US 11,035,707 B2

Sheet 18 of 32

Jun. 15, 2021

U.S. Patent

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US 11,035,707 B2

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230; subjecting analyte 222 to analytical light 232; produc-
ing photoanalyte 242 with analytical light 232, depending on
the intensity of analytical light; destroying photoanalyte
242; creating photoanalyte light 246; communicating pho-
toanalyte light 246 from photo interaction region 230 to
photodetector 248 via detection light path 244: and record-
ing detection light intensity versus time to determine the
flow rate of liquid 220

Analyte 222 can be converted to photoanalyte 242 by
photo damaging the analyte with light more powerful than is
used to excite fluorescence. In this process, the method for
determining flow rate involves measuring the intensity of
photoanalyte light 246 as a function of the intensity of
analytical light 232. The method depends on measuring the
remaining fluorescence from photoanalyte 242 that has not
been bleached, i.e., destroyed.

In the process for determining flow rate, the flow rate can
change the intensity of the photoanalyte light 246, which can
be used to determine relative flow rate changes. The mea-
surement process can also involve a calibration process,
whereby a scaling factor is determined to convert the
intensity of photoanalyte light 246 to flow rate. The scaling
factor can be determined by calibrating the photoanalyte
light 246 to a known flow standard, such as a gravimetric
flow meter. The intensity profile of photoanalyte light 246
can be mapped to flow rate or analytical light intensity. In
such a way, a scaling factor can be determined to relate the
measurements of the optical flow meter to a valid or trace-
able scale of volumetric flow rate. The scaling factor can
depend on the nature of analyte and photoanalyte.

In an embodiment, with reference to FIG. 19, a process
using analytical light can also include multiple photo inter-
action regions 230. Here, measurement is as above with a
scaling relationship determined separately for each photo
interaction region, based on dependencies described above.

In an embodiment, with reference to FIG. 20, a process
for determining the flow rate of liquid 220 with optical flow
meter 200, occurs via a time-of-flight determination and
includes: flowing liquid 220 including pre-analyte 221
through microchannel 212 and communicating liquid 220
with pre-analyte 221 to activation region 228; producing
activation light 226 from activation light source 236; com-
municating activation light 226 to activation region 228 via
activation light path 224 to produce analyte 222; commu-
nicating liquid 220 with analyte 222 to analytical interaction
region 230; producing analytical light 232 and communi-
cating activation light 232 through activation light path 244
to optical interaction region 230; subjecting activated ana-
lyte to activation light 232; producing photoanalyte 242 and
photoanalyte light 246 from activation light 232; commu-
nicating photoanalyte light 246 from optical interaction
region 230 to photo detection 248 via detection light path
244; recording intensity of detected light as a function of
time; and calculating flow rate based on ratio of distance, L,
between activation region 228 and analytical interaction
region 230, and time between creation of activated analyte
and measurement of photoanalyte light.

The process for determining flow rate of liquid 220 can
include pulses or continuous application of activation light
226 to the analyte 222 or pre-analyte 221.

The process for determining the flow rate of liquid 220
can including measuring the timing or change of activation
light 226 after interaction with analyte 222 or pre-analyte
221 with a light path and detector.

The process for determining the flow rate of liquid 220
can include determining a calibration factor from the flow
rate of liquid 220 as the function of time. The process for

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determining the flow rate of liquid 220 can include calibrat-
ing flow controller 250 from the calibration factor.

The process for determining the flow rate of liquid 220
can induce measuring the time difference between applica-
tion of activation light 226.

The process for determining the flow rate of liquid 220
can include detecting photoanalyte 242 with photodetector
248 through detection light path 244A and detection light
246A; detection light path 244B and detection light 246B;
detection light path 244C and detection light 246C; or a
combination thereof. A spectral filter can be optically inter-
posed between light path and detector to selectively remove
activation or analytical light from the detection light prior to
photodetector 248.

The process of determining the flow rate of liquid 220 can
include comparing measurement readings from photodetec-
tor 248 with either dimensional measurements or a math-
ematical description of the device. The measurements or
mathematical description can include, e.g., the microchannel
size and shape, the activation light 226 and photoanalyte
light 246, the flow rate of liquid 220, the physical properties
of the analyte 222 or pre-analyte 221, the properties of the
photodetector, light-transmitting properties of the activation
light path 224 and analytical light path 238, and properties

sof the liquid 220.

In the process for determining the flow rate of the liquid,
flowing liquid 220 including analyte 222 or pre-analyte 221
through microchannel 212 includes flowing the liquid 220
through microchannel 212 at a constant rate. A constant flow
rate can be achieved, e.g., by driving constant flow such as
from a syringe pump or by ensuring constant pressure at the
fluid receiver 214 or fluid transmitter 216. A constant
pressure can be achieved by connecting the fluid receiver
214, e.g. to a pressure source or gravimetric flow controller.
It should be appreciated that the volumetric flow rate of
liquid 220 in the fluid receiver 214 is the same as in the
microchannel 212, optical interaction region 228, photo
interaction region 230, and fluid transmitter 216.

In the process for determining flow rate of liquid, the
measurement of flow determined from the optical measure-
ments or mathematics can be calibrated against a pump or
flow meter, e.g., comparing the measurements with record-
ings of liquid mass over time on a gravimetric flow system
in a metrology laboratory.

In the process for determining the flow rate of the liquid,
communicating activation light 226 through activation light
path 224 to optical interaction region 228 using a measur-
able and known intensity of activation light 226 through the
activation light path 224 and into the optical interaction
region 228. The activation light 226 can be time changing or
constant. Moreover, the intensity of activation light 226 can
be controlled, e.g., by an electronic power supply controlling
the activation light sources 236 or filters that only transmit
a fraction of the light emitted by the activation light sources

5 236.

In the process for determining the flow rate of the liquid,
exposing analyte 222 or pre-analyte 221 with activation light
226 induces a reaction or other physical process that changes
the composition or physical properties of the analyte 222 or
pre-analyte 221. This transformation can, e.g., destroy the
pre-analyte 221 or produce analyte 222. Exposing photoana-
lyte 242 to analytical light 232 can induce the photoanalyte
to emit photoanalyte light 246 or scatter analytical light into
a detection light path 244,

In the process for determining the flow rate of the liquid,
communicating, in fluid member 218, pre-analyte 221, ana-
lyte 222, photoanalyte 242 and combinations thereof, from


US 11,035,707 B2

5
246A. Photoanalyte 242 can scatter or emit photoanalyte
light 246B, which is communicated via detection light path
244B to photodetector 248B.

According to an embodiment, with reference to FIG. 8,
optical flow meter 200 includes a plurality of flow readers
252 arranged in tandem along microchannel 212. In this
manner, optical flow meter 200 includes with respect to flow
of liquid 220 through microchannel 212 from optical inter-
action region 228 to fluid transmitter 216, consecutive photo
interaction regions 230, analytical light paths 238, and
detection light paths 244 disposed on substrate 210. That is
optical interaction region 228 is interposed between fluid
receiver 214 and photo interaction regions 230. Is contem-
plated that flow readers 252 provide determination from
analyte 222 or photo analyte 242 of a dynamic process (e.g.
biological cell flow cytometry), photoactive process (e.g.,
light absorption rate, scattering cross-section), chemical
kinetics (e.g., reaction rate, e.g., with surface-mounted oli-
gomers), chemical dynamics (e.g., cellular nutrient uptake
rates, diffusion processes), and the like.

