Note: Descriptions are shown in the official language in which they were submitted.
WO 2021/236735
PCT/US2021/033100
SPATIAL GRADIENT-BASED FLUOROMETER
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit to provisional patent application serial no.
63/027,587 (911-023.9-1-1/N-YSI-0045US01), filed 20 May 2020; 63/028,013 (911-
023.010-1-1/N-YSI-0046US02), filed 21 May 2020, and 63/028,723 (911-023.011-1-
1/N-YSI-0047US02), filed 22 May 2020, which are all incorporated by reference
in its
entirety.
BACKGROUND OF THE INVENTION
1. Field of Invention
This invention relates to a fluorometer for measuring the concentration of
species-of-interest in a liquid; and more particularly, to a fluorometer for
measuring the
concentration of fluorophores in a liquid using non-intensity (i.e.,
amplitude) based
measurements.
2. Description of Related Art
The phenomenon of optical fluorescence is commonly exploited for use in
environmental water quality monitoring as such technology can be realized as a
compact, field-rugged sensor. Fluorescence-based sensing consists of an
excitation
light source (at a specified optical wavelength), used to optically excite the
water
parameter of interest and re-emit light (at a longer optical wavelength)
specific to the
water parameter of interest.
1
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
Known fluorometers gauge the concentration of the water species by measuring
the amplitude of the return fluorescence signal. The amplitude-based
measurement is
plagued with multiple issues:
1) Degradation of excitation source
Typical excitation sources include LEDs, laser diodes or lamps, all of which
suffer
from intensity degradation through the course of use. Options for dealing with
source
degradation are limited. One option is to include a reference detector, which
will factor-
out/nullify the effects of degradation, but adds complexity to the sensor's
electrical
circuit and requires additional opto-mechanical space. A second option is to
periodically
re-calibrate the sensor which necessarily limits the duration of field
deployments.
2) Thermal drift of excitation source
All of the sources mentioned above have a non-negligible response to
temperature, i.e., the optical output power changes as the ambient temperature
changes. This poses a real problem in sensor performance relying again on
either a
reference detector, or some elaborate electrical or embedded software
compensation
scheme. Furthermore, temperature compensation requires some measurement of the
temperature sensor, usually enabled by an onboard (i.e., located internally
within the
electrical circuit) temperature sensor, requiring additional circuitry and
physical space.
2
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
3) Interfering Species
Fluorescence-based sensors can suffer from optical interferences in which the
presence of other competing species can absorb at the same respective target
excitation and/or emission wavelengths, resulting in a decrease of
fluorescence
amplitude.
4) Opto-mechanical configuration
Traditional fluorescence sensing techniques suffer from poor sensitivity
(especially field-deployable sensors) stemming from poor/inefficient capture
of the
fluorescence signal. Existing fluorescence sensors typically employ a single
excitation
light source and a single (point-like) emission receiver, utilizing a
photosensitive
element. Regardless of the particular photosensitive element or excitation
light source
used, known prior art is not opto-mechanically configured for efficient
capture of
fluorescence, resulting in compromised limit of detection.
5) Inner Filter Effect (IFE)¨a range limiting effect
Known prior art exhibits the following problem: At low concentrations, the
fluorescence signal is approximately proportional to the species
concentration.
However, as the concentration is increased, the signal reaches a maximum
followed by
a decrease in the signal with ever higher concentrations. In this regard,
traditional
fluorometers are ambiguously double-valued, meaning that for any particular
measured
fluorescence signal, there are two possible concentrations¨one high, one low.
For
3
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
these known fluorometers, there is no way to distinguish between the two
possible
outcomes.
Known Literature
There is known literature concerning fluorescence using 2-D arrays to estimate
concentration gradients, and a brief summary of the major findings is
presented below:
Known prior art discloses a 2-D array used for "the determination of
concentration gradients in space and time". Here, the ("diffusion driven")
concentration
gradients are being determined by the local distribution and amplitude of
fluorescence
signal. This is an amplitude-based technique as the signal reported by any
particular
array element is simply proportional to the amount of "local" fluorescence
(i.e., the local
signal at a particular single array element), where the local amplitude of the
fluorescence is understood to be proportional to the local fluorophore
concentration
density.
