FLUORESCENCE DETECTION SYSTEM
Abstract
An instrument for fluorometric assays in liquid samples is
disclosed. The instrument may include multiple optical channels for
monitoring a first fluorophore associated with a target analyte and
a second fluorophore associated with a control. The disclosed
instrument finds utility in any number of applications, including
microfluidic molecular biological assays based on PCR amplification
of target nucleic acids and fluorometric assays in general.
Inventors: |
Battrell; C. Frederick;
(Wenatchee, WA) ; Daiber; Troy D.; (Auburn,
WA) ; Hunter; William Samuel; (Jan Juc, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Micronics, Inc. |
Redmond |
WA |
US |
|
|
Assignee: |
Micronics, Inc.
Redmond
WA
|
Family ID: |
42060531 |
Appl. No.: |
14/219905 |
Filed: |
March 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13536592 |
Jun 28, 2012 |
8716007 |
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14219905 |
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13191120 |
Jul 26, 2011 |
8329453 |
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13536592 |
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PCT/US2010/022581 |
Jan 29, 2010 |
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13191120 |
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61148843 |
Jan 30, 2009 |
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Current U.S.
Class: |
435/287.2 ;
422/82.08; 435/288.7 |
Current CPC
Class: |
G01N 21/6452 20130101;
C12M 23/16 20130101; B01L 7/52 20130101; G01N 21/645 20130101; G01N
21/6456 20130101; G01N 21/6454 20130101; G01N 2201/0245
20130101 |
Class at
Publication: |
435/287.2 ;
435/288.7; 422/82.08 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1-36. (canceled)
37. An apparatus for performing a fluorescence assay on a sample
contained in a detection chamber of a microassay cartridge, the
apparatus comprising: a) a housing configured for receiving the
microassay cartridge, the housing comprising a host controller with
clock, memory and firmware, and a scanning detector head, wherein
the scanning detector head is configured to move in a series of
spatially resolved steps over the detection chamber according to
instructions executed by the host controller, the scanning detector
head comprising: i) one or more optical channels, each optical
channel having an excitation light source, an objective lens for
directing the excitation light onto the sample and for receiving
fluorescence emissions therefrom, an emissions sensor for detecting
the fluorescence emissions, and an amplifier circuit operatively
connected to the emissions sensor, wherein the amplifier circuit is
enabled to amplify a voltage from the sensor, thereby generating an
amplified voltage signal output; ii) an embedded microprocessor
with clock, memory, and firmware for performing a scan of the
microassay cartridge while the scanning detector head is moving in
the series of steps under control of the host controller, the
embedded microprocessor having an electronic input for receiving
the amplifier output, wherein the embedded microprocessor is
enabled to digitally process the amplifier voltage signal output at
each step of the scan across the detection chamber; b) further
wherein the embedded microprocessor generates a digital output
comprising digitally processed data collected during the scan and
conveys the digital output to the host controller; and c) further
wherein the embedded microprocessor is configured to compare the
amplified voltage signal output with a threshold value and score
the signal as a digital one if greater than the threshold value and
as a digital zero if less than the threshold value for each step in
the scan, and the digital output is a string of single-bit digital
scores for each step in the scan.
38. The apparatus of claim 37, wherein the host controller is
provided with instructions to receive the digital output from the
embedded microprocessor and to process the digital output to
generate an assay result, and wherein the apparatus is further
provided with a user interface for displaying or electronically
transmitting the assay result.
39. The apparatus of claim 38, wherein the host controller reports
a qualitative assay result for detection of a target analyte in the
sample.
40. The apparatus of claim 37, wherein the scanning detector head
is operatively driven in a series of linear steps by a stepper
motor under control of the host controller while scanning the
detection chamber with spatial resolution under control of the
embedded microprocessor.
41. The apparatus of claim 37, wherein the embedded microprocessor
is configured for strobing the excitation light source at a strobe
rate and with a selectable pulse width configured to filter
electrical or ambient interference.
42. The apparatus of claim 37, wherein the sensor and amplifier
circuit form a low noise three-stage amplifier circuit surrounded
by a faraday cage.
43. The apparatus of claim 42, wherein the three-stage amplifier
circuit is electronically shielded from electronic noise
originating outside the scanning detector head.
44. The apparatus of claim 42, wherein excitation electronics and
emission electronics are separately grounded in the scanning
detector head.
45. The apparatus of claim 37, wherein the excitation light source
is a spectrally specific LED configured with excitation filter for
transitioning an essentially monochromatic excitation beam onto the
detection chamber.
46. The apparatus of claim 45, wherein the LED is a surface mounted
LED.
47. The apparatus of claim 37, wherein the emissions sensor is a
photodiode, a CCD chip, a CMOS chip, a photomultiplier, an
avalanche diode, or a photosensitive electronic device.
48. The apparatus of claim 39, wherein the fluorescence assay is an
assay for a nucleic acid target or a protein target in the
sample.
49. A method for reducing noise during fluorescence assay of a
liquid sample, the method comprising digitally processing a signal
output at each step of a scan of the sample, wherein the signal
output is compared to a threshold value and scored as a digital one
if greater than the threshold value and as a digital zero if less
than the threshold value for each step in the scan.
50. The method of claim 49, wherein the digital output is a string
of single-bit scores for each step in the scan.
51. The method of claim 49, wherein the digital output comprises
the sum of all the scores for all steps in the scan.
52. The method of claim 49, wherein the digital output is a
single-bit digital score, a string of single-bit digital scores, or
a calculation based on the single-bit digital scores.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
13/191,120, filed Jul. 26, 2011, now pending, which is a
continuation of International PCT Patent Application No.
PCT/US2010/022581, filed Jan. 29, 2010, now pending, which claims
the benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent
Application No. 61/148,843, filed Jan. 30, 2009, which applications
are incorporated herein by reference in their entireties.
BACKGROUND
[0002] 1. Field
[0003] The present invention relates to a compact fluorescence
detection instrument with optics for use in assays performed in a
microfluidic cartridge.
[0004] 2. Description of the Related Art
[0005] Although the benefits of the use of fluorophores as probes
for in-vitro diagnostic assays are well known, the most commonly
available forms of equipment for such assays are large, complex to
use, relatively slow and rely on expensive confocal optics. These
attributes make much equipment unsuitable for fully integrated
"sample-to-answer" testing in remote locales and on-site at the
point of care, where such equipment is required to be rugged, fast,
compact, inexpensive, and easy to use. Although automated nucleic
acid amplification in a microfluidic cartridge was first proposed
some years ago (see Wilding, U.S. Pat. Nos. 5,304,487 and
5,635,358), detection of fluorescent assay targets outside
controlled laboratory conditions is still hampered by the lack of
portable and robust equipment. Two decades since their inception,
molecular diagnostics are still relatively uncommon in the absence
of advanced laboratory facilities because of these and other
unsolved problems.
[0006] Needed to promote broader access to molecular diagnostics
are self-contained assay systems designed to operate outside
specialized laboratory facilities. Nucleic acid assays are rapidly
becoming the "gold standard" for the detection of many different
disease types, including infectious diseases, because they offer
both higher sensitivity and specificity. Such assays have proven
highly specific to a broad range of pathogenic conditions and are
useful for tracking genetic strains of a particular disease as is
fundamental to epidemiology, for example in discriminating H5N1
avian influenza from other types of influenza A or B, in
determining whether a particular pathogen target is of a
drug-resistant strain or not, and in detecting toxigenic strains of
an enteric isolate such as E. coli O157:H7. Fluorescence-based
assays have also been shown to be useful for monitoring conditions
such as diabetes, cardiopathies, coagulopathies, immunoassays in
general, and for detection of endotoxin in foods or drug products
for example. Improved equipment is particularly needed for the
large numbers of remote health clinics in the developing world
where access to health care is limited and many infectious diseases
are endemic, and health and life expectancy are poor.
[0007] In a typical fluorescence assay system, a fluorescent probe
or fluorophore absorbs light having a wavelength or range of
wavelengths and becomes excited; and the fluorophore then emits a
fluorescent signal. The activity or inactivity of the fluorophore
is indicative of the assay result. The emission signal has a
wavelength or range of wavelengths that is generally longer than
the exciting light (but may be shorter as in "up-converting
fluorophores"). A dichroic beam splitter or band-pass filter, or
combination thereof, is then used to separate the fluorescent
signal from other light, and the signal is passed to a sensor. The
sensor is often a photodiode, and generates an electrical signal
that can be used to score the assay. Qualitative and quantitative
assays using real time or endpoint fluorometry are feasible.
[0008] In such systems, a liquid sample is conveyed via a
microfluidic channel into a detection chamber or channel of a
microfluidic cartridge where a fluorescent probe admixed with or
native to the sample is excited by an excitation source. Controls
may be run in parallel or multiplexed in the assay channel. Emitted
light is measured to determine the presence or absence of a target.
A plurality of detection channels may be arranged in the detection
region of the microfluidic cartridge. Assays involve making one or
more measurements of fluorescence; fluorophores may be used as
markers for nucleic acid amplicons formed in an amplification step,
or more generally for the presence or absence of a fluorescent
assay target. Real time fluorometry, FRET, qPCR, thermal melt
curves, kinetic and rate endpoints for assay scoring and validation
are also known in the art.
[0009] Prior art fluorescence detectors typically employ relatively
expensive optical components (such as confocal optics, lasers and
aspheric lenses) in order to pick up and localize fluorescent
emissions present within a microfluidic cartridge or microarray. WO
98/049543 to Juncosa, for example, teaches three dichroic beam
splitters in a single optical train, one for controlling excitation
source power and another for controlling reflectance signal; the
third dichroic beam splitter is used for discriminating
probe-specific fluorescent emission. One or more lenses serve to
focus the excitation beam on the sample. Juncosa further teaches
use of an aperture at the inlet of a photomultiplier and optical
objective lens components of a confocal microscope for controlling
an imaging beam with a resolution of "microlocations" at about
fifty microns. "By restricting the scope of the illumination to the
area of a given microlocation, or a fraction thereof, coupled with
restricting the field of view of the detector to the region of
illumination, preferably through use of an aperture, significant
improvements in signal-to-noise ratio may be achieved." [p 7, lines
10-15]. These teachings are presaged by U.S. Pat. No. 3,013,467 to
Minsky, U.S. Pat. Nos. 5,296,703 to Tsien, 5,192,980 to Dixon,
5,631,734 to Stern, 5,730,850 to Kambara, and are reiterated in
U.S. Pat. No. 6,614,030 to Maher and 6,731,781 to Shams, among
others. Maher uses lasers, fiber optics, a quartz plate and
aspherical lenses with mini-confocal optical system in order to
optimize focusing and emission at a ten micron-sized spot at the
center of the microfluidic chamber [Col 3, lines 23-38; Col 7,
lines 7-16, 43-48 and 58-63].
[0010] Similarly, in U.S. Pat. No. 6,635,487, Lee affirms that
focusing the cone of the excitation beam on the plane of the sample
"provides the greatest intensity to enhance analytical detection
measurements on the assay chips." [Col 1, lines 57-59]. This
teaching thus encapsulates the prior art.
[0011] In a more recent filing, US Patent Application 2008/0297792
to Kim teaches that an image of an LED serving as a light source
for fluorescence detection in a microfluidic chip is projected onto
a sample as an "optical spot" by an objective lens. The optical
spot is focused at the middle of the depth of a fluid in a chamber
in the microfluidic chip [para 0018, 0067, claim 5]. Fluorescence
emitted by the sample is collimated as nearly as possible to
parallel rays by the objective lens and focused on an avalanche
photodiode. The requirement for high precision in alignment relates
to the dichroic mirror because the stopband will be shifted for
light rays that do not enter the mirror at a 45.degree. angle [para
0071], as is well known. Thus the teachings of Kim reflect the
generally recognized state of the art.
[0012] In PCT Publication WO2008/101732 to Gruler, where is
described a fluorescence detector head for multiplexing multiple
excitation and detection wavelengths in a single light path, it is
stated that, "A confocal measurement means that the focus of the
illumination optics or the source, respectively intrinsically is
the same as the focus of the detection optics or sensor,
respectively." [p 7, lines 13-16]. Gruler goes on to state, "The
confocal optics [of the invention] . . . secures highest signal and
lowest background intrinsic features of confocal design" [p 32,
lines 1-5], i.e., according to Gruler the highest possible signal
and lowest noise are obtained with confocal optics.