In an embodiment, with reference to FIG. 9, FIG. 10, FIG.
1, FIG. 12, FIG. 13. FIG. 14, and FIG. 15, optical flow
meter 200 includes substrate 210 and microchannel 212
disposed in substrate 210. Microchannel 212 includes fluid
receiver 214 that receives liquid 220 including pre-analyte
221; fluid transmitter 216 that receives liquid 220 from fluid
receiver 214; and fluid member 218 interposed between fluid
receiver 214 and fluid transmitter 218. Fluid member 218
includes optical interaction region 228 that receives activa-

tion light 226 and photo interaction region 230. Photo 3

interaction region 230 receives analytical light 232 and
communicates photoanalyte light 246 out of fluid member
218. Microchannel 212 is bounded by substrate 210. Further,
fluid member 218 communicates liquid 220 from fluid

receiver 214 to fluid transmitter 216. Optical flow meter 200 3

also includes activation light path 224 disposed in substrate
210 and is arranged at an oblique angle or right angle to fluid
member 218 proximate to optical interaction region 228.
Activation light path 224 receives activation light 226 from
activation light source 236 and communicates activation
light 226 to optical interaction region 228, wherein activa-
tion light 226 interacts with pre-analyte 221 in optical
interaction region 228 to produce analyte 222. Additionally,
optical flow meter 200 includes analytical light path 238
disposed in substrate 210 and arranged at an oblique angle
or right angle to fluid member 218 proximate to photo
interaction region 230. Analytical light path 238 receives
analytical light 232 from analytical light source 240 and
communicates analytical light 232 to photo interaction
region 230, wherein analytical light 232 interacts with
analyte 222 in photo interaction region 230 subsequent to
the interaction of pre-analyte 221 with activation light 226
in optical interaction region 228 and produces photoanalyte
242 from analyte 222 in response to interaction of analyte
222 with analytical light 232 in photo interaction region 230.
Photoanalyte 242 produces photoanalyte light 246 that is
communicated through detection light path 244 disposed in
substrate 210. Detection light path 244 is arranged at an
oblique angle or right angle to fluid member 218 proximate
to photo interaction region 230 and receives photoanalyte
light 246 from photo interaction region 230 and communi-
cates photoanalyte light 246 from microchannel 212 to
photodetector 248.

Optical flow meter 200 can include flow controller 250
that provides liquid 220 to fluid receiver 214. Moreover,
activation light source 236 is in optical communication with
activation light path 224 and provides activation light 226 to

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activation light path 224. Further, analytical light source 240
can be in optical communication with analytical light path
238 and can provide analytical light 232 to analytical light
path 238.

In an embodiment, optical flow meter 200 includes pho-
todetector 248 in optical communication with detection light
path 244 and that receives photoanalyte light 246 from
detection light path 244, Moreover, analytical light source
240 can be included in optical flow meter 200, wherein
analytical light source 240 is in optical communication with
analytical light path 238 and provides analytical light 232 to
analytical light path 238.

According to an embodiment, with reference to FIG. 16,
optical flow meter 200 includes a plurality of detection light

5 paths 244 (e.g., 244A, 244B, 244C, and the like) that

transmit photoanalytical light (e.g., 246A, 246B, 246C, and
the like) to photodetectors 248 (e.g., 248A, 248B, 248C, and
the like). It is contemplated that interaction of analytical
light 232 with analyte 222 produces photoanalyte 242
(which may be the same or different chemical species as
analyte 222). Accordingly, analytical light 232 can be scat-
tered or emitted from analyte 222 to produce light that is
received by photodetector 248C via detection light path
244C as photoanalyte light 246C. Analytical light 232 can be

5 absorbed by analyte 222 to produce photoanalyte 242 (e.g..

an excited state of analyte 222), wherein a portion of
analytical light 232 is not absorbed or scattered and is
transmitted or emitted to photodetector 248A via detection
light path 244A as light referred to as photoanalyte light
246A. Photoanalyte 242 can scatter or emit photoanalyte
light 246B, which is communicated via detection light path
244B to photodetector 248B.

In an embodiment, with reference to FIG. 17, optical flow
meter 200 includes a plurality of flow readers 252 arranged
in tandem along microchannel 212. In this manner, optical
flow meter 200 includes with respect to flow of liquid 220
through microchannel 212 from optical interaction region
228 to fluid transmitter 216, consecutive photo interaction
regions 230, analytical light paths 238, and detection light
paths 244 disposed on substrate 210. That is optical inter-
action region 228 is interposed between fluid receiver 214
and photo interaction regions 230. Is contemplated that flow
readers 252 provide determination from pre-analyte 221,
analyte 222 or photo analyte 242 of a dynamic process (e.g.

5 biological cell flow cytometry), photoactive process (e.g.,

light absorption rate, scattering cross-section), chemical
Kinetics (e.g., reaction rate, e.g., with surface-mounted oli-
gomers), chemical dynamics (e.g.. cellular nutrient uptake
rates, diffusion processes), and the like.

Substrate 212 of flow meter 200 can include a material
that provides flow in microchannel 212. Moreover, substrate
212 can be disposed in or can include a microfluidic device.
Exemplary materials for the substrate include polymeric
materials such as elastomers (e.g. PDMS), epoxies and

5 adhesives (including tapes and photocurable adhesives),

acrylics (e.g. PMMA), polycarbonates, polystyrenes, poly-
esters, polypropylenes, cyclic olefin copolymers and hard
materials, such as glasses, semiconductor materials (e.g.
pure and doped silicon), and metals (e.g. aluminum). The
substrate can include a combination of transparent, opaque,
and reflective boundaries to control light propagation in or
through an interior of flow meter 200. In an embodiment, the
substrate includes a light scattering structure to absorb,
scatter, or reflect light out of the optical paths or to prevent

5 cross-talk between light paths (see FIG. 24).

It is contemplated that substrate 210 can include a flow
member also referred herein as a microchannel. In an


US 11,035,707 B2

23
detecting the photoanalyte light with the photodetector;
and
determining a flow rate of the liquid as a function of time.
16. The process for determining the flow rate of the liquid
of claim 14, further comprising determining a calibration
factor from the flow rate of the liquid as the function of time.
17. The process for determining the flow rate of the liquid
of claim 16, further comprising calibrating a flow controller
from the calibration factor.
18. A process for determining a flow rate of a liquid, with
the optical flow meter of claim 2, the process comprising:
flowing the liquid comprising an analyte through the
microchannel;
communicating activation light through the activation
light path to the optical interaction region:
subjecting the pre-analyte to the activation light;
exciting the pre-analyte with the activation light to pro-
duce analyte;
communicating, in the fluid member, the analyte from the
optical interaction region to the photo interaction
region, wherein the photo interaction region is a portion
of the microchannel that receives the analytical light
and where the analytical light interacts with the analyte;
communicating analytical light through the analytical
light path to the photo interaction region;
subjecting the activated analyte to the analytical light;

24

producing a photoanalyte from the activated analyte in
response to subjecting the analyte to the analytical
light;

producing photoanalyte light from the photoanalyte;

communicating the photoanalyte light from the photo

interaction region to the detection light path to deter-
mine the flow rate of the liquid.

19. The process for determining the flow rate of the liquid
of claim 18, further comprising communicating the pho-
toanalyte light from the photo interaction region to a pho-
todetector.

20. The process for determining the flow rate of the liquid
of claim 19, further comprising:

detecting the photoanalyte light with the photodetector:

and

determining a flow rate of the liquid as a function of time.

21. The process for determining the flow rate of the liquid
of claim 19, further comprising determining a calibration
factor from the flow rate of the liquid as the function of time.

22. The process for determining the flow rate of the liquid
of claim 21, further comprising calibrating a flow controller
from the calibration factor.

23. The process for determining the flow rate of the liquid
of 22, further comprising calibrating a flow controller from
the calibration factor.

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US 11,035,707 B2

Sheet 12 of 32

Jun. 15, 2021

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US 11,035,707 B2

Sheet 22 of 32

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optical interaction region 228 to photo interaction region
230 occurs directly through convection and diffusion of
pre-analyte 221, analyte 222, photoanalyte 242 and combi-
nations thereof, in liquid 220.

In the process for determining the flow rate of the liquid,
subjecting photoanalyte 242 to analytical light 232 includes
transmission of the analytical light 232 into the photo
interaction region 230.

In the process for determining the flow rate of the liquid,
communicating analytical light 232 through analytical light
path 238 to photo interaction region 230 can include using
a measurable and known intensity of analytical light 232
through the analytical light path 238 and into the photo
interaction region 230. The analytical light 232 can be time
changing or constant. Moreover, the intensity of analytical
light 232 can be controlled, e.g., by an electronic power
supply controlling the analytical light sources 240 or filters
that only transmit a fraction of the light emitted by the
analytical light sources 240.