Moreover, see PCT/US2008/059575, filed 7 April 2008, which discloses a system
and method for high-throughput turbidity measurements, as well as an article
by A
Singh et al., entitled "The performance of 2D array detectors for light sheet
based
fluorescence correlation spectroscopy."
4
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
SUMMARY OF THE INVENTION
The present invention is distinctly different from the prior art described
above:
For example, the present invention uses a spatial gradient (a consequence of
Beer's law) to determine a single, fixed /quasi-static concentration, where
changes in
concentration in time are understood to change much slower than the required
time for
signal acquisition. Put another way, the spatial gradients for the sensor
according to the
present invention are a consequence of Beer's law and are not a result of some
changing/varied spatial distribution of the fluorophore concentration.
In addition, the present invention circumvents many of the problems associated
with amplitude-based fluorescence measurements while providing an opto-
mechanical
configuration, capable of greatly enhanced signal capture and elimination of
IFE. The
present invention employs a linear photodiode array (however, the present
invention is
not limited to photodiode technology, e.g., a linear CCD or CMOS array could
also be
used as well). A linear array allows a non-intensity-based determination of
fluorescence. These measurements are spatially dependent, the main idea being
that
an optical signal will undergo attenuation across the linear array, following
Beer's law,
thereby creating a "spatial gradient". This spatial gradient contains
information
regarding the concentration of the fluorescent species.
The key element to the present invention deals specifically with the use of a
linear
sensor array to assess the spatial gradient of the signal along the length of
the linear
sensor array. The spatial gradient of the signal provides an assessment of the
fluorophore concentration that offers many advantages over the known amplitude-
based
methods including:
5
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
- Immunity to source degradation/drift,
- Calibration-free sensing,
- Immunity to florescence-band interference,
- Enhanced signal sensitivity, and
- IFE correction.
Other Implementation
The above "spatial gradient" method requires that each optical element in the
array
be individually addressable. However, there is a possible variant of the
design that
involves adding a transmission photodiode (located at the end of the array,
opposite of
the source) and connecting all of the linear array elements in an electrically
parallel
configuration. This design variant would further improve low signal
sensitivity thereby
further enhancing the minimum detection limit while retaining the sensor's
ability to
perform drift and IFE correction.
Finally, another variant could include the spatial gradient method in addition
to the
amplitude-based method to provide complementary information. Here the gradient-
based method could be used to identify excitation degradation, while the
amplitude
based method could be used to bolster low signal detection.
6
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
Specific Embodiments
According to some embodiments, the present invention may include, or take the
form of, apparatus featuring a signal processor or processing module
configured to:
receive signaling containing information about light reflected off
fluorophores in a liquid and sensed by a linear sensor array having a length
and
rows and columns of optical elements; and
determine corresponding signaling containing information about a
fluorophore concentration of the liquid that depends on a spatial gradient of
the
light reflected and sensed along the length of the linear sensor array, based
upon
the signaling received
The apparatus may include one or more of the following additional features:
The apparatus may include the linear sensor array.
The linear sensor array may include a linear photodiode array, a linear CCD
array, or a linear CMOS array, as well as a closed cylinder sensor array
having a three-
dimensional cylindrical array of the rows and columns of the optical elements.
The spatial gradient may be determined by a linear array algorithm that
defines a
relationship between the fluorophore concentration [c], the length or location
(I) along
the linear sensor array, a species absorption coefficient (a), and a signal
(S(I)) of an
array optical element along the linear sensor array.
7
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
The linear array algorithm takes the form of the equation:
y = mx +b,
where
y = -In (S(I)),
mx = a [c] I, and
b = -In ([c]ft To).
The linear array algorithm is based on Beer's law.
The apparatus may include, or take the form of, a spatial gradient-based
fluorometer.
The apparatus may include a quasi-collimated light source having a
corresponding length and being configured to provide the light, including
quasi-
collimated light, along the length of the linear sensor array.
The signal processor or processing module may be configured to determine the
fluorophore concentration based upon an attenuation of an optical signal
sensed across
the linear sensor array, including along the length and/or width of the linear
sensor
array.
The linear sensor array may include a two-dimensional array of the optical
elements that are individually addressable.