[0013] While the consensus teaching of the prior art arose out of
the specialized use of confocal optics for epifluorescence
microscopy, the teaching has been widely and uncritically applied
to microfluidic, lateral flow, capillary electrophoresis and
microarray applications. However, we have found that this approach
is not well suited to liquid phase microfluidic diagnostic assays
where detection of one or more molecular probes in a fluid-filled
channel is required. Due to effects such as photoquenching,
thermo-convection, and the occasional presence of bubbles or
gradients in a fluid-filled channel, colocalizing the focal point
of the excitation beam and emission cone in the plane of the sample
chamber can lead to unacceptable instability, loss of signal,
quenching, noise, irreproducibility and overall loss of sensitivity
in the results. Because of the higher temperatures of PCR, for
example, outgassing of reagents and sample is not an uncommon
problem, and interference from bubbles entrained in the liquid
sample is a frequent problem. The conventional approach also
requires more expensive optical components and thus is
disadvantageous for widespread application outside advanced
clinical laboratories.
[0014] A second problem is assay validation. Current standards for
validation of infectious disease assays by PCR, for example, have
come to rely on use of spiked nucleic acid templates or more
preferably, co-detection of endogenous normal flora, for example
ubiquitous non-pathogenic Escherichia coli in stools where
pathogens such as Salmonella typhi or E. coli O157 are suspected.
Another ubiquitous endogenous template is human 18S rRNA, which is
associated with higher quality respiratory and blood samples.
Co-amplification and detection of an endogenous template ensures
confidence in the assay results but is difficult to achieve in
practice because of possible crosstalk between the fluorophores
used as markers. When using high gain amplification, some level of
crossover in the spectra of the excitation and emission of
fluorophores commonly selected for multiplex PCR is typical and
expected. Thus a solution that would isolate fluorescent signals
with spectrally overlapping shoulders by using separate optical
channels within a scanning detector head having shared low-noise
electronics for downstream processing would be a technological
advance of benefit in the art.
[0015] A third problem is portability. Use of disposable cartridges
has proved beneficial because cross-contamination due to shared
reagent reservoirs and shared fluid-contacting surfaces is avoided.
However, configuring a precision optical instrument platform for
accepting disposable cartridges is problematic. Problems include
inaccuracies and stackup in mechanical tolerances that affect
cartridge alignment and detector head positioning, the need for
forming a highly conductive thermal interface between the plastic
disposable cartridges and heating sources in the instrument, the
need for sealing the pneumatic interface between control servos on
the apparatus and microvalves on the cartridge, and the necessarily
shorter light path available in a microfluidic cartridge (typically
about or less than 1 millimeter), which without optimization can
lead to loss in sensitivity. A simultaneous solution of these
interlocking problems is only achieved by extensive experimentation
and development, most often guided by trial and error in this
highly unpredictable art. Thus there is a need in the art for
numerous improvements, elements of which are the subject of the
disclosure herein.
BRIEF SUMMARY
[0016] The present invention addresses the problem of reliable and
sensitive detection of fluorescent probes, tags, fluorophores and
analytes in a microfluidic cartridge in the presence of bubbles and
other interfering inhomogeneities in a liquid sample, in a first
aspect of the invention, by providing a reflective mirror face
formed on a heating block that contactingly interfaces with a
thermo-optical window on the underside of the detection channel or
chamber containing the liquid sample. The mirror face is formed on
the top surface of a heating block and contacts the lower optical
window of the detection chamber during use, avoiding the complexity
and expense of manufacturing an integral mirror on the bottom of
each disposable cartridge, and allowing us to use thinner, more
compliant films with lower resistance to heat transfer and
transparent optical characteristics for the thermo-optical window
of the cartridge. The mirror face is optically flat and polished to
improve both heat transfer and fluorescence emission capture. A
scanning objective lens is positioned above an upper optical window
on the top of the microfluidic cartridge. Excitation light is
transmitted through both the upper optical window and the lower
thermo-optical window before striking the mirror and reflecting
back. Direct and reflected emissions are collected by the objective
lens and focused on a detection sensor such as a photodiode,
photocell, photovoltaic device, CMOS or CCD chip.
[0017] Also, and starkly in contrast to the teachings of the prior
art, the problem of fluorescence detection is shown to be solved by
configuring the optics so that the excitation optics are decoupled
from the emission optics on a common optical path. By trial and
error, when using the back mirror, we have found that it is
advantageous to place the focal point of the excitation cone near
or behind the plane of the reflective mirror and to independently
position the emission cone so that emissions are preferentially
focused on the detection sensor. Surprisingly, decoupling increases
sensitivity, improves limits of detection, and reduces noise or
interference of bubbles and other inhomogeneities in the
sample.
[0018] Contrary to the teachings of the prior art, we find that the
conventional confocal localization of the excitation and emission
signal is less effective in generating a robust signal over a wide
range of sample and operating conditions. Therefore, in one aspect
of the invention, it was found that optimization of signal
detection may be improved by displacement of the focal point of the
excitation light from the plane of the sample to a point behind the
sample, a technological advance in the field of low cost optics for
use with microfluidic fluorescence assays. Decoupling of excitation
and emission optics flies against decades of prior art dedicated to
the principles (first espoused by Minsky in U.S. Pat. No.
3,013,467) that form the foundation of conventional practice in
confocal microscopy, epifluorescence detection, and microfluidic
fluorescence assays. The prior art teachings have lead to the use
of aspherical lenses, laser diodes, and precise parfocal alignment
of the detection optics with the excitation optics. In contrast,
the optics required for delocalized focus of the excitation cone as
described here are fortuitously of very low cost and do not require
precision assembly or maintenance, as is desirable for
manufacturing a low cost, portable instrument.
[0019] The mirror face on the top surface of the heating block
under the detection chamber is used to increase sensitivity by
improving the light-gathering capacity of the objective lens. As
the objective lens is placed closer to the detection chamber, a
lens of defined angular aperture and numerical aperture becomes
more efficient in collecting emissions. Without the back mirror,
collection efficiency of a typical system of the prior art is less
than 2.5% (assuming for example a 5 mm planoconvex lens). Adding a
back mirror can improve this by as much as 200%, and theoretically
as much as 400%. And focusing the excitation beam behind the sample
chamber can add synergically to any gain in sensitivity by
increasing the excitation pathlength by using a mirror. We have
found that this is especially advantageous in low aspect ratio
microfluidic cartridges, where the optical path length on a z-axis
of a cartridge is typically sub-millimeter in length, a significant
reduction relative to a standard optical cuvette. Happily, this
combination was also found to reduce interferences due to
irregularities in the sample chamber such as the presence of small
bubbles.
[0020] In one embodiment, the mirror is a chromed or polished metal
surface on an aluminum or copper heating bock, and is also used to
transmit heat or cooling for temperature controlled assays, thus
achieving another synergy of design. In a preferred embodiment, the
mirror is an electropolished chrome surface on an optically flat
aluminum block, the aluminum selected for its superior heat
transfer characteristics and scaleable thermal inertia. The block
is heated by a resistive heating element in contact with the base
of the block. The smoothness and flatness of the mirror face favors
optimal heat transfer. In this aspect of the invention, the
mirrored face is the upper surface of the heating element used for
example for FRET detection or thermal melting analysis of
fluorescent probes for PCR amplicons. In one embodiment, a combined
application of the optical and thermal properties of the
mirror-faced heating block is illustrated by construction of FRET
melt curves taken by monitoring fluorescence while ramping the
temperature of the assay fluid. In another embodiment, the mirror
faced heating block is used to adjust or control a reaction
temperature in the detection chamber of a microfluidic card while
the cartridge is scanned for fluorescence emission. A mirror-faced
heating block for use with microfluidic cartridges in real-time and
temperature modulated fluorescence assays demonstrates a technical
advance in the art.
[0021] According to another aspect of the present invention, we
have employed a high gain multi-stage amplifier with noise
elimination augmented through the use of downstream signal
processing firmware compactly mounted in the scanning head. Very
high gain amplification and out-of-plane delocalization of the
excitation light cone were found to be synergic in optimizing assay
discrimination and sensitivity, even in the presence of bubbles
which disrupt specular reflection from the mirror face behind the
liquid sample, and happily were implemented with no increase in
cost.
[0022] The complete optical path uses three lenses, the first for
collimating excitation from a light source, the second for
projecting the excitation source onto the mirror and for collecting
a fluorescent emission from any fluorophore in the sample as
collimated emissions, and the third for focusing the emissions on a
detector. Each fluorophore is optically isolated by a separate
optical channel for measurement. A combination of
spectrally-specific LEDs, dichroic mirrors, and barrier filters are
used to achieve near monochromatic excitation light in each optical
channel. The lenses and related optical components, including the
dichroic mirror intersecting the light path for separating
excitation and emissions wavelengths and filters, are provided in a
guiderail-mounted scanning head that moves laterally across the
detection chambers. To minimize noise, the head also includes all
electronic components for amplifying the signal and an on-board
embedded microprocessor for analog and digital signal processing.
Even in the presence of bubble foam interferences that defeat
signal averaging and baseline subtraction methods of data
acquisition, assay scanning data from each optical channel may be
accurately evaluated and reported by conversion to a single bit
output (ie. a 1 or a 0). This has been found to be a simple and
remarkably effective means for qualitative scoring for the presence
or absence of a signal from a particular fluorophore in a liquid
sample mixture while the detector head is scanned over the
detection chamber or chambers and across the mirror face.
[0023] The scanning head and rails are configured with a drive
chain coupled to a stepper motor for accurate spatial resolution
during scanning The entire microfluidic cartridge docking bay and
optical bench is mounted in an instrument housing at a pitched
angle, which we have found advantageous in decreasing bubble
entrainment and improving venting during loading, wetout, and
mixing operations on the microfluidic cartridge. In a preferred
embodiment, the entire optical bench is mounted at an angle of
about 15 degrees so that bubbles are displaced from the
microfluidic circuitry, necessitating a complete suspension mount
for the floating optical bench and the docking bay, and a
spring-biased clamping mechanism to ensure active formation of a
thermoconductive interface between on-board heating elements
mounted in the docking bay on the bottom of the optical bench
assembly and the insertable cartridge when the cartridge is loaded
into the instrument. A sealed interface between the angled
cartridge and a gasketed pneumatic interface port must also be
established during docking.
[0024] The detector head may be scanned across the detection
chamber, or conversely, the microfluidic cartridge may be scanned
across the detector. As implemented here, the detector is mounted
with a worm gear- or rack and pinion gear-driven stage under
control of a stepper motor on twin, paired guiderails. Samples may
be scanned on demand by synchronizing data acquisition with motor
control; this may be performed using an on-board or external host
controller in communication with the embedded microprocessor in the
detector head. Surprisingly, such a detector head with integral
co-processor, mounted on a linear motion stage for sampling,
filtering and averaging measurements on the fly, improved the
capability of the system to validate and report assay results
despite many potential interferences.
[0025] It was expected that there will be variations in fluorescent
intensity across the microfluidic chamber, channel or detection
field, which can be several millimeters in width. These variations
arise, for example, as a result of inhomogeneities in mixing, from
differences in well thickness, from imperfections in the optical
windows, from small temperature variations across a heated
detection well (which can cause accompanying variations in
hybridization-dependent fluorescence of amplicons being detected,
for example) and from bubbles or foam which arise from degassing
and mixing. It was found that these variations can be minimized by
signal digitization over a sampling transect across the detection
chamber using a threshold to discriminate positive and negative
test results.
[0026] In order to further provide noise elimination, extraneous
noise is removed by strobing the excitation beam at a frequency
known to prevent interference by AC line fluctuations and other
ambient electric or RF interference; resulting in cleaner signal
modulation, a modulated signal that also can be filtered to remove
the effects of any ambient light leaking into the instrument
housing. We have achieved this result by configuring the optics
printed circuit board with a dedicated co-processor, and using
independent clock frequency and firmware, which efficiently
minimizes traffic on a databus connected to the host instrument.