In the process for determining the flow rate of the liquid,
subjecting photoanalyte 242 to analytical light 232 includes
absorption of a photon by photoanalyte 242, which can
release a photon. In fluorescence, the emitted photon is
red-shifted or has longer wavelength than analytical light
232. Alternatively, photoanalyte 242 can dissipate energy
absorbed from analytical light 232 by a non-radiative pro-
cess or can scatter analytical light 232.

In the process for determining the flow rate of the liquid,
producing photoanalyte light 246 from photoanalyte 242,

emission of fluorescent light from photoanalyte 242, in the 3

event that analytical light 232 is absorbed by photoanalyte
242 includes producing an excited state and subsequently
emitting light from the excited state.

In the process for determining the flow rate of the liquid,

communicating photoanalyte light 246 from photo interac- 3

tion region 230 to detection light path 244 includes trans-
mitting photoanalyte light 246 to light detection path 244.

In the process for determining the flow rate of the liquid,
communicating photoanalyte light 246 from photo interac-
tion region 230 to photodetector 248 includes transmitting
light along an optical path, such as a fiber optic cable
inserted into the photoanalyte light path 244

In the process for determining the flow rate of the liquid,
detecting photoanalyte light 246 with photodetector 248
includes converting the photoanalytical light into a signal,
e.g., an electrical signal, with a photodiode, spectrometer, or
the like, and recording the intensity or wavelength of the
photoanalytical light as a function of time.

In the process for determining the flow rate of the liquid,
determining the flow rate of liquid 220 as a function of time
includes measuring the time, intensity, or wavelength
changes between activation of pre-analyte 221, analyte 222,
or photoanalyte 242, and recording of photoanalytical light
246. The process can include estimating the arrival time or
peak emission of the photoanalyte at the photo interaction
region 230. The process can also include estimated adjust-
ments for time delays associated with chemical processes or
interactions with the analyte or photoanalyte with objects in
the microchannel. The process can include measurements or
mathematical calculations of the distance or volume of fluid
between the optical interaction region 228 and the photo
interaction region 230. Calculations can include, e.g.. the
microchannel size and shape, the activation light 226 and
photoanalyte light 246, the flow rate of liquid 220, the
physical properties of the pre-analyte 221, analyte 222,
photoanalyte 242, or the liquid 220. The process can also
include calibration of the measurements of volume and time

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to a known flow standard, such as a gravimetric flow meter.
In such a way, a calibration factor can be determined to
relate the measurements of the optical flow meter to a valid
or traceable scale of volumetric flow rate.

In the process for determining the flow rate of the liquid,
calibrating flow controller 250 from the calibration factor
includes adjusting the input flow parameters, e.g. pressure or
pump rate, in order to match the desired flow rate to the
measured flow rate.

The process for measuring flow-rate can be extended to
the case of multiple photo interaction regions 230. In this
case, the measurement proceeds as described above but the
calculation for each photo interaction region 230 is based on
the dependencies described above.

Optical flow meter 200 has numerous advantageous and
beneficial properties. The optical flow meter provides quan-
titative microscale chemical and biological measurement
and can be integrated with sensors for synergistic function
and performance. Moreover, the optical flow meter provides
optofluidics that integrate optical fibers into microfluidic
waveguides. Optical components transmit and collect light
from substances in microflows such as fluorescent com-
pounds. Components for splitting light into multiple paths,
focusing light into the microchannel, and collecting fluo-

Ss rescence emission exclude excitation light as described

below. An integrated system of the optical flow meter
provides real-time and continuous flow-monitoring using
caged fluorophores. The optical flow meter also probes
dynamic fluid properties and measures samples using micro-
fluidic cytometry.

The optical flow meter can be used for investigations in
chemistry, biology, and physics because it provides unique
fluid transport regimes, dynamic control of small fluid
volumes (femtoliters to milliliters), and high-throughput
analysis of materials in flow. Integration of on-chip optical
components of the optical flow meter provides miniaturiza-
tion, portability, and speed of sensitive and multimodal
measurement capabilities. The optical flow meter includes
optical sensing elements with microfluidics to accurately
and reproducibly measure microflows and characterize opti-
cal properties of materials in flow using sensitive chemical
and biological assays. The optical flow meter can be used for
cytometry, drug delivery, and diagnostic assays. Further, the
optical flow meter can perform rapid, high quality measure-
ments in both industrial and distributed or point-of-use
applications.

In an aspect, optical flow meter 200 determines a flow rate
of analyte 222 that is from 0.1 nanoliter per minute (nL/min)
to 1 milliliter per minute (mL/min), specifically from 1
nl/min to 100 L/min, and more specifically from 1 nL/min
to 10 L/min. Moreover, optical flow meter 200 has the
beneficial property in that the volumetric flow rate can be
directly related to the dimensions of the microchannel, and
that it can be precisely determined from calibration or
modeling. Further, optical flow meter 200 has the advantage
in that it is easy to integrate optical elements into the device
using a coupling fluid or optical adhesive, and the perfor-
mance of the system appears to be rather robust to variations
in assembly. Optical flow meter 200 uses fluorescence in that
the output photoanalyte light is a different wavelength than
the analytical light, which straightforwardly separates, i.e.,
filters, it from analytical light. Also, optical flow meter 200
advantageously places the photoanalyte light path (e.g.,
alternate path 244B) on a different axis than the analytical
light, the coupling of the analytical light entering the pho-
toanalyte light path is reduced, increasing the signal to noise
ratio. Also, optical flow meter 200 advantageously deter-



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US 11,035,707 B2

1
OPTICAL FLOW METER FOR
DETERMINING A FLOW RATE OF A
LIQUID

CROSS REFERENCE TO RELATED
APPLICATIONS

This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/576,709 filed Oct. 25, 2017,
the disclosure of which is incorporated herein by reference
in its entirety.

S

STATEMENT REGARDING FEDERALLY
SPONSORED RESEARCH

This invention was made with United States Government
support from the National Institute of Standards and Tech-
nology (NIST), an agency of the United States Department
of Commerce. The Government has certain rights in the
invention. Licensing inquiries may be directed to the Tech- 2
nology Partnerships Office, NIST, Gaithersburg, Md.,
20899; voice (301) 301-975-2573; email tpo@nist.gov; ref-
erence.

8

BRIEF DESCRIPTION 2

Disclosed is an optical flow meter comprising: a substrate;
a microchannel disposed in the substrate comprising: a fluid
receiver that receives a liquid comprising an analyte: a fluid
transmitter that receives the liquid from the fluid receiver; 3
and a fluid member interposed between the fluid receiver
and fluid transmitter and that: comprises: an optional optical
interaction region and that receives activation light; and a
photo interaction region and that: receives analytical ligl
and communicates photoanalyte light out of the fluid mem-
ber; is bounded by the substrate; and communicates the
liquid from the fluid receiver to the fluid transmitter; an
optional activation light path disposed in the substrate,
arranged at an oblique angle or right angle to the fluid
member proximate to the optical interaction region, and that:
receives activation light from an activation light source; and
communicates the activation light to the optical interaction
region, such that the activation light interacts with the
pre-analyte in the optical interaction region to produce an
analyte; an analytical light path disposed in the substrate,
arranged at an oblique angle or right angle to the fluid
member proximate to the photo interaction region, and that:
receives the analytical light from an analytical light source;
and communicates the analytical light to the photo interac-
tion region, such that the analytical light: interacts with the
analyte in the photo interaction region subsequent to the
interaction of the analyte with the activation light in the
optical interaction region; and produces a photoanalyte from
the analyte in response to interaction of the analyte with the
analytical light in the photo interaction region, the photoana- 55
lyte producing photoanalyte light; and a detection light path
disposed in the substrate, arranged at an oblique angle or
right angle to the fluid member proximate to the photo
interaction region, and that: receives the photoanalyte light
from the photo interaction region; and communicates the
photoanalyte light from the microchannel to a photodetector,
the optical flow meter determines a flow rate of the analyte
that is from 0.1 nanoliter per minute (nL/min) to 5 mL/min.