Either the rows or the columns of the optical elements may be connected in
parallel and addressable by the signal processor or processing module; the
apparatus
8
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
may include a transmission photodiode located at an end of the linear sensor
array,
opposite the light source, configured to respond to the light reflected off
the fluorophores
and provide transmission photodiode signaling containing information about the
same;
and the signal processor or processing module may be configured to receive the
photodiode signaling and correct the corresponding signaling for drift or the
inner filter
effect (IFE).
A Spatial Gradient-based Fluorometer
By way of further example, and according to some embodiments, the present
invention may include, or take the form of, a spatial gradient-based
fluorometer
featuring a quasi-collimated light source, a linear sensor array and a signal
processor or
processing module.
The quasi-collimated light source has a length and may be configured to
provide
quasi-collimated light to a liquid sample.
The linear sensor array has a corresponding length and rows and columns of
optical elements and may be configured to sense light reflected off
fluorophores in the
liquid sample along the length of the collimated light source and provide
signaling
containing information about the light reflected off the fluorophores.
The signal processor or processing module may be configured to:
receive the signaling; and
determine corresponding signaling containing information about a
fluorophore concentration of the liquid that depends on a spatial gradient of
the
9
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
light reflected and sensed along the corresponding length of the linear sensor
array, based upon the signaling received
The spatial gradient-based fluorometer may also include one or more of the
features set forth above.
The Method
According to some embodiments, the present invention may include a method,
featuring:
receiving, with a signal processor or processing module, signaling containing
information about light reflected off fluorophores in a liquid and sensed by a
linear
sensor array having a length and rows and columns of optical elements; and
determining, with the signal processor or processing module, corresponding
signaling containing information about a fluorophore concentration of the
liquid that
depends on a spatial gradient of the light reflected and sensed along the
length of the
linear sensor array, based upon the signaling received
The method may also include one or more of the features set forth above.
Computer-readable Storage Medium
According to some embodiments of the present invention, the present invention
may also take the form of a computer-readable storage medium having computer-
executable components for performing the steps of the aforementioned method.
The
computer-readable storage medium may also include one or more of the features
set
forth above.
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
Advantages
The present invention offers distinct advantages over the current known
techniques in the prior art, as follows:
1) The present invention determines fluorophore concentrations through a
spatial
gradient (a fluorescence signal that changes across the length of the linear
array
detector in keeping with Beer's law) (See Figure 1), and not by the amplitude
of the
fluorescence signal (algorithm for concentration determination (See Figure
5)). As
such, it is unaffected by moderate changes in the intensity of the source.
This means
that the spatial gradient is immune to source degradation, source thermal
response, or
change in source drive conditions (such as LED drive current). However, it is
necessary
that a non-negligible signal be present, i.e., there has to be some measurable
amount of
light incident upon the array to form the spatial gradient. Additionally, the
elements
need to be individually addressable to resolve the spatial information. The
present
invention is not limited to any specific linear array detector technology; a
linear
photodiode, CCD or CMOS array could be used.
2) The present invention, being immune to source degradation/drift, is capable
of
calibration-free deployments thereby extending the length of each deployment.
3) A linear sensor array provides a much larger overall active area to capture
the
return fluorescence. More importantly, the active area is larger in the
dimension that
matters most--along the optical axis (a quasi-collimated excitation source is
often used
which emits radiation predominantly along a single axis commonly referred to
as the
"optical" axis) (See Figure 1). The increased capture of fluorescence greatly
enhances
11
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
the signal sensitivity which, in turn, leads to a significant improvement in
the minimum
limit of detection the fluorescence species.
4) Just as the gradient-based method is impervious to moderate changes in
excitation power, it is also impervious to certain type of interferences. Any
interfering
species which absorbs the fluorescence signal, but not the excitation signal
(fluorescence-band interference), will not affect the signal gradient and
therefore not
hinder any assessment of the fluorophore concentration. Note, the spatial
gradient
method cannot address any interfering species that does absorb the excitation
signal
(excitation-band interference) as this would affect the signature of the
signal gradient.
5) While the fluorescence amplitude of traditional fluorometers suffers from
an
ambiguous double-valued response (due to IFE), such is not the case for the
spatial
gradient method whose response is monotonic with increasing concentration (See
Figure 4). The spatial gradient method enables real-time, inner filter effect
(IFE)
correction. [for the known prior art, the common method of inner filter
correction
involves post processing via lab analysis after a field deployment]. The IFE
correction
greatly enhances high-concentration sensing range (See Figure 3).