The on-board optical signal processor has dedicated instructions
stored in EEPROM resident on the optics card, and synchronizes
pulses sent to the source LED with interrogation of the sensor
photodiode at a frequency selected to limit electromagnetic
interference. The firmware is designed to evaluate the difference
in signal between strobe illumination bursts of excitation light
and background between strobe bursts, and any background due to
ambient or extraneous light is readily subtracted by this
method.
[0027] The resulting optics modules are packaged in a sealed
detector housing and can be expanded as a series of optics modules
with multiple optical channels for simultaneously scanning multiple
samples or fluorophores in series or in parallel. Signal processing
is routed to a single co-processor embedded in the detector head,
and from there to the host instrument controller. Thus the
invention comprises single head/single channel embodiments, dual
head/dual channel embodiments, and multi-head/multi-channel
embodiments and may perform single and multiplex assays on single
samples or on multiple samples in parallel.
[0028] The detectors were found to function well when housed as
dual head and multi-head detectors, where two or more channels in a
single housing were configured with fully independent optics,
fluorophore-specific filters, dichroic mirrors and source LEDs,
reducing crosstalk between multiple fluorophores. The head thus
will optionally contain a plurality of light sources mounted on a
first circuit board and a plurality of objective lens and
associated detectors mounted on a second circuit board, and will
collect signals for each of a plurality of fluorophores
independently using shared signal processing capability and
firmware embedded in the detector head. To reduce noise, no analog
signals are transmitted from the detector head to the host
instrument.
[0029] In this way, light sources for excitation in each channel
can be matched to the individual fluorophores. It is no longer
necessary to provide white light and an excitation filter to ensure
a narrow pass beam of excitation light striking the sample. This
simplification proved useful in assay protocols calling for paired
collection of "biplex" or multiplex target and control signals.
Where, as for FDA CLIA waiver requirements, both target and control
templates are amplified in parallel, a positive control signal must
be present before an assay result on a test sample can be reported
or billed. In the absence of a detectable control signal, any
target signal detected is not a valid result. In order to meet CLIA
waiver requirements, it is necessary that the fluorescence detector
be able to detect not only the presence, for example, of a target
infectious organism amplified by PCR but also of an endogenous
human control organism co-existing with the target and amplified by
the same PCR reaction or a PCR reaction conducted in parallel in
the instrument, for example.
[0030] Such an approach requires that the fluorescent detector have
the capacity to determine the presence of both the target and the
control fluorophores as a "biplex" ("duplex") amplification
reaction mixture in a common detection chamber. A positive
amplification control fluorophore is typically used which has
fluorescent excitation and emission spectra which is shifted (in
wavelength) so as to be well resolved by selective band pass
filters from the fluorescent excitation and emission spectra for
the target fluorophore (see FIG. 7 for example). However, according
to the present invention, it proved possible to achieve superior
resolution by using a dual head detector and by scanning each
detection chamber twice in series, once with each optical channel,
detecting first the control fluorescence signature, then the test
sample fluorescence signature--each scanning pass utilizing
separate excitation and emission optics as described above.
[0031] A benefit was found by configuring these detectors with
fully separated and independent light paths. In this aspect of the
invention, the use of a duplex head design ensures that the
presence of an amplification control fluorophore in a sample does
not inadvertently create a signal in the target channel due to
"crosstalk". Such a situation would result in the sample being
classed as a "false positive". Conversely, it is also important
that there is no crosstalk from the target channel to the control
channel, which is likely when multiple signals share a common
optical path. Such a situation could result in the positive
amplification control being inadvertently deemed present, when this
may not be the case, and would lead to reporting of an invalid
assay, an unacceptable outcome.
[0032] These principles are exemplified by the use of fluorescein
or equivalent fluorophore as a molecular probe for the target, and
Texas Red or equivalent fluorophores as a molecular probe for the
control. A dual head detector, with one detection channel optimized
for detection of fluorescein and the other detection channel
optimized for Texas Red, each with separate excitation and
detection optics, was found to be surprisingly sensitive, accurate
and robust. The detector head was moved so as to position each
optical channel in turn over the sample and separate fluorescence
readings were made. Surprisingly, this improved resolution and
minimized cross talk but did not contribute to higher noise or loss
of sensitivity due to the mechanics of moving the head. Because
excitation is not performed with white light, but is instead
performed at a wavelength specific for an individual fluorophore,
quenching of the second fluorophore is not an issue.
[0033] In another aspect of the invention, we have found that
multiple microfluidic channels can be scanned by sequential
traverse of a multihead detector across multiple sample wells, each
head being configured with independent optics for excitation and
emission of a single fluorophore. Although the excitation and
detection optics are separated for each optical channel, signal
processing is performed in circuitry shared within the detector
head.
[0034] Thus in yet another embodiment the invention provides a
robust multi-head independent channel fluorescent detection system
for a point of care molecular diagnostic assay which has a high
degree of specificity directed towards the presence of both a
target and an nucleic acid amplification control co-existing in a
common or parallel detection chamber. The cleaner signals permit
higher gain electronic amplification without a corresponding
decrease in signal-to-noise ratio. Although a first embodiment in
this invention describes a dual channel detector (for the presence
of a single target and a control), the invention may also be
applied to a fluorescent detection system having more than two
channels, for example to detect a multiplex of targets and a
control co-existing in a single detection chamber. Optionally, the
heads may be positioned side-by-side, in an array, or radially, as
in a cylinder.
[0035] Use of the independent optical pathways fortuitously
resulted in reduced need for precision in assembly of lenses,
dichroic beam splitters, and associated filter elements, improving
the manufacturability of the apparatus. Embodiments of the present
invention incorporate inexpensive non-precision optics, plastic
lenses, a mirror on the heating block behind the sample window,
moveable stage elements, strobed excitation and emission, noise
suppression, on-board continuous signal processing over a movable
detection field, and more than one optical channel for biplex assay
validation by use of paired target and control fluorophores.
Despite its high amplification gain, the instrument has proven
advantageously resistant to interferences such as electrical noise
and bubbles in the detection chamber.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a perspective view of an instrument of the
invention and a microfluidic cartridge in the docking bay.
[0037] FIG. 2 is an animated view showing the insertion of the
microfluidic cartridge in the docking bay.
[0038] FIG. 3A is a simplified representation of a floating stage
with docking bay, optical bench, and clamping mechanism for
thermally contacting the microfluidic cartridge with the heating
module and scanning the microfluidic cartridge.
[0039] FIG. 3B demonstrates conceptually that the floating stage,
docking bay, optical bench and microfluidic cartridge are mounted
in the instrument chassis at a defined angle "theta" relative to
the ground plane, where the ground plane is horizontal.
[0040] FIGS. 4A and 4B are front and rear interior perspective
views from above, showing the suspension-mounted stage with docking
bay, optical bench, and scanning detector head.
[0041] FIG. 5A is a front interior perspective view from below the
docking bay, showing the underside heating module and cooling
fan.
[0042] FIG. 5B is a detail of the heating module with heating block
elements and mirror face.
[0043] FIGS. 6A and 6B are perspective views of the floating stage
with insertable cartridge in place in the docking bay. For clarity,
the docking saddle and accessory mounting elements have been
removed.
[0044] FIG. 7 is a detailed view of the docking bay and floating
stage suspended from the underside of the docking saddle. The
floating stage is fitted with a four point spring suspension.
[0045] FIGS. 8A and 8B are anterior subassembly views of the
clamping mechanism. FIG. 8B illustrates the worm drive operation on
the clamping gear rack.
[0046] FIGS. 9A and 9B are posterior subassembly views of the
clamping mechanism. FIG. 9B illustrates the worm drive operation of
the clamping gear rack and the platen arm.
[0047] FIG. 10 is a block diagram providing an overview of the
functional units, software and firmware of the apparatus.
[0048] FIGS. 11A and 11B are perspective views of an insertable
microfluidic cartridge for use with the apparatus of the
invention.
[0049] FIG. 12 is an exploded view showing the internal components
of a microfluidic cartridge of FIG. 11.
[0050] FIG. 13 is a perspective view of a detector head with dual
optical channels and electronically isolated circuit boards for
excitation and emissions detection. One half of the housing is
removed in order to view the internal components.
[0051] FIGS. 14A and 14B are schematic views of the internal
optical components of a fluorescence detector with dual optical
channels, heating block-mounted mirror and microfluidic cartridge.
Excitation optics are mounted on one circuit board and detection
optics on another to reduce noise interference.
[0052] FIG. 15 is a representation of an excitation cone and
planoconvex objective lens relative to the cartridge detection
chamber and mirror-faced heating block.
[0053] FIG. 16 is a representation of emission collection with a
planoconvex objective lens at short working distance relative to
the cartridge detection chamber and mirror-faced heating block.
Shown are primary and reflected fluorescent emissions.
[0054] FIG. 17 is a schematic representation of an optical pathway
with decoupled excitation and emission optics.
[0055] FIG. 18 is a block diagram of the detector head electronics
used for controlling the fluorescent excitation, and receiving,
processing, and digitally communicating fluorescence emission
signals to the host instrument.
[0056] FIGS. 19A and 19B are representations of raw input and
digitized output showing digital removal of bubble
interference.
[0057] FIGS. 20A and 20B show emission and excitation wavelengths
for two fluorophores in a liquid sample, and illustrate dual head
optical isolation for removal of crosstalk.
[0058] FIG. 21A plots experimental results demonstrating
enhancement of signal output by varying the height of the objective
lens above the mirror. FIG. 21B graphs the integrated output signal
strength with and without the back mirror. Output signal was found
to be optimized by focusing the excitation beam at a focal point
behind the mirror as shown in FIG. 15 and collecting emissions at a
shorter working distance as shown in FIG. 16.
[0059] FIGS. 22 and 23A and 23B demonstrate a thermal melt curve of
a molecular beacon hybridized to an amplicon.
DETAILED DESCRIPTION
[0060] Although the following detailed description contains
specific details for the purposes of illustration, one of skill in
the art will appreciate that many variations and alterations to the
following details are within the scope of the claimed invention.
The following definitions are set forth as an aid in explaining the
invention as claimed.
Definitions
[0061] "Angular aperture"--is the angle between the most divergent
rays from a single point that can enter the objective lens and
participate in image formation.
[0062] "Back focal length"--is defined for a lens with an incident
beam of collimated light entering the lens as the distance L from
the back surface of the lens to the focal point of a cone of
focused light. "Back focal positions" indicates that non-collimated
rays may be focused at alternate distances from the back of the
lens by decoupling the optics.
[0063] Target analyte: or "analyte of interest", or "target
molecule", may include a nucleic acid, a protein, an antigen, an
antibody, a carbohydrate, a cell component, a lipid, a receptor
ligand, a small molecule such as a drug, and so forth. Target
nucleic acids include genes, portions of genes, regulatory
sequences of genes, mRNAs, rRNAs, tRNAs, siRNAs, cDNA and may be
single stranded, double stranded or triple stranded. Some nucleic
acid targets have polymorphisms, deletions and alternate splice
sequences. Multiple target domains may exist in a single molecule,
for example an immunogen may include multiple antigenic
determinants. An antibody includes variable regions, constant
regions, and the Fc region, which is of value in immobilizing
antibodies. Target analytes are not generally provided with the
cartridge as manufactured, but are contained in the liquid sample
to be assayed; in contrast, "control analytes" are typically
provided with the cartridge and are assayed in order to ensure
proper performance of the assay. Spiked samples containing target
assay may be used in certain quality control testing and for
calibration, as is well known in the art.
[0064] Means for Amplifying: The grandfather technique was the
polymerase chain reaction (referred to as PCR) which is described
in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159,
Ausubel et al. Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1989), and in Innis et al., ("PCR
Protocols", Academic Press, Inc., San Diego, Calif., 1990).
Polymerase chain reaction methodologies require thermocycling and
are well known in the art. Briefly, in PCR, two primer sequences
are prepared that are complementary to regions on opposite
complementary strands of a target sequence. An excess of
deoxynucleoside triphosphates are added to a reaction mixture along
with a DNA polymerase, e.g., Taq polymerase. If the target sequence
is present in a sample, the primers will bind to the target and the
polymerase will cause the primers to be extended along the marker
sequence by adding on nucleotides. By raising and lowering the
temperature of the reaction mixture, the extended primers will
dissociate from the template to form reaction products, excess
primers will bind to the template and to the reaction products and
the process is repeated. By adding fluorescent intercalating
agents, PCR products can be detected in real time.