Also disclosed is a process for determining a flow rate of
a liquid, with the optical flow meter, the process comprising: 6:
flowing the liquid comprising an analyte through the micro-
channel; communicating analytical light through the ana-

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lytical light path to the photo interaction region; subjecting
the analyte to the analytical light; producing a photoanalyte
from the analyte in response to subjecting the analyte to the
analytical light; producing photoanalyte light from the pho-
toanalyte; communicating the photoanalyte light from the
photo interaction region to the detection light path to deter-
mine the flow rate of the liquid.

Further disclosed is a process for determining a flow rate
of a liquid, with the optical flow meter, the process com-
prising: flowing the liquid comprising an analyte through the
microchannel; communicating activation light through the
activation light path to the optical interaction region: sub-
jecting the pre-analyte to the activation light; exciting the

s Pre-analyte with the activation light to produce analyte;

communicating, in the fluid member, the analyte from the
optical interaction region to the photo interaction region;
communicating analytical light through the analytical light
path to the photo interaction region; subjecting the activated
analyte to the analytical light; producing a photoanalyte
from the activated analyte in response to subjecting the
analyte to the analytical light; producing photoanalyte light
from the photoanalyte; communicating the photoanalyte
light from the photo interaction region to the detection light
path to determine the flow rate of the liquid.

BRIEF DESCRIPTION OF THE DRAWINGS.

The following descriptions should not be considered
limiting in any way. With reference to the accompanying
drawings, like elements are numbered alike.

FIG. 1 shows a perspective view of an optical flow meter;

FIG. 2 shows a top view of the optical flow meter shown
in FIG. 1;

FIG. 3 shows a side view of the optical flow meter shown
in FIG. 1;

FIG. 4 shows a cross-section along line A-A of the optical
flow meter shown in FIG. 2;

FIG. 5 shows a cross-section along line C-C of the optical
flow meter shown in FIG. 2:

FIG. 6 shows a cross-section along line D-D of the optical
flow meter shown in FIG. 3;

FIG. 7 shows a cut-away view of an optical flow meter;

FIG. 8 shows a cut-away view of an optical flow meter
that includes repeating elements;

FIG. 9 shows an optical flow meter;

FIG. 10 shows a top view of the optical flow meter shown
in FIG. 9;

FIG. 11 shows a side view of the optical flow meter shown
in FIG. 9;

FIG. 12 shows a cross-section along line A-A of the
optical flow meter shown in FIG. 10;

FIG. 13 shows a cross-section along line B-B of the
optical flow meter shown in FIG. 10;

FIG. 14 shows a cross-section along line C-C of the
optical flow meter shown in FIG. 10;

FIG. 15 shows a cross-section along line D-D of the
optical flow meter shown in FIG. 11;

FIG. 16 shows a cut-away view of an optical flow meter;

FIG. 17 shows a cut-away view of an optical flow meter
that includes a plurality of repeating elements;

FIG. 18 shows a flow chart for measuring flow;

FIG. 19 shows a flow chart for measuring flow with a laser
and an optical flow meter that includes repeating elements;

FIG. 20 shows a flow chart for measuring flow with an
activation laser;


US 11,035,707 B2

Sheet 19 of 32

Jun. 15, 2021

U.S. Patent

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U.S. Patent Jun. 15, 2021 Sheet 25 of 32 US 11,035,707 B2

300 nifmin 40 nisin No bleaching (100 pl/min)
pprox. Photobleached Area

Fluor. intensity (a.u.)

3 2500 5000 7500
Flow Rate (ni/min)

Figure 25


U.S. Patent

Jun. 15, 2021

Sheet 21 of 32

US 11,035,707 B2

Produce analytical light 232
at-each analytical light

Flow liquid 220 witb pre-analyte
221 through microchannel 212 at

Produce activation light and
record time of first. production

interaction region 230

source unknown constant rate
Communicate analytical Communicate flow liquid 220 Communicate activation light
light 282 into each with pre-analyte 221 into oF mane alton 8

activation region

into activation region

Create analyte

Communicate flow liquid 220
with activated analyte into each
interaction region 230

Create photo-analyte 242

Communicate photo-analyte
light 246 te photodetector 248
via each detection light path 244

Record first: detection time for
photoanalyte light ineach
photo-interaction region.

Compute flow-rate as ratio of
distance and time traveled

between interaction regions for

each photo interaction region

Figure 21


US 11,035,707 B2

3

FIG. 21 shows a flow chart for measuring flow with an
activation laser and an optical flow meter that includes
repeating elements;

FIG. 22 shows an optical flow meter;

FIG. 23 shows an optical flow meter that includes a
plurality of light collection paths;

FIG. 24 shows an optical flow meter;

FIG. 25 shows an optical flow meter in which (upper left
panel) an analyte has photobleaching under a moderate flow
condition; (upper middle panel) an analyte has more pho-
tobleaching under a low flow condition; (upper right panel)
an analyte has no photobleaching; and (lower panel) a graph
of fluorescence intensity versus flow rate for emission of
light by an analyte in the optical flow meter;

FIG. 26 shows an optical flow meter;

FIG. 27 shows an optical flowmeter;

FIG. 28 shows an optical flow meter;

FIG. 29 shows a graph of optical power versus time for
emission produced by a pulsed activation light:

FIG. 30 shows a graph of optical power versus time for
steady-state emission from a photoanalyte subjected to acti-
vation light;

FIG. 31 shows a graph of time until a 50% rise in optical
power versus flow rate; and

FIG. 32 shows a graph of flow rate versus time.

DETAILED DESCRIPTION

A detailed description of one or more embodiments is

presented herein by way of exemplification and not limita- 3

tion.

It has been discovered that an optical flow meter and a
process for determining a flow rate of a liquid unexpectedly
and advantageously provide a determination of convection

or diffusion of a liquid, e.g., a liquid that includes a contrast 3

agent (analyte) (e.g., a fluorescent marker) at a measurement
point located within a photo interaction region, wherein the
liquid flows in a microchannel of the optical flow meter.
Further, a pre-contrast agent (pre-analyte) can be converted
to a contrast agent at an activation region upstream of a
measurement point. Beneficially, the optical flow meter can
be integrated ina microfluidic system and can be part of flow
control, chemical analysis, particle or cell measurement
system, and the like.

The optical flow meter is part of the NIST on a Chip
program, which is creating portable, SI-traceable measure-
ment devices that can be used directly on factory floors, in
hospital diagnostic centers, and the like. These NIST-pio-
neered technologies will be manufactured and distributed by
the private sector, opening new tech-transfer and lab-to-
market opportunities in accordance with NIST’s goal of
strengthening U.S. economic competitiveness by supporting
advanced manufacturing. In the optical flow meter, volu-
metric flow and flow velocity are traceable to a dimension of
microchannels and waveguide spacing (meter) and timing of
light activation pulses and power measurements (second).
Compared to conventional devices based on convection or
diffusion to visualize or measure microflows, the optical
flow meter can be used in an absence of a microscope or
computation of tracer displacements in a field (eg.,
microparticle velocimetry). Moreover, determination of
flow rate with the optical flow meter is continuously moni-
tored by recording the amount of light given off by a contrast
agent with respect to a maximum amount of light or repeated
by controlling the activation light (e.g., by pulsing the power
or using a waveform) and measuring the time-of-flight or
swept volume from the activation to a measurement point.

S

2

40

4

Optical waveguides can be hard-wired onto a chip such that
operation of the optical flow meter is independent of laser
positioning, focusing, or alignment of the laser and laser
light.