12
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
BRIEF DESCRIPTION OF THE DRAWING
The drawing, which are not necessarily drawn to scale, includes Figures 1 - 8,
as
follows:
Figure 1 is a side view of fluorescence "spatial gradient" following Beer's
law
(simulated in TraceProTm).
Figure 2 includes Figures 2A and 2B that show a spatial mapping and intensity
plot of fluorescence gradient (simulated in TraceProTm).
Figure 3 is a graph of sensor response vs. relative concentration with and
without
IFE correction [illustrating 10X enhanced detection range] (simulated in
TraceProTm).
Figure 4 is a graph of sensor response vs. relative concentration with and
without
IFE correction [elimination of double value problem] (simulated in
TraceProTm).
Figure 5 is an algorithm to determine concentration from the spatial gradient,
according to some embodiments of the present invention.
Figure 6 is a block diagram of apparatus, including a spatial gradient-based
fluorometer, according to some embodiments of the present invention.
Figure 7 is a block diagram of a linear sensor array having a length and rows
and
columns of optical elements, according to some embodiments of the present
invention.
Figure 8 is a three dimension perspective view of a quasi-collimated light
source
that provides a quasi-collimated light in relation to a linear sensor array,
according to
some embodiments of the present invention.
To reduce clutter in the drawing, each Figure in the drawing does not
necessarily
include every reference label for every element shown therein.
13
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
DETAILED DESCRIPTION OF BEST MODE OF THE INVENTION
Figure 6 shows apparatus 10, including a spatial gradient-based fluorometer,
according to the present invention having a quasi-collimated light source 20,
a linear
sensor array 30, and a signal processor or processing module 40.
The signal processor or processing module 40 may be configured to
receive signaling containing information about light Lr (Fig. 8) reflected off
fluorophores in a liquid and sensed by the linear sensor array 30 having a
length
L and rows and columns of optical elements (r1, c1; r1, c2; r1, c3; r1, c4;
r1, c5;
r1, c6; r1, c7; r1, c8; ...; r1, cn; r2, c1; r2, c2; r2, c3; r2, c4; r2, c5;
r2, c6; r2, c7;
r2, c8; ...; r2, cn; r3, c1; r3, c2; r3, c3; r3, c4; r3, c5; r3, c6; T3, c7;
r3, c8; ...; r3,
cn; ...; rn, c1; rn, c2; rn, c3; rn, c4; rn, c5; rn, c6; rn, c7; rn, c8; ...;
rn, cn), e.g., as
shown in Figure 7; and
determine corresponding signaling containing information about a
fluorophore concentration of the liquid that depends on a spatial gradient of
the
light reflected and sensed along the length L of the linear sensor array 30,
based
upon the signaling received
The Linear Sensor Array 30
By way of example, the apparatus 10 may include the linear sensor array 30,
e.g., such as a linear photodiode array, a linear charge-coupled device (CCD)
array, a
linear CMOS array. By way of further example, the linear sensor array 30 may
include
a two-dimensional array of rows and columns of optical elements, e.g., like
that shown
in Figure 7, that are individually addressable. Linear sensor arrays are known
in the art,
14
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
and the scope of the invention is not intended to be limited to any particular
type or kind
thereof either now known or later developed in the future.
By way of example, linear sensors arrays are disclosed in the following US
Patent nos. 9,020,202; 8,022,349; 7,956,341; 7,040,538; 5,252,818; and
4,193,057,
which are all hereby incorporated by reference.
The Light Source 20
By way of example, the apparatus 10 may include the light source 20 configured
to provide the light Lc (Fig. 8), including quasi-collimated light, along the
length L of the
linear sensor array 30 through a liquid sample arranged in relation to the
light source 20
and the linear sensor array 30 so as to reflect the light Lr off the
fluorophores in the
liquid sample being monitored or tested onto the linear sensor array 30. See
Figure 8.
For example, the light may be reflected radially and backwards, i.e.,
backscattered
reflected light or radiation.
As a person skilled in the art would appreciate, quasi-collimated light
sources are
known in the art, and the scope of the invention is not intended to be limited
to any
particular type or kind thereof either now known or later developed in the
future.