[0065] Other amplification protocols include LAMP (loop-mediated
isothermal amplification of DNA) reverse transcription polymerase
chain reaction (RT-PCR), ligase chain reaction ("LCR"),
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA), "Rolling Circle", "RACE"
and "one-sided PCR", also termed "asymmetrical PCR" may also be
used, having the advantage that the strand complementary to a
detectable probe is synthesized in excess.
[0066] These various non-PCR amplification protocols have various
advantages in diagnostic assays, but PCR remains the workhorse in
the molecular biology laboratory and in clinical diagnostics.
Embodiments disclosed here for microfluidic PCR should be
considered representative and exemplary of a general class of
microfluidic devices capable of executing one or various
amplification protocols.
[0067] Typically, nucleic acid amplification or extension involves
mixing one or more target nucleic acids which can have different
sequences with a "master mix" containing the reaction components
for performing the amplification reaction and subjecting this
reaction mixture to temperature conditions that allow for the
amplification of the target nucleic acid. The reaction components
in the master mix can include a buffer which regulates the pH of
the reaction mixture, one or more of the natural nucleotides
(corresponding to A, C, G, and T or U--often present in equal
concentrations), that provide the energy and nucleotides necessary
for the synthesis of nucleic acids, primers or primer pairs that
bind to the template in order to facilitate the initiation of
nucleic acid synthesis and a polymerase that adds the nucleotides
to the complementary nucleic acid strand being synthesized.
However, means for amplication also include the use of modified or
"non-standard" or "non-natural" bases such as described in U.S.
Pat. No. 7,514,212 to Prudent and U.S. Pat. Nos. 7,517,651 and
7,541,147 to Marshall as an aid to detecting a nucleic acid
target.
[0068] Means for detection: as used herein, refers to an apparatus
for displaying an endpoint, ie. the result of an assay, and may
include an instrument equipped with a spectrophotometer,
fluorometer, luminometer, photomultiplier tube, photodiode,
nephlometer, photon counter, voltmeter, ammeter, pH meter,
capacitative sensor, radio-frequency transmitter,
magnetoresistometer, or Hall-effect device. Magnifying lenses in
the cover plate, optical filters, colored fluids and labelled
probes may be used to improve detection and interpretation of assay
results. "Labels" or "tags" include, but not limited to, dyes such
as chromophores and fluorophores; and chemoluminescence as is known
in the prior art. QDots, such as CdSe coated with ZnS, decorated on
magnetic beads, or amalgamations of QDots and paramagnetic Fe3O4
microparticles, are a convenient method of improving the
sensitivity of an assay of the present invention. Fluorescence
quenching detection endpoints are also anticipated. A variety of
substrate and product chromophores associated with enzyme-linked
immunoassays are also well known in the art and provide a means for
amplifying a detection signal so as to improve the sensitivity of
the assay, for example "up-converting" fluorophores. Fluorescence
and optical detectors may include photodiodes, photovoltaic
devices, phototransistors, avalanche photodiodes, photoresistors,
CMOS, CCD, CIDs (charge injection devices), photomultipliers, and
reverse biased LEDs. Detection systems are optionally qualitative,
quantitative or semi-quantitative.
[0069] "Molecular beacon"--is a single stranded hairpin-shaped
oligonucleotide probe designed to report the presence of specific
nucleic acids in a solution. A molecular beacon consists of four
components; a stem, hairpin loop, end-labelled fluorophore and
opposite end-labelled quencher. When the hairpin-like beacon is not
bound to a target, the fluorophore and quencher lie close together
and fluorescence is suppressed. In the presence of a complementary
target nucleotide sequence, the stem of the beacon opens to
hybridize to the target. This separates the fluorophore and
quencher, allowing the fluorophore to fluoresce. Alternatively,
molecular beacons also include fluorophores that emit in the
proximity of an end-labelled donor. `Wavelength-shifting Molecular
Beacons` incorporate an additional harvester fluorophore enabling
the fluorophore to emit more strongly. Current reviews of molecular
beacons include Wang K et al, 2009, Molecular engineering of
DNA:molecular beacons. Angew Chem Int Ed Engl, 48(5):856-870;
Cissell K A et al, 2009, Resonance energy transfer methods of RNA
detection, Anal Bioanal Chem 393(1):125-35 and Li Y, et al, 2008,
Molecular Beacons: an optimal multifunctional biological probe,
Biochem Biophys Res Comm 373(4):457-61. Recent advances include
Cady, N.C., 2009, Quantum dot molecular beacons for DNA detection.
Methods Mol Biol 554:367-79.
[0070] Fluorescence nucleic acid assays include amplification with
tagged primers and probe-based detection chemistries. Fluorescent
products can be assayed at the end of the assay, or by measuring
the amount of amplified product in real time. While not limiting,
TaqMan Probe (Applied Biosystems) which relies on displacement and
polymerase-mediated hydrolysis of a 5' reporter dye with 3'
quencher construct, FRET hybridization probes, dual oligo
FRET-based probes (Roche), minor groove binder-conjugated
hybridization probes (MGB probes, Applied Biosystems), Eclipse
probes, Locked NA Probes (Exiqon/Roche), Amplifluor primer
chemistries, Scorpions primer chemistries, LUX primers, Qzyme
primers, RT-PCR, among others, are all suitable in the present
invention. Intercalation dyes may also be used. Reverse
transcriptase is used to analyze RNA targets and requires a
separate step to form cDNA. Recent advances include Krasnoperov L N
et al. 2010. Luminescent probes for ultrasensitive detection of
nucleic acids. Bioconjug Chem 2010 Jan. 19 epub.
[0071] In addition to chemical dyes, probes include green
fluorescent proteins, quantum dots, and nanodots, all of which are
fluorescent. Molecules such as nucleic acids and antibodies, and
other molecules having affinity for an assay target, may be tagged
with a fluorophore to form a probe useful in fluorescent assays of
the invention.
[0072] "FRET" (Fluorescence Resonance Energy Transfer)--is a
fluorescence technique that enables investigation of molecular
interactions. It depends on the transfer of energy from one
fluorophore to another fluorophore (ie. a donor and a quencher)
when the two molecules are in close proximity such a when
hybridized. Recent advances include Carmona A K et al, 2009, The
use of fluorescence resonance energy transfer (FRET) peptides for
measurement of clinically important proteolytic enzymes, An Acad
Bras Cienc 81(3):381-92.
[0073] Unless the context requires otherwise, throughout the
specification and claims which follow, the word "comprise" and
variations thereof, such as, "comprises" and "comprising" are to be
construed in an open, inclusive sense, that is as "including, but
not limited to". Reference throughout this specification to "one
embodiment", "an embodiment", "one aspect", or "an aspect" means
that a particular feature, structure or characteristic described in
connection with the embodiment or aspect may be included one
embodiment but not necessarily all embodiments of the invention.
Furthermore, the features, structures, or characteristics of the
invention disclosed here may be combined in any suitable manner in
one or more embodiments. "Conventional" is a term designating that
which is known in the prior art to which this invention relates.
"About" and "generally" are broadening expressions of inexactitude,
describing a condition of being "more or less", "approximately", or
"almost" in the sense of "just about", where variation would be
insignificant, obvious, or of equivalent utility or function, and
further indicating the existence of obvious minor exceptions to a
norm, rule or limit.
[0074] "Crosstalk"--in fluorescence imaging occurs when the
excitation and/or emission spectra of two or more fluorophores
(and/or autofluorescence) in a specimen overlap, making it
difficult to isolate the activity of one fluorophore alone.
[0075] Turning now to the figures, FIG. 1 is a perspective view of
the instrument 100 with a microfluidic cartridge 200 in the docking
bay. Shown are membrane panel 104 and touch screen display surfaces
108 and compact chassis or housing 106. Because all reagents are
provided in the microfluidic cartridge, the instrument has full
standalone operability. FIG. 2 complements this exterior view by
animating the insertion of the microfluidic cartridge anterior nose
105 into the docking bay 103. The docking bay is suspension-mounted
and tilted at an angle relative to the instrument base, as will be
discussed in more detail below.
[0076] The angled, tilted, floating stage with on-board optical
bench and docking bay is a distinctive feature of the instrument.
This feature is introduced conceptually in FIG. 3. A tilt sensor
may be used in conjunction with the instrument host controller in
order to ensure the proper angle is maintained for improved
performance. The mounting angle of the inclined mounting plate
determines the angle at which the microfluidic cartridge is held
during the assay. This angle "theta" has been found to be
advantageous in the range of 10-45 degrees from the ground plane,
more preferably 10-20 degrees, and is most preferentially about 15
degrees. The angular mount has been found to relieve bubble
interference that may be associated with deterioration in PCR
amplification results, a technological advance in the art.
[0077] FIG. 3A is a schematic diagram of the primary
optothermomechanical subsystems of the instrument. The floating
stage 300 consists of a tray-like chassis 301 that is suspended on
an inclined plane by a four-point spring-mounted suspension
(indicated by 302,303) and supports a docking bay 103 for receiving
a microfluidic cartridge 200 and scanning detector head 311 mounted
on paired guiderails (308,309). The cartridge is not part of the
instrument 100, but interfaces with the instrument after insertion
into the floating docking bay 103.
[0078] During operation, the floating stage is clamped (indicated
by 320) against inclined mounting plate (330) and engages
contacting surfaces of zone heating blocks (341,342,343,344) of a
heating module 340 and associated resistive heating elements and
circuits. A fan 345 is provided to dissipate excess heat during
cooling.
[0079] The inclined mounting plate is also provided with a
pneumatic interface port 350 for sealedly docking to the base of
the microfluidic cartridge. Pneumatic pressure is delivered to the
cartridge through the pneumatic interface port from an integral
pneumatic distribution "manifold" or system embedded in the
inclined mounting plate 330. The pneumatic manifold supplies
negative and positive pressure from sources mounted on the inclined
mounting plate. A motherboard-mounted, programmable host controller
directs pneumatic driving pressure, vacuum, and control pulses to
pumps and valves on the cartridge via the internal manifold in the
base plate 330 and pneumatic interface port 350.
[0080] The detector head is motorized and scanning of the cartridge
is performed under the control of the central host controller. To
scan the detector head along paired guiderails (308,309) the host
controller engages a worm-gear driven by stepper motor 307. The
detector head is fitted with an external window with objective lens
315 which scans optical windows in the anterior nose 105 of the
microfluidic cartridge and collects raw optical signals. The
detector head has its own embedded microprocessor as described in
FIGS. 10 and 18. The programmable host controller also regulates
temperature in one or more heating elements in the heating module
and a set of solenoid valves and positive pressure and vacuum pump
reservoirs linked to the pneumatic interface. The instrument is
supplied with a display panel and touch panel for user
interactions. Power input is flexible, and is optionally supplied
by an AC adaptor, car adaptor, or from a rechargeable battery
mounted under the instrument. Also included are optional wireless
IO and digital IO ports.
[0081] FIG. 3B demonstrates conceptually that the floating stage
301, docking bay 103, detector head 311, and microfluidic cartridge
200 are mounted in the instrument chassis 106 at a defined angle
relative to the ground plane. Tilting the cartridge at an angle
from the ground plane improves venting during fluid loading and
minimizes air entrainment during wetting and mixing operations.
Bubble accumulation, which interferes with heat transfer and
optical interrogation of assay results, is avoided by this and
other innovations disclosed here. The inclined mounting plate 330
establishes the angle of the floating stage 301, cartridge 200, and
mechanical components of the clamping 800 and optical scanning 310
subassemblies. We also found that bubble accumulation interfered
with nucleic acid amplification, and was limited by the angular
mount of the stage.