In an embodiment, with reference to FIG. 1, FIG. 2, FIG.
3, FIG. 4, FIG. 5, and FIG. 6, optical flow meter 200
includes substrate 210 and microchannel 212 disposed in
substrate 210. Microchannel 212 includes fluid receiver 214
that receives liquid 220 including analyte 222; fluid trans-
mitter 216 that receives liquid 220 from fluid receiver 214;
and fluid member 218 interposed between fluid receiver 214
and fluid transmitter 218. Fluid member 218 includes photo
interaction region 230. Photo interaction region 230 receives

s analytical light 232 and communicates photoanalyte light

246 out of fluid member 218. Microchannel 212 is bounded
by substrate 210. Further, fluid member 218 communicates
liquid 220 from fluid receiver 214 to fluid transmitter 216.
Additionally, optical flow meter 200 includes analytical light
path 238 disposed in substrate 210 and arranged at an
oblique angle or right angle to fluid member 218 proximate
to photo interaction region 230. Analytical light path 238
receives analytical light 232 from analytical light source 240
and communicates analytical light 232 to photo interaction

S region 230, wherein analytical light 232 interacts with

analyte 222 in photo interaction region 230 and produces
photoanalyte 242 from analyte 222 in response to interaction
of analyte 222 with analytical light 232 in photo interaction
region 230. Photoanalyte 242 produces photoanalyte light
246 that is communicated through detection light path 244
disposed in substrate 210. Detection light path 244 is
arranged at an oblique angle or right angle to fluid member
218 proximate to photo interaction region 230 and receives
photoanalyte light 246 from photo interaction region 230
and communicates photoanalyte light 246 from microchan-
nel 212 to photodetector 248.

Optical flow meter 200 can include flow controller 250
that provides liquid 220 to fluid receiver 214. Further,
analytical light source 240 can be in optical communication
with analytical light path 238 and can provide analytical
light 232 to analytical light path 238.

In an embodiment, optical flow meter 200 includes pho-
todetector 248 in optical communication with detection light

5 path 244 and that receives photoanalyte light 246 from

detection light path 244, Moreover, analytical light source
240 can be included in optical flow meter 200, wherein
analytical light source 240 is in optical communication with
analytical light path 238 and provides analytical light 232 to
analytical light path 238.

According to an embodiment, with reference to FIG. 7,
optical flow meter 200 includes a plurality of detection light
paths 244 (e.g., 244A, 244B, 244C, and the like) that
transmit photoanalytical light (e.g., 246A, 246B, 246C, and

5. the like) to photodetectors 248 (e.g., 248A, 248B, 248C, and

the like). It is contemplated that interaction of analytical
light 232 with analyte 222 produces photoanalyte 242
(which may be the same or different chemical species as
analyte 222). Accordingly, analytical light 232 can be scat-
tered or emitted from analyte 222 to produce light that is
received by photodetector 248C via detection light path
244C as photoanalyte light 246C. Analytical light 232 can be
absorbed by analyte 222 to produce photoanalyte 242 (e.g.,
an excited state of analyte 222), wherein a portion of

5 analytical light 232 is not absorbed or scattered and is

transmitted or emitted to photodetector 248A via detection
light path 244A as light referred to as photoanalyte light


US 11,035,707 B2

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U.S. Patent

QT emnsty



US 11,035,707 B2

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Jun. 15, 2021

U.S. Patent



U.S. Patent

Time to 50% rise

Jun. 15, 2021 Sheet 31 of 32 US 11,035,707 B2

1 1

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¥
10! . %
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10 10° 10° 10! 10°

Flow rate (ni/min)

Figure 31


US 11,035,707 B2

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U.S. Patent Jun. 15, 2021 Sheet 23 of 32 US 11,035,707 B2

e—

Figure 23


a2 United States Patent

Cooksey et al.

US011035707B2

(10) Patent No.:
(45) Date of Patent:

US 11,035,707 B2
Jun. 15, 2021

(54)

(71)

(72)

(73)

(*)

(21)

(22)
(65)

(60)

GD

(52)

OPTICAL FLOW METER FOR
DETERMINING A FLOW RATE OF A
LIQUID

Applicant: Government of the United States of
America, as represented by the
Secretary of Commerce, Gaithersburg,
MD (US)

Inventors: Gregory Alan Cooksey, Gaithersburg,

MD (US); Paul Nathan Patrone, Silver

Spring, MD (US); Anthony Jose

Kearsley, Hanover, MD (US)

GOVERNMENT OF THE UNITED
STATES OF AMERICA, AS
REPRESENTED BY THE
SECRETARY OF COMMERCE,
Gaithersburg, MD (US)

Assignee:

Notice: Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
US.C. 154(b) by 446 days

Appl. No.: 15/967,966

Filed: May 1, 2018

Prior Publication Data
US 2019/0120673 Al Apr. 25, 2019

Related U.S. Application Data

Provisional application No. 62/576,709, filed on Oct.
25, 2017

Int. Cl.
GOIS 1166 (2006.01)
GOLF 1466 (2006.01)
(Continued)
US. Cl
cpc GOLF 1/661 (2013.01); C23C 16/00

(2013.01); GOIF 1/704 (2013.01); GOIP 5/26
(2013.01); GOSD 7/00 (2013.01)

(58) Field of Classification Search
CPC .... GOIN 21/6428; GOIN 21/53; GOIN 15/14;
GOIN 21/645; GOIN 15/1459;

(Continued)

(56) References Cited

U.S. PATENT DOCUMENTS

2005/0068536 AL* 3/2005 Schwabe GOIN 21/6428
356/436
2013/0004987 AL* 1/2013 Lo BOLL 3/502715
435/34

OTHER PUBLICATIONS

D. Ross, et al., “Imaging of electroosmotic flow in plastic microchan-
nels”, Anal. Chem., 2001, p. 2509-2515, 73

(Continued)

Primary Examiner — Luke D Ratcliffe
(74) Attorney, Agent, or Firm — Office of Chief Counsel
for National Institute of Standards and Technology

(7) ABSTRACT

An optical flow meter includes a substrate; a microchannel
with a fluid receiver; a fluid transmitter; a fluid member with
an optical interaction region; a photo interaction region; an
analytical light path, such that analytical light interacts with
an analyte in a photo interaction region subsequent to an
interaction of a pre-analyte with activation light in an optical
interaction region to produce analyte; and a detection light
path disposed in the substrate, arranged at an oblique angle
or right angle to the fluid member proximate to the photo
interaction region, and that: receives the photoanalyte light
from the photo interaction region; and communicates the
photoanalyte light from the microchannel to a photodetector,
the optical flow meter determines a flow rate of the analyte.

23 Claims, 32 Drawing Sheets



US 11,035,707 B2

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Jun. 15, 2021

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OT eansty

i 886 ——



U.S. Patent Jun. 15, 2021 Sheet 9 of 32 US 11,035,707 B2

210

Figure 9


US 11,035,707 B2

19

measure flow rate using a time-of-flight principle. In this
example, a non-fluorescent pre-analyte is activated, or con-
verted to analyte, using an activation light at a particular
position in a flow channel. The activation light is pulsed to
produce a narrow band of contrast (e.g. fluorescent analyte)
in the flow. At a measured distance (which corresponds to a
known volume when channel cross section dimensions are
known), the arrival of the now-fluorescently-excitable ana-
lyte as it crosses an analytical light path, and emits light,
which is collected and transmitted to a detector. The fabri-
cation method for the flow meter and measurement process
for the analytical light occur in a similar manner as described
in Example 1. In this example, photobleaching (correspond-
ing to loss of measured intensity) is not a concern because
the measurement is based on how long it takes for the
analyte, once produced at the activation laser, to reach the
analytical light. Photobleaching, as well as the amount of
pre-analyte converted to analyte, can be utilized for changes
in flow rate in real time if the activation laser is operated in
continuous mode rather than in pulsed mode (see FIG. 32).

As in Example 1, optofluidic devices were prepared using
photolithography and cast in  poly(dimethylsiloxane)
(PDMS) against SU8 features on silicon masters. Optical
waveguides were filled with optical adhesive followed by
insertion of optical fibers and UV curing. Fiber-coupled
LEDs (350 nm and 490 nm wavelengths) from ThorLabs
were used with a power meter to deliver and measure light,
respectively. Liquid flow in the optical flow meter were
controlled by a syringe pump (Harvard Apparatus Pico Plus

Elite) and monitored in series with a commercial flow meter 3

(Sensirion LG16-0150D). Flow within the optical flow
meter was visualized with fluorescein (analyte) and CMNB-
caged fluorescein (pre-analyte)

To focus light from the waveguides across the microchan-

nel, lenses with approximately 50 um radius of curvature 3

(Rc) were placed on the ends of the waveguides for activa-
tion and analytical light paths. Lenses with 100 um Re were
placed on the waveguides for detection light. The shape of
the illumination, the width of contrast produced when the
activation light pulse converted pre-analyte to analyte, and
the shape of contrast as it flowed down the microchannel
were imaged with a fluorescence microscope, as shown in
FIG. 27.