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
The Signal Processor or Processing Module 40
By way of example, the signal processor or processing module 40 may be
configured to determine the fluorophore concentration based upon a spatial
gradient of
the optical signal sensed across the linear sensor array, e.g., consistent
with that set
forth in relation to Figure 5.
In an alternative embodiment, either the rows or the columns of the optical
elements may be connected in parallel and addressable by the signal processor
or
processing module 40; the apparatus 10 may include a transmission photodiode
30a
located at an end of the linear sensor array 30, opposite the light source 20,
configured
to respond to the light reflected off the fluorophores and provide
transmission
photodiode signaling containing information about the same; and the signal
processor
or processing module 40 may be configured to receive the photodiode signaling
and
correct the corresponding signaling for drift or the inner filter effect.
Implementation of Signal Processing Functionality
By way of example, the functionality of the signal processor or processing
module 40 may be implemented using hardware, software, firmware, or a
combination
thereof. In a typical software implementation, the signal processor 40 would
include
one or more microprocessor-based architectures having, e. g., at least one
signal
processor or microprocessor. One skilled in the art would be able to program
with
suitable program code such a microcontroller-based, or microprocessor-based,
implementation to perform the signal processing functionality disclosed herein
without
undue experimentation.
16
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
The scope of the invention is not intended to be limited to any particular
implementation using technology either now known or later developed in the
future.
The scope of the invention is intended to include implementing the
functionality of the
signal processor(s) as stand-alone processor, signal processor, or signal
processor
module, as well as separate processor or processor modules, as well as some
combination thereof.
By way of example, the apparatus 10 may also include, e.g., other signal
processor circuits or components generally indicated 50, including random
access
memory or memory module (RAM) and/or read only memory (ROM), input/output
devices and control, and data and address buses connecting the same, and/or at
least
one input processor and at least one output processor, e.g., which would be
appreciate
by one skilled in the art.
By way of further example, the signal processor 40 may include, or take the
form
of, some combination of a signal processor and at least one memory including a
computer program code, where the signal processor and at least one memory are
configured to cause the system to implement the functionality of the present
invention,
e.g., to respond to signaling received and to determine the corresponding
signaling,
based upon the signaling received.
Inner Filter Effect (IFE)
As a person skilled in the art would appreciate, the IFE is a fluorescence
spectroscopy phenomenon, e.g., where there is a decrease in fluorescence
emission
seen in concentrated solutions due to the absorption of exciting light by the
fluorophore
17
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
that is close to the incident beam and which significantly diminishes light
that reaches
the sample further away from it.
As a person skilled in the art would appreciate, techniques for correcting for
the
IFE are known in the art, and the scope of the invention is not intended to be
limited to
any particular type or kind thereof either now known or later developed in the
future.
Beer's Law
As a person skilled in the art would appreciate, Beer's law is defined by the
relationship, as follows:
A = 6 b C,
where
A = absorbance,
C = molar absorptivity,
b = length of the light path, and
C = concentration,
Fluorophores
As a person skilled in the art would appreciate, a fluorophore is a
fluorescent
chemical compound that can re-emit light upon excitation. Fluorophores
typically
contain several combined aromatic groups, or planar or cyclic molecules with -
Fr bonds.
18
CA 03178549 2022- 11- 10
WO 2021/236735
PCT/US2021/033100
By way of example, fluorophores are sometimes used as a tracer in fluids, as a
dye for staining of certain structures, as a substrate of enzymes, or as a
probe or
indicator (when fluorescence is affected by environmental aspects such as
polarity or
ions).
The scope of the invention is not intended to be limited to any particular
type or
kind of fluorophore either now known or later developed in the future.
Applications
The present invention has applications, e.g., in the basic parameter of water
quality monitoring for freshwater applications, as well as drinking water
monitoring.
The Scope of the Invention
While the invention has been described with reference to exemplary
embodiments, it will be understood by those skilled in the art that various
changes may
be made, and equivalents may be substituted for elements thereof without
departing
from the scope of the invention. In addition, modifications may be made to
adapt a
particular situation or material to the teachings of the invention without
departing from
the essential scope thereof. Therefore, it is intended that the invention not
be limited to
the particular embodiment(s) disclosed herein as the best mode contemplated
for
carrying out this invention.
19
CA 03178549 2022- 11- 10