[0082] As shown in FIG. 3B, the detector head is mounted in a
clamshell housing with mating half shells (312,313). The detector
head slides on lateral guide rails 308 and 309 and is under host
control of a stepper motor 307 with worm drive. The floating stage
chassis 301 is springedly mounted in a four-point suspension and
has no direct connection to the inclined mounting plate 330 until
clamped. The clamping mechanism is indicated here figuratively by
an arrow 320 and will be discussed in more detail below. All
mechanical components of the clamping mechanism are attached to the
inclined mounting plate so that the entire docking bay and clamping
mechanism are rotated at a fixed angle theta from the ground
plane.
[0083] One of the two scanning guiderails 308 is readily visible in
this view, and is supported at either end by the floating stage
chassis or tray 301. The docking bay (103) is indicated by a dotted
line and marks the opening for insertion of the nose of the
microfluidic cartridge under the detector head 311, which scans
from side to side as shown (double arrow). The pneumatic interface
port 350, shown here as a raised platform under the docking bay,
obscures the position of the heating module 340 and heating blocks
341-344 immediately inferior and in line with the docking bay.
Power conditioning, AC adaptors and battery storage functions are
mounted beneath the inclined mounting plate 330 above the underside
of the 360, which is designed to rest on a flat surface.
[0084] FIGS. 4A and 4B are front and rear interior CAD views from
above, showing the suspension-mounted stage with floating chassis
301 with optical bench and detector head 311, and docking bay
occupied by a microfluidic cartridge 200. The floating stage is
suspended from a saddle shaped support, docking saddle 400, which
is rigidly bolted to the inclined mounting plate 330. Also shown is
a gear rack 401 that provides clamping pressure, as will be
described in more detail below. In FIG. 4B, the guiderails
(308,309) and stepper motor 307 of the optical bench are readily
recognized. The inclined mounting plate 330 is populated with
pumps, vacuum and pressure storage tanks, solenoids and pneumatic
control circuitry (not shown), but will not be described in detail
here.
[0085] FIG. 5A is a front interior perspective view from below the
docking bay, showing the underside heating module 340 and cooling
fan 345. It can be seen that on insertion of the microfluidic
cartridge 200 into the docking bay, the heating module 340 is
brought into alignment with the underside of the cartridge.
[0086] FIG. 5B is a detail of the heating module 340 with heating
block elements ("thermal elements" 341,342, 343,344) and mirror
face (500). The superior aspect of the heating module consists of
one or more heating blocks, each of which forms a thermal interface
with a defined zone on the underside of the microfluidic cartridge
for proper operation of the biochemical or molecular biological
reactions that occur in the enclosed channels and chambers of the
cartridge during the assay. These reactions can be as simple as
immunobinding or hybridization, or as complex as nucleic acid
amplification or enzymatic dehydrogenation coupled to the formation
or consumption of nicotinamide adenine dinucleotide and adenosine
triphosphate, or cascading clotting factors, and generally require
relatively stringent temperature control for optimal reactivity and
specificity. The heating blocks (341,342,343,344, although the
invention is not limited to this configuration) may be
spring-mounted and are urged upward in opposition to the downward
pressure of the clamping mechanism so as to establish high thermal
diffusivity contact zone for heat transfer. Each heating block is
in thermal contact with a resistive (Coulombic) heating element,
generally by means of a compliant thermal pad for good thermal
conductivity. Each heating block contacts a thermal window in the
microfluidic cartridge. Each window is generally a thin layer of a
flexible plastic film, may be less than 3 mils in thickness, and
most commonly of a compliant transparent material such as
polyethylene terephthalate (Mylar.RTM.), although optionally of a
cyclic polyolefin or polyimide with good optical transparency,
while not limited thereto (see U.S. Pat. No. 7,416,892, which is
co-assigned), and also having good thermal conductivity. Thus in
one embodiment the invention is a thermo-optical interface for
reflective transillumination of a detection chamber in a
microfluidic card while controlling or modulating the temperature
of a liquid sample in the detection chamber. This feature is of
benefit for performing a reaction or reactions associated with an
analyte while monitoring an optical signal associated with the
analyte. In one exemplary application, thermal melt curves are used
to verify FRET hybridization results.
[0087] Also shown is the fan housing 345, which is used to
dissipate heat from heat sinks below the heating blocks and in PID
control of temperature in the blocks in combination with resistance
heating circuits (not shown).
[0088] Heating block 341 in this case is modified by fabrication
with a polished chromium mirror face 500 on the upper aspect which
contacts and aligns with thermo-optical windows in the microfluidic
card 200 during the assay. The thermo-optical window in this case
corresponds to a detection chamber enclosed in the cartridge body.
The mirror face reflects light from the detector head 311, which
scanningly transilluminates the cartridge, back into the objective
lens 315, and also reflects any fluorescent emission from the
cartridge detection chambers back into the detector head and from
there to the detection sensor, which is typically a photodiode, as
will be discussed in more detail below. Heating block 341 in this
example differs from the other heating blocks, and is generally
machined from aluminum, then polished and coated with an underlayer
of copper under nickel before application of the chromium mirror
face. Electropolishing and/or buffing may be used to form a highly
reflective optical finish on the chrome surface. The optically flat
superior surface of the block aids in heat transfer and improves
sensitivity of fluorescence assays. Happily, the use of the mirror
permits simultaneous heating and optical interrogation of the fluid
contents of the detection chamber, as is useful for example in
optically assaying melting curves.
[0089] Other heating zones may be modified similarly to permit
optical monitoring with simultaneous temperature control or
modulation. The configuration of heating zones and mirrors may be
modified or adapted for particular assay/cartridge requirements,
and is not limited to the configuration shown here.
[0090] FIG. 5B also shows pneumatic interface port 350, here with
ten outlets, each independently ported to a source of positive or
negative pressure from the pneumatic distribution manifold of the
host instrument and independently under the control of a
programmable host controller. These outlets interface and seal to
mated inlets in the underside of the microfluidic card, and a timed
pattern of intercommunicating pneumatic pressure, vacuum and
pressure pulses are routed through the pneumatic interface to drive
and control the assay in the cartridge.
[0091] FIGS. 6A and 6B are perspective views of the floating stage
subassembly 300 with insertable cartridge in place in the docking
bay 103. For clarity, the docking saddle 400 and accessory mounting
elements have been removed. It can be seen in FIG. 6A how the
inferior surface of the microfluidic cartridge 200 is shaped to be
contacted with the mated superior surfaces of the heating module
340 of FIG. 5B. The cartridge is secured and supported under the
floating stage 301 by two attached lateral flanges 609 and 610,
which are bolted in place and guide insertion.
[0092] In FIG. 6B, four vertical posts (601,602,603,604) forming
the male elements of the four-point suspension 600 are apparent on
the anterior section of the floating stage 300. These posts are
fitted with coil springs (603a in FIG. 7) and inserted into
cylindrical suspension housings (605, 606,607,608 in FIG. 7) formed
with in the docking saddle 400. They serve to suspend the floating
stage 300 and optical scanning assembly as will be described in
more detail in the next figure, FIG. 7. The entire optics bench and
docking bay subassembly shown in FIGS. 6A and 6B floats on this
suspension and is rigidly brought into contact with the rest of the
instrument only on downward action of the clamping mechanism as
will be shown in FIGS. 8 and 9.
[0093] FIG. 7 is an exploded view of the floating stage 300 with
detector head 311 docking bay 103 suspended from the underside of
the docking saddle 400. The floating stage is fitted with a four
point spring suspension with coil springs (replicates of 603a) on
each of the four supporting posts (601,602,603,604). Each post is
received in a mated suspension housing (605, 606,607,608) of the
docking saddle. The microfluidic cartridge 200 is not shown, but it
can be seen in this view that the docking bay is configured for
receiving the nose of the cartridge at the projecting nose 515 of
the docking bay, so that the cartridge rests on lower lateral
flanges (609,610). Alignment pins (616,617) ensure that the docking
bay seats true when pressed down against the heating module 340.
The posterior section of the floating stage, which contains the
guiderails (308,309) and detector head 311 for fluorescence
scanning, is free of any support other then at the docking saddle
and is cantilevered from the docking saddle during operation. The
role of guiderails (308,309) in supporting motion of the scanning
detector head 311 is apparent in this view.
[0094] The docking saddle is provided with brackets 712 and 713 for
attaching the clamping mechanism 800 as will be discussed below,
and bar code readers as are useful in automated operation. Linker
arm 710 with slot 711 is engaged by the clamping gear mechanism as
discussed below and the floating stage 300 raised or lowered as a
single assembly. Docking saddle 400 and linker arm 710 also
operatively fix the floating stage at the theta angle of the
inclined mounting plate.
[0095] FIG. 8A is a frontal view of the clamping mechanism. The
bridging shape of the docking saddle 400 is seen to rest above the
anterior nose 515 of the floating docking bay 103 Immediately under
the docking bay mouth is the pneumatic interface port 350, visible
between lateral flanges 609 and 610 forming the channel for
receiving the microfluidic cartridge 200. During docking of the
loaded cartridge, the function of the clamping mechanism is to urge
the floating stage and spring-mounted chassis 301 with inserted
cartridge downward onto the pneumatic interface port and heating
module as described above.
[0096] Docking saddle 400 is bolted on inclined mounting plate 330,
and floating stage chassis 301 is suspended on the four-point
suspension 600. The suspension springs apply a downward pressure on
the floating stage, which is opposed by the suspending action of
clamping assembly 800 in the raised position. When clamping the
cartridge, clamp gear piece 804 is driven by worm gear 801 and worm
gear motor 802, driving travelling axle 803 in a downward arc. The
axle pin 803a is attached to a cam block (901, visible in FIG. 9B).
Linker arm (710, visible in FIGS. 7 and 9A) follows the cam-action
of the clamping gear 401 and slider block 901 up or down on the
four-point suspension. When disengaging the cartridge, the action
is reversed. Worm gear motor 802 is run clockwise, raising pin 803a
and cam block 901, thus lifting the linker arm 710, which is part
of the stage chassis assembly 301, and then reversed (double
arrow). When the stage chassis is in the uppermost resting
position, the microfluidic cartridge can be removed from the
instrument. Mechanical fiducials and alignment pins are used to
register the cartridge in the instrument docking bay during the
assay.
[0097] FIG. 8B is a detailed view of the front side of the clamp
gear piece 804 and worm drive gear 801, showing also cam follower
surfaces 805 and 806 on the anterior edge of the gear member that
are used by pressure switches to monitor the position of the gear
and actuate coordinated mechanical functions by the host
controller. For simplicity, the pressure switches are not shown.
Axle 810 is the center of rotation for the piece and rotates on pin
810a. Axle 803 travels during rotation of the gear piece, driving a
slider block which engages with the stage chassis as shown in the
following figure.
[0098] FIG. 9B is a detailed view of the rear side of the clamp
gear piece 804 and worm drive gear 801 showing the central axle
810a and acentric cam block axle 803a and cam slider block
(901).
[0099] FIGS. 9A and 9B are mechanical drawings showing the action
of the clamping mechanism assembly 800. The purpose of the clamp
gear-driven cam is to raise and lower the floating stage 301. The
clamping gear piece 804 pivots on stationary axle 810, so that
travelling axle 803 scribes an arc upward or downward, propelling
the linker arm 710 up or down vertically (double arrow). Slider
block 901 captive on pin 803a slides left to right in a slot 711 in
the linker arm (710, see FIG. 7) to accommodate the lateral vector
of the motion of the clamp gear while raising or lowering the
floating stage 300. The upward movement of the floating stage is
opposed by springs 603a as shown in the preceding figures.
[0100] The mechanism illustrated here is not limiting, insofar as
the invention can be realized in alternate ways, for example by
clamping up from the bottom rather than down from the top, or by
magnetically clamping rather than mechanically clamping. Other
spring means may be selected from coil spring, leaf spring, torsion
spring, helical spring, and alternatives such as pneumatic
canisters (e.g. gas springs) and elastomeric materials or other
equivalent means known in the art.