FIG. 28 shows an optical flowmeter with repeated optical
elements that can measure the displacement of pre-analyte,
analyte, or photoanalyte as flows occur down the channel. In
such an embodiment, accuracy and knowledge of the time-
dependent nature of the fluid flow is improved by repeated
determination of the flow at multiple points along the
channel. Repeated measurement elements also allow for
observation of time-dependent changes in the photoanalyte,
as was mentioned above.

FIG. 29, FIG. 30, and FIG. 31 show data acquired with the
optical flow meter. In the optical flow meter, CMNB caged
fluorescein in the flow was activated by crossing an activa-
tion region containing UV light. In FIG. 29, the two separate
UV activation pulses (350 nm light) are shown followed by
the corresponding arrival of the analyte as it arrived at the
photo interaction region, as measured by emitted fluores-
cence on the photodetector. Fluorescence emission was
excited and collected by a pair of waveguides at a fixed
distance downstream. Under continuous operation of the
activation light with stable flow, as shown in FIG. 30, the
intensity of the emitted fluorescence reaches a steady state.
The timing of the arrival of the fluorescence signal or other
properties such as the time-to-half-maximum intensity can
be used to calculate the flow rate given the timing of the

S

2

40

&

20

activation pulse and the dimensions of the channel between
the activation and analytical light paths, among other con-
siderations. FIG. 29 shows how using the measurement of
the time for the fluorescence to reach 50% of maximum
(given a continuous or long exposure activation light such as
in FIG. 30) is used to create a calibration of flow rates based
on time-of-flight measurements of the analyte (fluorescein)
after its creation from pre-analyte at the optical interaction
region (activation zone). Measurements were reproducible
with greater than 10% accuracy over approximately 3 orders
of magnitude in flow, from 20 nL/min to about 20 wL/min.

Additionally, as shown in FIG, 31, the optical flow meter can
provide a continuous flow measurement when the activation
light is continuous, but the flow is not stable. Changes in the

5 flow rate, such as occurs in syringe pumps as the motor

advances through individual steps, can be easily visualized
in the fluorescence intensity measurements.

The optical flow meter efficiently delivers and collects
light across a microfluidic channel and can also be used to
measure dynamic fluid properties and to characterize par-
ticles and cells in microfluidic cytometry as they flow past
activation and analytical light paths.

While one or more embodiments have been shown and
described, modifications and substitutions may be made
thereto without departing from the spirit and scope of the
invention. Accordingly, it is to be understood that the present
invention has been described by way of illustrations and not
limitation. Embodiments herein can be used independently
or can be combined.

Reference throughout this specification to “one embodi-
ment,” “particular embodiment,” “certain embodiment,” “an
embodiment,” or the like means that a particular feature,
structure, or characteristic described in connection with the
embodiment is included in at least one embodiment. Thus,
appearances of these phrases (e.g., “in one embodiment” or
“in an embodiment”) throughout this specification are not
necessarily all referring to the same embodiment, but may.
Furthermore, particular features, structures, or characteris-
tics may be combined in any suitable manner, as would be
apparent to one of ordinary skill in the art from this
disclosure, in one or more embodiments.

All ranges disclosed herein are inclusive of the endpoints,
and the endpoints are independently combinable with each
other. The ranges are continuous and thus contain every
value and subset thereof in the range. Unless otherwise
stated or contextually inapplicable, all percentages, when
expressing a quantity, are weight percentages. The suflix
“(s)” as used herein is intended to include both the singular
and the plural of the term that it modifies, thereby including
at least one of that term (e.g. the colorant(s) includes at least
one colorants). “Optional” or “optionally” means that the
subsequently described event or circumstance can or cannot
occur, and that the description includes instances where the
event occurs and instances where it does not. As used herein,
“combination” is inclusive of blends, mixtures, alloys, reac-
tion products, and the like.

As used herein, “a combination thereof” refers to a
combination comprising at least one of the named constitu-
ents, components, compounds, or elements, optionally
together with one or more of the same class of constituents,
components, compounds, or elements.

All references are incorporated herein by reference.

The use of the terms “a” and “an” and “the” and similar
referents in the context of describing the invention (espe-
cially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by con-


U.S. Patent Jun. 15, 2021 Sheet 30 of 32 US 11,035,707 B2

0.8

0.6

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6: : : : .
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Figure 30


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U.S. Patent Jun. 15, 2021 Sheet 20 of 32 US 11,035,707 B2

Flow liquid 220 with pre-analyte Produce activation light’and

Produce analytical light 232 221 through microchannel 212 at ee ek ap
record time of first. production

unknown constant rate

Communicate analytical Communicate flow liquid 220 Communicate activation light
light 282 into interacti with pre-analyte 221 into vommuanieale ACHN@HOD AB
ae nin : into activation region
region 230 activation region

Create analyte 222

Communicate flow liquid 220
with activated analyte into
interaction region 230

Create photo-analyte 242

Communicate photo-analyte
light 246 te photodetector 248
via detection light path 244

Record first detection time for
analytical tight

Compute flow-rate as ratio of
distance and time traveled
between interaction regions

Figure 20


US 11,035,707 B2

9

to emit fluorescent light and some of photoanalyte 242 to
bleach (e.g., be destroyed by the analytical light and stop
emitting fluorescence). The intensity of the analytical light
with the amount of time the photoanalyte spends in the photo
interaction region 230 (e.g., the speed of the photoanalyte)
can determine an amount of bleaching versus emitted fluo-
rescence.

In optical flow meter 200, photoanalyte 242 can be a
deactivated state of analyte 222, an excited state of analyte
222, or a modified variant of analyte 222. The type of
photoanalyte 242 can be selected based on an interaction
time with analytical light 232, color of emitted or scattered
light, or an increase in signal to noise ratio in photodetector
248.

In optical flow meter 200, analytical light path 244 carries
detection light from photo interaction region 230. The light
path can include filters or bifurcations to reduce the intensity
of light or direct part of the light to a light intensity meter or
spectrometer. In an embodiment, detection light path 244 is
a waveguide filled with optical material that permits total
internal refection of light. In an embodiment, the optical
material is a photocurable adhesive that has a higher index
of refraction than the substrate. In an embodiment, the
detection light path includes a lens element to focus light
into the light path and is slightly wider than the analytical
light path in order to capture more light from photo inter-
action region as shown in FIG. 24.

In optical flow meter 200, analytical light 246 can be
fluorescence, scattered light, or a change in transmission of
the analytical light 232 resulting from interaction of ana-
lytical light 232 with photoanalyte 242 in optical interaction
region 230. In an embodiment the detection light is emitted
light from a fluorescent photoanalyte. In an embodiment, the
detection light is loss of fluorescence due to photobleaching

of the photoanalyte. Detection light 246 can be modified by 3

components of output light path 244. This light can have a
wavelength from 240 nm to 1000 nm, and specifically from
350 nm to 750 nm.

In optical flow meter 200, photodetector 248 records
properties of detection light 246. In an embodiment, pho-
todetector 248 is connected to the detection light path by an
optical fiber. In an embodiment, the optical fiber is a
125-micrometer diameter multimode fiber that carries a
visible wavelength of light. In an embodiment, the photo-
detector is a broadband photodiode connected to a power
meter. In an embodiment, the photodiode had a spectral
range from 200 nm to 1100 nm. In an embodiment, the
photodiode is preceded by an optical filter that excludes the
analytical light to enhance collection of emitted fluorescence
light over analytical light. In an embodiment the filter blocks
wavelengths outside the range from 510 nm to 530 nm,
allowing only fluorescence emitted from fluorescein to reach
the detector. In an embodiment, the combined photodiode/
power meter has a sensitivity in a power range from 10 pW
to 100 mW. In an embodiment, the photodetector is a
spectrometer that measures a spectrum of detection light
246. The photodetector can be a photodiode or the like
integrated into the flow meter or interfaced with the sub-
strate.

In an embodiment, optical flow meter 200 is connected to
a flow controller 250 that serves to drive fluid and analyte
through the system. In an embodiment, the flow controller is
a syringe pump that delivers volumetric flow in the range of
100 picoliter per minute (pL/min) to 100 microliters/min. In
an embodiment, the flow controller is a vessel filled with
fluid at a fixed height above the outflow of the meter. Gravity
can control pressure on the fluid and drive the flow rate as

0

30

40

&

0

10

a function of the fluidic resistance through the system. In an
embodiment, external pressure is applied to the fluid-filled
vessel in order to provide more pressure drop than can be
reasonably achieved with gravity alone. Other flow control-
lers can be attached to the flow meter and modified with or
without feedback from the flow meter to control volumetric
flow. Exemplary flow controllers include peristaltic and
rotary pumps, piston pumps, diaphragm pumps, and the like.

Optical flowmeter 200 can include additional light paths
for light collection. In an embodiment, detection light path
244 communicates detection light 246.4 to a power meter
248A in order to acquire a change in intensity or spectrum
of light that is transmitted through the photoanalyte. In an
embodiment, this measurement estimates power of the ana-
lytical light incident on the photoanalyte and measures
time-dependent changes in the interaction between the ana-
lytical light and the photoanalyte through processing of
acquired light.

In an embodiment, a supplementary detection light path
244B collects and communicates light 246B from the photo
interaction zone to a photodetector 248B. In an embodiment,
light path 244B is on the same side of the microchannel as
the analytical light and oriented at some angle and shape in
order to maximize collection of emitted or scattered light

s from the photoanalyte while minimizing the amount of

analytical light reflected into light path 244B.

In an embodiment, a supplementary detection light path
244C collects and communicates light 246C from the photo
interaction zone to a photodetector 248C. In an embodiment,
light path 244C is on the opposite side of the microchannel
as the analytical light and oriented at some angle and shape
in order to maximize collection of emitted or scattered light
from the photoanalyte while minimizing the amount of
analytical light reflected into light path 244C.

Optical flowmeter 200 can include repeated optical units
252 that independently can include an analytical light 232
and analytical light path 238; detection light 246A and
detection light path 244A; detection light 246B and detec-
tion light path 244B; or detection light 246C and detection
path 244C.

Inan embodiment, repeated optical elements 252 measure
the displacement of photoanalyte as it flows down the
channel. In such a way, accuracy and knowledge of the
time-dependent nature of the fluid flow is improved. In
addition, convention and diffusion of the photoanalyte is
determined. Moreover, time-dependent changes in the pho-
toanalyte, including photoanalyte disposed as a droplet or
vesicle containing a time-dependent process such as an
enzymatic reaction, or a photoanalyte that appear or degrade
as a result of a biological process contained in a cell, are
measured as particles move, with time, past subsequent
repeated optical elements. Furthermore, repetitive measure-
ments of photoanalytes such as those just mentioned that are
not changing in time would provide additional validation of
the measurements and produce means to assess the statistical
variation from repeat measurements

It is contemplated that flow meter 200 includes a plurality
of microchannels to measure flow rates from a plurality of
samples in a monolithic apparatus. A bifurcation in the flow
member scales measured flow from one split of the fluid to
another. In such a way, ratios of a large flow are provided as
smaller volumetric components to determine flow ina single
apparatus.

In optical flow meter 200, activation light source 236 can
include light-emitting diodes or lasers to emit light at a
desired wavelength. The properties of the source light and
type can be selected based on packaging of the flow meter,


US 11,035,707 B2

11

level of interaction with pre-analyte 221 or analyte 222, and
transmission of light by activation light path 224.

In optical flow meter 200, activation light 226 can be used
to imprint contrast to analyte 222 or pre-analyte 221. In an
embodiment, pre-analyte 221 is a caged fluorophore. In an
embodiment, the analyte is fluorescein. The activation light
can be selected based on whether activation light interacts
with the pre-analyte or analyte. A wavelength of light can be
from 240 nm to 1000 nm, and specifically from 350 nm to
750 nm. In an embodiment, the wavelength of the activation
light is 375 nm. The intensity of the activation light is
selected based on optical properties for modifying the ana-
lyte or pre-analyte, the nature of the flow, and selected
characteristics of detected light. Intensity of the activation
light is from 1 nW to 100 mW. In an embodiment, the
intensity of the activation light is from 1 microwatt to 70
mW.

In optical flow meter 200, analytical light path 224
includes a waveguide that transmits light from light source
236 to optical interaction region 228. Moreover, the light
path can contain filters or bifurcations to reduce the intensity
of light or direct part of the light to a light intensity meter.
In an embodiment, activation light path 224 was a wave-
guide filled with optical material that permits total internal
refection of light. In an embodiment, the optical material
was a photocurable adhesive that has a higher index of
refraction than the substrate. In an embodiment, the activa-
tion light path had a curved end or lens to shape the light in

the optical interaction region 228. In an embodiment, the °

light was approximately uniform intensity across the optical
interaction region.

In optical flow meter 200, optical interaction region 228
is where activation light 226 interacts with the analyte 222

S

8

2

or pre-analyte 221 to modify the analyte 222 or pre-analyte ©

221. In an embodiment, modification of pre-analyte 221
produces analyte 222. A modification of pre-analyte 221 can
include a chemical modification such as uncaging, fluores-
cence activation, photobleaching, and the like. In an
embodiment, the analyte 222 or pre-analyte 221 are a
fluorophore or a caged-fluorophore such as fluorescein or
caged-fluorescein.

‘A process for making optical flow meter 200 includes
defining the microflow channel and light paths in the sub-
strate. The process for defining the flow channel can involve
subtractive manufacturing (e.g. laser ablation, engraving,
cutting, etching, etc.) or additive manufacturing (e.g. 3D
printing, controlled deposition, templated substrate growth,
and the like) or photolithography to define topographic
features that will become or will be used to produce con-
stituent elements of the flow meter. In an embodiment,
topographic features were produced on silicon wafers using
photolithography. A negative photoresist polymer (e.g.,
SU8) was used as the photosensitive polymer to produce the
topographic patterns on a silicon wafer. In an embodiment,
the SU8 features were approximately the same height as the
optical fiber diameter. The SU8 features are typically treated
with a silane to facilitate removal of substrates after casting.
Alternative methods to produce topographic features include
milling and embossing of metals or plastics or etching
materials such as glass and silicon.

In an embodiment, features in substrate 210 are produced
by casting and curing a liquid material, such as poly(dim-
ethylsiloxane) (PDMS) against the topographic features.
Other exemplary casting materials include epoxy, adhesives
or curable polymers or melts. Alternative means of fabri-

40

&

12

cating a substrate in the milli or micro or nanoscale size
range include 3D printing, cutting, milling, laser writing,
and embossing.

In an embodiment, the substrate is bonded to another
substrate so as to form a bottom of the channel features and
to form open-ended microchannels that include the flow
member and light paths. In an embodiment, the substrate is
bonded to a flat surface of the same material as the cast
substrate so that the index of refraction of the material is the
same around the exterior of the light guides. In an embodi-
ment, the substrate and bonded layer were PDMS and the
bonding was performed by oxygen plasma treatment of both
layers prior to placing them together. In an embodiment, the
PDMS layers are bonded together using a layer of liquid

s PDMS and the device bond following heat treatment to cure

the liquid PDMS. Other methods to form the enclosed
channels include using an adhesive such as epoxies, double-
sided tape, or a mechanical force provided by a device such
as a clamp or vacuum pump.