[0101] FIG. 10 is a block diagram providing an overview of the
functional units, software and firmware of the apparatus. As
described above and presented schematically here, a floating stage
(1000, dotted line) within the instrument supports a docking bay
1001 for receiving a microfluidic cartridge and is provided with a
scanning detector head 1002. The scanning detector head contains
subassemblies for providing excitation light and sensors for
detecting, amplifying and processing fluorescent emission signals
under control of an embedded microprocessor. Interfacing with the
floating stage are a heating module with separately controllable
heating zones under control of the host controller, a pneumatics
interface connected to pneumatic servos mounted on the base plate
330, which also serves as a pneumatic distribution manifold, a wire
harness connecting the stepper motor and the host controller, and
wiring harnesses for the clamp motor, and related sensors,
including pressure switches for measuring the position of the clamp
and the microfluidic cartridge, a barcode reader, and temperature
monitors. Optionally, a tilt gauge is also supplied to measure
instrument orientation before controls are activated.
[0102] Power is supplied to all systems by a rechargeable battery,
or by direct connection to an AC converter or to a DC source such
as an automobile.
[0103] The host controller 1003 is mounted on a motherboard which
also contains a touch pad panel for operation of the instrument and
an LCD display panel. The instrument may transmit data to an
outside network or device via a variety of digital serial I/O
links, including a wireless networking card. A special digital
junction is provided for service access to the RAM registers and
programming, which is software encoded in solid state ROM.
[0104] General instructions for operation of the instrument, such
as the sequence of pneumatic pulses and valve logic required to
operate a particular microfluidic card having the capability to
diagnose a particular disease or pathology from a liquid sample,
are provided by programmable software in the host instrument. If
for example, the barcode reader detects a particular microfluidic
cartridge, the device is programmed to perform a particular assay
and interpret and display the results in a designated format.
However, the operation of the optics, including modulation of
source intensity, signal amplification and filtering, is controlled
by an embedded microprocessor on the sensor PCB within the detector
head. Thus analog operations that are highly sensitive to noise are
shielded in the detector head from the more noisy environment of
the host instrument, and transmission of analog signals from
detector head to host A/D converters is completely avoided. This
unconventional separation of functions has happily proved highly
advantageous in reducing noise susceptibility of the instrument, as
is needed for full portability and field operation.
[0105] FIGS. 11A and 11B are perspective views of an insertable
microfluidic cartridge for use with the apparatus of the invention.
The cartridge shown here consists of a housing 1102 and coverplate
1103 with internal workings. Port 1104 is for receiving a liquid
sample and anterior nose 1105 is for inserting into the docking bay
of the host instrument. Housing cutout 1101 is for exposing optical
windows formed in internal subcomponents of the cartridge, as shown
in FIG. 12. Gasket 1106 is for sealedly interfacing with the
pneumatic interface port 350 of the host instrument.
[0106] FIG. 12 is an exploded view showing the internal components
of a microfluidic cartridge of FIG. 11. While not limiting in scope
by this single embodiment, this particular cartridge 1100 is
designed for PCR with FRET or molecular beacon detection. An
optical window 1101 on the nose of the housing 1102 inserts into
the host instrument and aligns the optical windows of the FRET or
molecular beacon detection chambers 1201 of the microfluidic
inboard circuit card 1200 with the optical path scanned by the
detector head. Added microfluidic processing related to sample
preparation is supplied on outboard card 1204. All liquid reagents
are enclosed in sealed frangible pouches 1206 and are dispensed
when needed under pneumatic control. Other reagents are provided
on-card in dry form. Fluid waste is sequestered in an adsorbent
batting 1207 that is sealed in place under the plastic coverplate
1103. The details are beyond the present scope, but microfluidic
circuit 1200 with internal microfluidic channels and wells for
thermocycling and amplifying a nucleic acid target includes
detection chambers 1201 for FRET detection of any resultant
amplicon. The cartridge as shown is a disposable cartridge. Gasket
material 1106 serves as a single-use sealing gasket between
pneumatic control ports on the microfluidic cartridge and a
corresponding pneumatic control manifold and interface 350 on the
inclined mounting plate 330.
[0107] FIG. 13 is a perspective view of a dual channel detector
head 1300 with two optical channels and electronically isolated
circuit boards (1301,1302) for excitation and for emissions
detection respectively. In this view, the upper half of the housing
1303 is removed in order to show the internal components of the
detector head. The dual channels are marked by objective lenses
(1310, 1330) and optic pathways A and B (open arrows). The SMD LED
excitation light sources (1311, 1331) are mounted on a source LED
printed circuit board (1301), which is connected at right angles to
sensor PCB (1302) via an edge-type resistive pin-connector (1304).
The photodetection components are mounted on the sensor PCB (1302).
A Faraday cage element (1306) is used to shield the photodiodes
(1317) and (1337) and surrounding high gain amplification
circuitry.
[0108] Fluorescent excitation is provided in the target channel
(Arrow A) by a surface mounted LED (1331) which is chosen to match
the excitation spectrum of the target fluorophore. Source LED
(1331) emits a divergent light beam, and the radiated light beam is
then collimated by source excitation lens (1332). Source lens
(1332) is a planoconvex lens having its flat surface facing the
LED. The collimated light beam may then be passed through an
excitation bandpass filter (1333), the purpose of which is further
explained in the description associated with FIG. 20. The
collimated, filtered excitation light beam is then reflected from a
dichroic mirror element or beamsplitter (1334), which is installed
at a forty-five degree angle to the incident beam, and is passed
through a planoconvex objective lens (1330) and through an external
window in the detector housing (Arrow A). After passing lens 1330,
the excitation light is focused through a detection chamber (not
shown, see FIGS. 14-17) embedded in a microfluidic cartridge, which
contains a sample liquid with any target fluorophore. The path
length of the excitation light through the sample liquid is doubled
by use of a back mirror behind the microfluidic cartridge. The
target fluorophore is excited by the incident light beam. The
emission of the fluorophore is generally at a longer wavelength
than the excitation wavelength and is shifted by an amount equal to
the Stokes shift of the target fluorophore.
[0109] A portion of the returning emission from the target
fluorophore in the detection chamber is collected by planoconvex
sampling lens 1330 and is collimated before striking dichroic
mirror 1334. Optionally, a Fresnel lens may be use to further
reduce the working distance between the lens and the sample so as
to optimize collection of emitted light, which is further enhanced
by back mirror mounted on a heating block behind the detection
chamber. Because dichroic beamsplitter 1334 has a wavelength cutoff
between the excitation and emission wavelength, the dichroic mirror
1334 now acts as a pass-band beam splitter for the emitted
fluorescent light beam and a stopband filter for the excitation
light. It transmits the emitted fluorescent light while reflecting
reflected excitation light and any ambient light entering the light
path through the objective lens window. Emitted light passing
through the dichroic beamsplitter 1334 then passes through an
emission filter 1335, the purpose of which is further explained in
the description associated with FIG. 20. Light exiting emission
filter 1335 then passes through planoconvex sensor lens 1336, where
it is focused onto the surface of a photo-sensor 1337 which is
surface mounted to PCB 1302 and is protected from electrical noise
by Faraday cage 1306.
[0110] The above described optical pathways are repeated in a
second (control) channel having control excitation LED 1311,
planoconvex excitation lens 1312, excitation filter 1313, dichroic
beamsplitter 1314, objective lens 1310, control emission filter
1315, planoconvex sensor lens 1316, and control photodiode 1317.
Outputs from both photodiodes are amplified by three-stage
trans-impedance amplifiers built into the board next to the
photodiodes and grounded to an embedded microprocessor on the
sensor PCB via carefully shielded pins from the amplifiers.
[0111] In one embodiment, as exemplified by the use of fluorescein
and Texas Red as fluorophores, excitation LED 1331 is a 470 nm LED
with band-pass excitation filter 1333 for delivering essentially
monochromatic light of 485.+-.12 nm used for the target channel and
a 590 nm LED 1311 with band pass filter 1313 was used for the
control channel. The excitation LEDs are modulated or "strobed" on
and off using a strobe rate of 130 Hz so as to filter AC
power-related noise at 50 or 60 Hz and at harmonic frequencies
associated with fluorescent overhead illumination, also filtering
phantom signal related to stray ambient light and electrical noise
that may be present at 30 or 60 Hz. Local feedback sensors are used
to monitor and stabilize source LED output intensity. Detection
monitoring of fluorophore emission is coordinated with movement on
rails of the detector head under power of a stepper motor
controlled by a host controller. An embedded microprocessor and
associated circuitry in the detector head is provided with RAM
memory, ROM memory, an A-D converter, a three-stage trans-impedance
amplifier, and signal processing and command sequence firmware to
handle these functions.
[0112] Each of the photo-sensors 1317 and 1337 are mounted on a
common PCB 1302. The output signal legs from each of these
photo-sensors are connected directly to the first stage of
respective tri-stage trans-impedance amplifiers (not shown). PCB
1302 makes extensive use of hardware noise-reduction components, in
particular an embedded ground plane and a Faraday Cage 1306 to
minimize the unwanted effects of any RF or electromagnetic
interference on the input signals. The combination of the use of
these hardware noise-reduction elements with a digital signal
processing (DSP) method, leads to a detector design which is
essentially immune from the effects of unwanted noise.
[0113] FIGS. 14A and 14B are schematic views of the internal
optical components of a fluorescence detector head 1300, showing
the external optical interface with optical windows in a
microfluidic cartridge 1402 and back mirror 1400 mounted behind the
cartridge on the surface of a heating block 1410 that is used to
control or ramp the temperature in detection chambers enclosed in
the cartridge. Unconventionally, multiple independent optic
pathways or "channels" are formed in a single detection head and
share electronic PCBs and downstream signal processing circuitry,
but excitation optics are mounted on one circuit board and
detection optics on another to reduce noise interference. The two
boards are electrically coupled by a corner mounted pin junction
1304 and are electronically isolated.
[0114] Shown in FIG. 14A is the optical transition for the
excitation of a fluorophore in a detection well (1403a or 1403b)
embedded within a microfluidic cartridge 1402. The head is a
scanning head and moves across microfluidic cartridge 1402 (double
arrow). Light from excitation LED 1331 on PCB 1301 is collimated by
lens 1332 and made essentially monochromatic by band-pass filter
1333. Any fluorophore or fluorophores in detection well 1403a
(whether the control or the target fluorophore) are excited by
incident light 1420 focused on the sample by objective lens 1330.
In FIG. 14B, the emission of the fluorophore(s) is collected by
objective lens 1330 and transmitted to sensor 1337 after passing
through dichroic beamsplitter 1334, emission filter 1335 and sensor
lens 1336. Sensor 1337 is in direct electrical contact with the
base of a high gain transistor that amplifies the output signal and
is shielded in a Faraday cage 1306. The emitted fluorescent light
is generally at a longer wavelength according to the Stokes shift
of the fluorophore, enabling the emitted light to pass through
dichroic bandpass mirror 1334 and emission band-pass filter 1335
without losses. Mirror face 1400 is used to increase the amount of
excitation light on the target, doubling the excitation path
length, and to improve emission collection efficiency. The light
returned from sample chamber 1403a to objective lens 1330 is thus a
mixture of emitted and reflected fluorescence 1421 and reflected
excitation light 1420. Light traps (not shown) are provided to
capture stray reflections. Reflected light 1420 does not pass
dichroic mirror 1334 and is returned to the source, and does not
interfere with the measurement of emission intensity at sensor
1337. The optic elements of a single channel, including excitation
source, source collimating lens, excitation filter, dichroic
mirror, objective lens, excitation filter, sensor lens, and
detector with amplifier make up an optics module having an
essentially monochromatic source wavelength and a highly specific
sensor for detecting fluorescence at a particular wavelength
characteristic of the target (or control) fluorophore. One optics
module or channel may be used for an assay target, the other module
for a control channel. Tandem mounted optics channels may be used
to collect data on a plurality of fluorophores, where electrical
processing is shared by common embedded microprocessor before
transmission to the host instrument. Optionally additional channels
may be use. Each channel shares the two PCB but has separate
optics.
[0115] The microfluidic cartridge 1402 is movable (double arrow)
relative to detector head 1300 and motorization of the detector
head or cartridge tray or mounting chassis permits scanning: a
transect across cartridge 1402 permits measurements to be made on
sample chambers 1403a and 1403b, for example. By using multiple
detection optics modules mounted side-by-side in a detector head,
the sample chambers can be scanned for multiple fluorophores in
series.
[0116] According to one embodiment, the excitation electronics are
mounted on a printed circuit board (1301) and the detection
electronics are mounted on a second PCB (1302). An edge-connector
1304 electrically joins the boards. Faraday cage 1306 protects the
sensor and associated high gain amplifier from stray
electromagnetic noise. Mirror 1400 is fabricated on the upper
surface of heating block 1410, which also functions in heat
transfer and controls the temperature of the sample fluid during
the assay. The temperature of heating block 1410 can be ramped, for
example as in a FRET melt determination under control of the host
controller.
[0117] FIG. 15 is a representation of a planoconvex objective lens
1500 and excitation cone 1501 relative to the cartridge detection
chamber 1403 and heating block 1410 with mirror face 1400. The
excitation cone is formed by diverging rays illuminating the lens
so that the distance L2' is greater than the native focal length L2
of the lens. By convention, the native back focal length L2 of the
lens is determined using collimated light. Shifting the focal
position is termed "decoupling".
[0118] Interposed between the lens 1500 and the mirror face 1400 is
a microfluidic cartridge 200 with detection chamber 1403. The
detection chamber is bounded by an upper optical window 1502 and a
lower thermo-optical window 1503. In operation, the intervening
volume is taken up by a liquid sample, shown here with two
entrained bubbles 1505. The focal cone is seen to reflect from the
mirror face, forming a real image (1510, solid rays) of the source
in the detection chamber and a virtual image (1511, dotted rays) of
the source below the mirror face. The back focal position L2' is
thus generally equal to or greater than the distance between the
lens and the mirror. Excitation light striking the mirror is
reflected as a focused beam in the fluid volume of the detection
chamber, thus doubling the length of the light path of the
excitation light through the sample and increasing the excitation
fluorescence yield. The back focal position L2' is not equal to the
back focal length L2 of lens 1500; the two are decoupled, generally
by illuminating the lens with a divergent beam from the source.
[0119] FIG. 16 is a representation of the objective lens of FIG. 15
in emission collection mode. Shown are primary and reflected
fluorescent emissions (solid and dotted lines from a real image
1510 and a virtual image 1511). Again shown are bubbles in the
chamber 1403. Rays striking the planar back surface of the lens
will be collimated and transmitted to a detector. The quantity of
fluorescence signal captured depends on the angular and numerical
apertures of the objective lens 1500. The native back focal length
of the lens 1500 is L2. The back focal length and back focal
position of the lens (L2 versus L2') can be manipulated or
"decoupled" by repositioning the source as shown below.
[0120] FIG. 17 is a schematic representation of an optical pathway
1700 with decoupled excitation and emission optics. In this figure,
excitation rays are shown as solid lines and emission rays are
shown as dotted lines. Aspects of integration of the emission and
detection elements into an integrated optical system are discussed.
Here the light source 1701, may be an LED as shown, an SMD LED
without lens housing and reflector ring, an SLED, a pumping laser
diode, a ridge-waveguide (Fabry Perot) laser diode, a tuneable
laser, and so forth, such source of illumination preferably having
a narrow bandwidth and serving as a directed source of collimated
light. LEDs of various narrow bandwidths are available, for example
with peak emission at 630 nm (red), 470 nm (blue), 525 nm (green),
601 nm (orange), 588 nm (yellow) and so forth. While LEDs may also
be used if desired, but generally the LED output is matched with
the fluorophore of the target in the assay and an optional
excitation filter (not shown) may be used to sharpen the bandpass
as required. Light output of light source 1701 is transmitted by
source lens 1702, shown here as a planoconvex lens, although molded
aspheres may also be used, and optionally passed through a
excitation filter (not shown) before striking dichroic mirror
element 1704, where the excitation beam is redirected to objective
lens 1705. The excitation cone striking sample fluid volume 1720 in
microfluidic cartridge 1402 is shown with solid lines.
Unconventionally, the focal point of the excitation cone has been
projected past the sample chamber and back mirror 1400 by moving
the source 1701 closer to the source lens 1702 (ie. shortening
distance L1' in order to increase distance L2', where distance L1
would be the native back focal length of lens 1702). As distance
L1' is shortened, the source rays striking the objective lens are
caused to diverge, thus increasing distance L2'. The native back
focal length of objective lens 1705 is L2 and emissions from a
fluorophore in the sample chamber and reflected rays from the
mirror are collected as a virtual image of the fluorophore that
enter the objective lens in a cone having a focal point decoupled
from the focal point of the excitation light (which is focused
behind the mirror). The reflected virtual image of a population of
fluorophores in the chamber is bounded by dotted lines crossing the
heating block 1410. Fluorescent emissions within the focal length
L2 are effectively collimated by the objective lens; are
transmitted through dichroic mirror 1704, bandpass filter 1706, and
are then focused by sensor lens 1707 onto sensor 1708, which is
generally mounted on a PCB or other solid support 1709. Lens 1707
has a back focal length L3 that generally is equal to back focal
length L2 of objective lens 1705. However, a larger lens 1707 may
be used to better utilize the surface area of the sensor 1707,
which is for example a photodiode or CCD chip. Optimization of
signal may require independent adjustment of each lens according to
these principles.
[0121] The excitation light emerging from objective lens 1705 and
the emission light entering objective lens 1705 are operably
decoupled in different focal cones (L2' versus L2 respectively). A
distance separates the actual focal plane of the excitation light
and the native focal length of the objective lens, which can
capture light in a broad plane of origin of the fluorescent
emissions when excited using a mirror and an extended focal
position of the excitation cone 1501. This phenomenon, termed
"decoupling" was found to increase capture of fluorescent emissions
when a back mirror is used, and controverts earlier teachings in
favor of the confocal approach of the prior art.
[0122] While the teachings of the prior art strongly support making
the excitation and emission confocal, there is in fact an
hithertofor unseen advantage in decoupling the focus of the source
from the emission cone and using a back mirror 1400. Emitted light
arises from a greater area and depth throughout the sample
cartridge, thus overcoming any lack of signal from dead spots or
inhomogeneities as would be due to small bubbles, unmixed areas, or
quenched probe. Greater reliability is achieved at the expense of
some selectivity in the excitation at the point of focus. This is a
technological advance in the art.
[0123] To summarize, generally, L2' may be greater than L2 and L3.
L1 advantageously may be configured so that the cone of excitation
light falls behind the sample chamber 1403 and most preferentially
close to or behind the back mirror 1400. In a preferred embodiment,
the focal point of the excitation cone falls on or behind the back
mirror. The objective lens is configured, generally, so that
emitted light is efficiently collected and collimated for
projection onto the detection sensor by a symmetrical cone of
emitted light from sensor lens 1707 (i.e. L2=L3).
[0124] Accordingly, in another embodiment the apparatus of the
invention employs lenses configured so that excitation optics and
the emission optics are decoupled. In a first embodiment of this
apparatus, the light source is positioned at a distance L1' from
the source lens, where L1'<L1, whereby the excitation optics and
emission optics are decoupled by transitioning the excitation cone
to a focal position L2' at or behind the mirror face, such that
L2'>L2. In a second embodiment, the source lens is configured to
form a diverging beam of light incident on the objective lens,
thereby positioning the excitation cone at a focal position L2',
whereby L2'>L2.
[0125] Advantageously, L2 is configured to be symmetrical to L3
(i.e., L2=L3), so that the operation of detector 1708 is optimized
and robust. The sensor photodiode is preferentially configured to
be large enough with reference to the cone of focus of lenses 1705
and 1707 to accommodate some degree of misalignment without loss of
assay validity.
[0126] Performance is improved not only in assays where
differentiation of a positive or negative assay result is required,
but also in assays were some level of quantitation is required, as
for example schizont or merozoite copy number in the case of
Plasmodium falciparum. It should be recalled that the original
purpose of confocal optics, as articulated by its inventor, Minsky
in 1957 (U.S. Pat. No. 3,013,467), was to create a three-dimension
image of a thick solid specimen by rastoring a focal point of
excitation across and through the specimen (xyz axis rastoring)
while monitoring emission only from the area of the specimen where
the excitation cone is focused at any given time. In contrast, in a
fluid mixing specimen that is generally homogeneous, an opposite
effect is desired, that of measuring the cumulative fluorescence of
the entire specimen, and suppressing any localized variance in that
fluorescence. Thus by reformulating the problem, we have been able
to design a novel optical system with back mirror, with decoupling
of the focal plane of excitation and emission, with the happy
result that fluorescence detection is more sensitive and robust in
the presence of occasional optical interferences.
[0127] FIG. 18 is a block diagram of the detector head electronics
and optics used for controlling the fluorescent excitation and for
receiving, processing, and digitally communicating fluorescence
emission signals to the host instrument. Optical channels are again
identified by open arrows A and B. Channel A is taken as a control
channel and channel B as a target analyte channel, but the roles
are interchangeable. The electronic functional blocks in each
channel, source excitation circuits (1811,1831) and sensor circuits
(1817,1837) are identical and are driven by detection optics
control circuitry of the embedded microprocessor 1841, which has a
dedicated on-board instruction set as firmware, typically as a
socketed EEPROM chip 1842. The circuit board 1801 for the source
LEDs can support multiple LEDs, and the circuit board 1802 for the
sensor circuit can support multiple photodiodes. Each photodiode is
intimately associated with a multiple stage high gain amplifier
(1818,1838) and the two circuit boards are electronically isolated
with separate grounds.
[0128] The stepper motor and worm drive module controls scanning of
the detector head as directed by the host controller. Thus the
detector head operates as a self-contained optics and signal
analysis module while scanning under control of the host
controller. The host also handles the data tabulation and display
functional block (1820), including preparation of test result
reports and any I/O functions.
[0129] Within the detector head, each of the LEDs associated with
source excitation circuits (1811, 1831) are modulated by a square
wave at a frequency of 130 Hz. The reason for this modulation is
related to noise reduction measures from the following potential
noise sources: [0130] 1. 50/60 Hz mains [0131] 2. 100/120 Hz second
mains harmonic from the fluorescent lights. [0132] 3. Third and
higher harmonics of 50/60 Hz. [0133] 4. Differential frequencies
(rumble) of 130 Hz and all the above of the [0134] 5. Photodiode
sensor, first stage feedback resistor and amplifier electrical
noise. This noise is wide band white noise.
[0135] In order to retrieve the useful signal from the noisy
source, the following methods are employed: [0136] 1. Fast sampling
and averaging of taken data in order to avoid aliasing and at the
same time limit noise bandwidth. [0137] 2. Modulation of the LED
light at 130 Hz and correlation with the detected fluorescence
signal at 130 Hz in order to reject all uncorrelated components.
The 130 Hz modulating frequency was selected to provide at least 10
Hz difference from the 50/60 Hz mains and its harmonics (mainly
100, 120, 150 and 180 Hz). [0138] 3. Further filtering and
processing of the correlated data to eliminate electromagnetic
noise from mains power supplies of either 50 Hz or 60 Hz or
harmonics thereof.
[0139] The advantage of using the embedded microprocessor 1841 with
the fluorescence detector is that a proprietary method of digital
signal processing (DSP) may be programmed into the firmware of an
embedded microprocessor in the detector to eliminate noise before
transferring a digitized signal to the host controller.
[0140] The excitation LEDs are modulated at 130 Hz with 100% AM
modulation depth (on/off). The modulating frequency was selected to
provide sufficient separation from 50/60 Hz and its harmonics (50,
60, 100, 120, 150, 180 Hz, et seq.). Any intermodulation product
frequency is at least 10 Hz and can be filtered out during signal
processing. The LEDs are driven by FAN5612 LED-drivers. Each driver
is capable of sinking current up to 120 mA (40 mA on each of three
outputs) at the required frequency.
[0141] The host controller 1800 is responsible for the operator
interface, including display of results, and for operation of
mechanical and pneumatic functions required to perform an
on-cartridge assay and detector head scan. The embedded
microprocessor 1841 in the detector head is responsible for
controlling the excitation and detection circuits, which are
electronically isolated on separate PCB boards (1801,1802) and for
signal filtering, and has its own instruction set which is
programmable as a socketed EEPROM chip 1842 on the sensor board.
The clock frequency of the embedded detector head microprocessor is
used to strobe the excitation LEDs and to synchronize pulse
collection in the sensor diodes. A separate clock in the host
controller is used to drive the stepper motor during scanning.
Multiple excitation and detection optics can be housed in a single
detector head so that signal excitation and fluorescent emission
detection can be multiplexed in the embedded processor. We have
found that on-board packaging of signal processing achieves a low
noise environment with improved signal-to-noise ratio and
sensitivity by minimizing signal pathlengths and permitting
effective use of faraday shielding where necessary, such as around
the sensor diode leads and at the junction between the excitation
and sensor circuit boards.
[0142] Isolation of dual channels proved an advantageous means to
implement multiplex assays where target fluorophore and internal
control fluorophore are mixed in a common liquid sample. By
separating target and control channel optics, crosstalk that could
lead to false positive tests or test rejection due to invalid
control results was eliminated.
[0143] The host controller also controls pneumatic valve logic and
pulse trains for operating diaphragm pumps in the microfluidic
cartridge during the assay, any resistive or Peltier-type heating
elements associated with thermal cycling of the sample, and
optionally may perform melt curves in the detection chamber. Other
optional components include fiducials for aligning detector heads
and a bar code reader for sensing information printed on the
insertable microfluidic cartridges.
[0144] The host controller program with program coding means is
designed to perform all the steps of a fluorescence assay process
and to transform and format a signal or other data from the
fluorescence detector into a machine-readable or graphical report
of significance to the user. The program can be integrated into the
fluorescence detector instrument as shown here or can be connected
to it through data lines or wireless interfaces as part of a
network, intranet or internet. Generally, a serial asynchronous
communications interface is provided for communication with the
host controller on the instrument motherboard or on an external
network.
[0145] Similarly, results, data, error codes, status updates, and
so forth can be sent via common electronic interfaces and data
lines such as USB, RS232 or FireWire and via a wireless
transmission system such as IR-transmission, Bluetooth, GSM, GPRS,
RSID, etc. Programming, reprogramming, calibration, and
recalibration as well as system diagnosis of the device is possible
via common electronic interfaces and data lines such as USB, RS232
or FireWire and via a wireless transmission system such as
IR-transmission, Bluetooth, GSM, GPRS, RSID, etc.
[0146] The apparatus can be configured for wavelengths in the UV,
visible region, and near infrared spectrum. For applications in
fluorescence mode, which is one of the preferred operating modes,
the device can be configured for specific fluorescence dyes with
excitation spectrum in the UV and visible spectrum and for emission
in the UV, visible and near infrared spectrum. While a red shift is
more typical, up-converting fluorophores may also be used. Today
available light sources filters and available dyes allow for
customizing in the range of 300 to 900 nm.
[0147] FIGS. 19A and 19B are representations of raw input and
digitized output showing digital removal of bubble interference. In
FIG. 19A, output (1900) from the photodiode after amplification is
represented. The level of noise is generally low, but signal
deteriorates at 1901 and 1902 due to the presence of two small
bubbles in the detection chamber (see FIG. 15), for illustration.
The processor applies a threshold to the output signal and scores
the signal "high" (i.e. a one) if the signal is above the threshold
and "low" (i.e. a zero) if the signal is below the threshold. Since
no signal output can be above the threshold except in the presence
of a fluorophore matched to the emission optics and filters, any
positive signal (1903) is a positive assay for the presence of
fluorophore. The threshold comparator can be adjusted based on
experience with clinical samples in the assay. Thus a "1-bit"
digital transformation of the scanning image data removes any
interference from bubbles. We have found surprisingly that when a
fluorophore is present but multiple bubbles fill the chamber, light
refracted around or through the bubbles will result in a positive
signal. The system is thus very error resistant and robust for
qualitative testing, such as is needed in diagnostic assays for
infectious disease. The signal comparator is a digital function of
the microprocessor and firmware embedded in the detector head and
is independent of host controller function.
[0148] Should the presence of a positive fluorescence signal
indicate a negative assay result, the system can be easily
configured to score the test that way. Thus the use of a 1-bit
digital transformation is a remarkably simple and effective
solution for the presence of bubbles in a microfluidic assay.
Because of the nature of the mixing and heating operations in
microfluidic assays, and the frequent use of surfactants, the
presence of bubbles is not uncommon. The systems described here use
a combination of physical methods (venting, tilting of the stage,
wetout under low dead volume conditions, accentric channels between
mixing chambers) and signal processing methods to achieve robust
assay performance such as is needed for reliable operation outside
the controlled environment of a clinical laboratory.
[0149] FIGS. 20A and 20B show the excitation and emission spectra
of a typical system of mixed fluorophores for the target and
control, here illustrated by fluorescein and Texas Red. Shown in
FIG. 20A, curve 2001 is the excitation spectrum for fluorescein
(dotted line); curve 2002 is the emission spectrum (solid). Shown
in FIG. 20B, curve 2003 is the excitation spectrum for Texas Red
(dotted line); curve 2004 is the corresponding emission spectrum
(solid). Here control is Channel B (FIG. 20B) and target is Channel
A (FIG. 20A), but the assignment is arbitrary.
[0150] Boxed area ExA indicates the wavelength band that is allowed
to pass through the target excitation bandpass filter 1333 (cf FIG.
13). Box EmA indicates the wavelength band that is allowed to pass
through the target emission bandpass filter 1335. Box ExB indicates
the wavelength band that is allowed to pass through the control
excitation bandpass filter 1313. Box EmB indicates the wavelength
band that is allowed to pass through the control emission bandpass
filter 1315. The boxes indicate the presence of stopbands on either
side of the maxima. It can be seen from FIGS. 20A and 20B that,
given the spectra for these two fluorophores and optical filters
having the passband characteristics configured as shown, the
following error conditions are corrected: [0151] a. Long wavelength
excitation light from the target LED 1331 (greater than the
wavelength of the LED peak excitation) cannot be mistakenly
confused for target fluorescent emission, due to these longer
wavelengths being cut off by the LED excitation filter 1333. [0152]
b. Long wavelength excitation light from the control LED 1311
(greater than the wavelength of the LED peak excitation) cannot be
mistakenly confused for control fluorescent emission, due to these
longer wavelengths being cut off by the LED excitation filter 1313.
[0153] c. Target fluorescent emissions cannot be inadvertently
triggered by the (excitation filtered) control LED 1311. This error
condition would otherwise lead to the control photosensor 1317
receiving unwanted contemporaneous signals from both the target and
control fluorophores. [0154] d. Control fluorescent emissions
cannot be inadvertently triggered by the (excitation filtered)
target LED 1331. This error condition would otherwise lead to the
target photosensor 1337 receiving unwanted contemporaneous signals
from both the target and control fluorophores.
[0155] FIG. 21A plots experimental results demonstrating
enhancement of fluorescent signal output by varying the height of
the objective lens above the mirror. Briefly, fluorescent beads
(Thermo Fisher Scientific, p/n G0300, Pittsburg, Pa.) were inserted
into a microfluidic detection chamber and the detection chamber
mounted under the objective lens 315 of the detector essentially as
shown in FIG. 3A. Using a digital micrometer, the height of the
detector head above the microfluidic cartridge was then varied to
construct the plot. In a second paired experiment, the mirrored
surface was removed. The solid line (2100) shows the effect of
varying the lens height in the presence of a mirror on the heating
block; the dotted line (2101) shows the effect of varying lens
height in the absence of a back mirror. As can be seen, the
presence of the mirror seems to shift the optimal emission maximum
behind the mirror plane (i.e. a composite of the real and virtual
fluorescent emissions captured in the lens).
[0156] FIG. 21B graphs the integrated output signal strength with
(2103) and without (2102) the back mirror. Output signal was found
to be optimized by focusing the excitation beam at a focal point
behind the mirror as shown in FIG. 15 and collecting emissions at a
shorter working distance as shown in FIG. 16.
[0157] In a second experiment, the detection chamber is filled with
a liquid sample containing a soluble fluorophore and pumped through
the chamber at constant rate so as to avoid quenching artifact. The
detector head height is then varied as before and the optimal
detector height determined In related experiments, the working
distance of the sensor lens and objective lens are also varied so
as to optimize sensitivity and limit of detection. We learn that
optimal configuration is not achieved when the objective lens is
centered in the detection chamber and the other lenses are made
confocal. When a mirror is used, decoupled optics achieve
advantageous results, a technological advance in the field.
[0158] FIG. 22 shows scanning data collected for a molecular beacon
hybridized to an amplicon. The scanning axis transects detection
wells (2200,2201) representing positive and negative test
conditions respectively, and it can be seen that signal is limited
to the detection wells. In the figure, the sample is scanned
repetitively as the temperature in the detection chamber is
systematically varied. The scans are overlaid in the plot to
illustrate the spatial resolution of the data. Fluorescence scans
for 35.degree. C., 65.degree. C., 70.degree. C., 75.degree. C. and
80.degree. C. test conditions are marked. Test plots at 40, 45, 50,
55, and 60.degree. C., and the 85 and 90.degree. C. plots were not
well differentiated, as expected, and are not individually marked.
It can be seen that fluorescent signal is a function of
temperature. Fluorescence quenching is observed to increase as the
double stranded probe-target is melted, ie. signal is greatest at
35.degree. C. and is essentially not present at 80.degree. C. In
FIG. 23A, the data is plotted for signal versus temperature for the
positive (2301, solid line) and negative (2302, dotted line) test
conditions. In FIG. 23B, a first derivative is plotted, indicating
a FRET melt temperature of about 70.degree. C.
EXAMPLE I
[0159] In this example, the apparatus of the invention is shown to
be useful in diagnosis of infectious disease by detection of the
nucleic acids of a pathogen in a human sample such as blood. Using
on-board dry and liquid reagents, a blood sample is processed and
DNA associated with Plasmodium falciparum is detected in about 30
minutes or less. DNA purified from the sample is subjected to PCR
using two microfluidic chambers with dual temperature zones as
described in U.S. Pat. No. 7,544,506 and U.S. patent application
Ser. No. 11/562,611 (Microfluidic Mixing and Analytical Apparatus),
which are coassigned. Amplicon is then detected using a FAM
fluorescence-tagged molecular beacon directed at the amplified
target. Optionally, a control consisting of a California Red-tagged
RNAaseP leukocyte exon sequence, with multiplex amplification, is
used to validate the assay. A representative thermal melt curve
obtained using the thermo-optical interface of the invention is
shown in FIG. 22A.
EXAMPLE II
[0160] The apparatus of the invention is useful in the diagnosis of
coagulopathies. Using on-board dry and liquid reagents, a blood
sample is assayed for Coagulation Factor VIIa deficiency by
incubating plasma with a fluorogenic substrate such as
(D-Phe-Pro-Arg-ANSNH-cyclohexyl-2HCl; F.W.=777.81, Haematologic
Technologies, Essex Junction, Vt.) where ANSN is fluorophore
6-amino-1-naphthalene-sulfonamide, which lights up when the amide
bond between the dye and the peptide is cleaved. Tissue Factor (TF)
is obtained from Calbiochem (LaJolla, Calif.) and incorporated into
phosphatidylcholine or phosphatidylserine vesicles before use. TF
is used in excess. A 100 uL substrate reaction mixture consisting
of 20 mM Hepes buffer, pH 7.4, 0.15 M NaCl, with 5 nM TF and
containing 20 uM EDTA is incubated with a plasma sample for 10 min
to form the active enzyme complex. The ANSH substrate is then
added. The rate of hydrolysis of substrate is linear over the
normal range of Factor VIIa, and can be determined from a standard
curve. Descriptions of assay development may be found in US Patent
Application 2009/0325203 and other experimental literature.
[0161] While the above is a description of the preferred
embodiments of the present invention, it is possible to use various
alternatives, modifications and equivalents. Therefore, the scope
of the present invention should be determined not with reference to
the above description but should, instead, be determined with
reference to the appended claims, along with their full scope of
equivalents. The appended claims are not to be interpreted as
including means-plus-function limitations, unless such a limitation
is explicitly recited in a given claim using the phrase "means
for."
[0162] All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or cited in accompanying submissions, are
incorporated herein by reference, in their entirety. Aspects of the
embodiments can be modified, if necessary to employ concepts of the
various patents, applications and publications to provide yet
further embodiments.
* * * * *