In an embodiment, the light paths are filled with an
appropriate material such as an optical liquid or adhesive or
matching fluid to produce total-internal reflection and carry
light along the microchannel. Alternatively, the light paths
can be formed by modifying the optical properties of the

substrate, e.g, by chemical, thermal, or optical manipulation,

such that these regions become capable of confining and
transmitting light as a waveguide. As waveguides, the light
paths then function to deliver light to and collect light from
material in the photo interaction region of the flow member.
In an embodiment, the light paths are filled with PDMS that
has a higher crosslinker ratio (e.g. 1:5). In an embodiment,
the material filling the light paths was a UV curable optical
adhesive (e.g., commercially available as Norland Optical
Adhesive 88), which has a higher index of refraction com-
pared to PDMS.

In an embodiment, a process for making optical flow
meter 200 includes incorporating an optical fiber to transmit
light from sources and detectors to and from the device. In
an embodiment, the height of channel for the light paths was
set to match the diameter of bare fiber, and the width at the
entrance of the light path channel where the fiber is inserted
is set to be a small factor (e.g. 3 times) larger than the
diameter of the fiber. In an embodiment, the width of the
light path channel tapers down to width of the fiber to stop

5 the fiber insertion at a certain point. In an embodiment, the

width of the light path was narrowed further (e.g., 50
micrometers wide) in order to more carefully pinpoint the
light impinging on the flow member (see FIG. 9) or widened
(e.g. 150 um) to collect more light from the flow member.
Following insertion of the fibers into the light path, an
embodiment was to cure the coupling fluid with ultraviolet
light or heat to lock the fibers in place. Alternatively, the
waveguides can be solid, and light can be launched into the
light paths by gluing fibers to the ends of light paths or

5 focusing light into light paths using optical elements.

Optical flow meter 200 has numerous beneficial uses,
including determining a flow rate of liquid 220. In a process
for determining flow rate of the liquid, analyte 242 is
produced at 230 from analyte 222. In this process, the
analyte 222 can be fluorescent.

In an embodiment, with reference to FIG. 18, a process
for determining the flow rate of liquid 220, with optical flow
meter 200, includes: flowing liquid 220 including analyte
222 through microchannel 212; communicating liquid 220

5 with analyte 222 to photo interaction region 230; producing

analytical light 232; communicating analytical light 232
through analytical light path 224 to photo interaction region


US 11,035,707 B2

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US 11,035,707 B2

21

text. “Or” means “and/or.” Further, the conjunction “or” is
used to link objects of a list or alternatives and is not
disjunctive; rather the elements can be used separately or
can be combined together under appropriate circumstances.
It should further be noted that the terms “first,” “second,”
“primary,” “secondary,” and the like herein do not denote
any order, quantity, or importance, but rather are used to
distinguish one element from another. The modifier “about”
used in connection with a quantity is inclusive of the stated
value and has the meaning dictated by the context (e.g., it
includes the degree of error associated with measurement of
the particular quantity).
What is claimed is:
1. An optical flow meter that determines a flow rate of a
liquid and comprising:
a substrate;
a microchannel disposed in the substrate comprising:
a fluid receiver that receives a liquid comprising an
analyte;
a fluid transmitter that receives the liquid from the fluid
receiver; and
a fluid member interposed between the fluid receiver
and fluid transmitter and that:
comprises:
an optical interaction region and that receives
activation light; and
a photo interaction region and that:
receives analytical light: and
communicates photoanalyte light out of the
fluid member;
is bounded by the substrate; and
communicates the liquid from the fluid receiver to
the fluid transmitter;
an analytical light path disposed in the substrate, arranged

at an oblique angle or right angle to the fluid member 3

proximate to the photo interaction region, and that:
receives the analytical light from an analytical light
source; and
communicates the analytical light to the photo interac-
tion region,
such that the analytical light:
interacts with the analyte in the photo interaction
region subsequent to the interaction of the analyte
with the activation light in the optical interaction
region; and
produces a photoanalyte from the analyte in response
to interaction of the analyte with the analytical
light in the photo interaction region,
the photoanalyte producing photoanalyte light; and
a detection light path disposed in the substrate, arranged
at an oblique angle or right angle to the fluid member
proximate to the photo interaction region, arranged on
a same side of the fluid member as the analytical light
path, and that:
receives the photoanalyte light from the photo interac-
tion region;
communicates the photoanalyte light from the micro-
channel to a photodetector, the optical flow meter
determines a flow rate of the analyte in the liquid;
and
maximizes collection of emitted or scattered light from
the photoanalyte while minimizing an amount of
analytical light collected into the detection light path.
2. The optical flow meter of claim 1, further comprising:
an activation light path disposed in the substrate, arranged
at an oblique angle or right angle to the fluid member
proximate to the optical interaction region, and that:

45

50

60

22

receives activation light from an activation light source;
and
communicates the activation light to the optical inter-
action region,

such that the activation light interacts with the analyte in

the optical interaction region.

3. The optical flow meter of claim 1, further comprising
a flow controller that provides the liquid to the fluid receiver.

4. The optical flow meter of claim 2, further comprising
the activation light source in optical communication with the
activation light path and that provides the activation light to
the activation light path.

5. The optical flow meter of claim 1, further comprising
the analytical light source in optical communication with the
analytical light path and that provides the analytical light to
the analytical light path.

6. The optical flow meter of claim 1, further comprising
the photodetector in optical communication with the detec-
tion light path and that receives the photoanalyte light from
the detection light path.

7. The optical flow meter of claim 1, further comprising
the analytical light source in optical communication with the
analytical light path and that provides the analytical light to
the analytical light path.

8. The optical flow meter of claim 2, wherein the activa-
tion light path comprises a waveguide,

9. The optical flow meter of claim 1, wherein the ana-
lytical light path comprises a waveguide.

10. The optical flow meter of claim 1, wherein the
detection light path comprises a waveguide.

11. The optical flow meter of claim 1, wherein the
photoanalyte light comprises emitted light, scattered light,
transmitted light, or a combination comprising at least one
of the foregoing types of light.

12. The optical flow meter of claim 1, wherein the
detection light path comprises an emission waveguide, a
scattered light waveguide, a light obscuration waveguide, or
a combination comprising at least one of the foregoing types
of light paths.

13. The optical flow meter of claim 1, wherein a plurality
of photo interaction regions, a plurality of analytical light
paths, and a plurality of detection light paths are disposed on
the substrate,

wherein the optical interaction region is interposed

between the fluid receiver and the photo interaction
regions.

14. A process for determining a flow rate of a liquid, with
the optical flow meter of claim 1, the process comprising:

flowing the liquid comprising an analyte through the

microchannel;

communicating, in the fluid member, the analyte to the

photo interaction region;

communicating analytical light through the analytical

light path to the photo interaction region, wherein the
photo interaction region is a portion of the microchan-
nel that receives the analytical light and where the
analytical light interacts with the analyte;

subjecting the activated analyte to the analytical light;

producing a photoanalyte from the activated analyte in

response to subjecting the analyte to the analytical
light;

producing photoanalyte light from the photoanalyte;

communicating the photoanalyte light from the photo

interaction region to the detection light path to deter-
mine the flow rate of the liquid.

15. The process for determining the flow rate of the liquid
of claim 14, further comprising:


US 11,035,707 B2

Sheet 13 of 32

Jun. 15, 2021

U.S. Patent

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US 11,035,707 B2

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US 11,035,707 B2

7

embodiment, the flow member is made in a curable polymer
such as PDMS cast from a photoresist pattern provided in a
photolithographic process. In an embodiment, the flow
member is formed by laser oblation or chemical etching of
a material such as glass. The flow member can be made by
milling, cutting, embossing, and the like. Printing, e.g., 3D
printing of the substrate can form the flow member or other
structural element of flow meter 200.

Dimensions of the flow member are selected based, e.g.,
on a type of flow through flow meter 200. Dimensions such
as width (W) or height (H) of the flow member (in direction
X or Z) can be from 10 nm to 1 mm, specifically from 1
micrometer to 500 micrometers for water. Dimensions can
depend on speed of fluid through the flow meter as well as
pressure on the fluid. A shape of the flow member can be,
e.g., rectangular, circular, or a combination thereof and can
have a uniform or non-uniform cross section or can be
confined to a straight line or fixed within a single plane along
its length. For example, the flow member can include a wall
that bounds the flow member and that can curve, bend, split
or include or a junction that routes liquid in the flow system.
A length (L) of microchannel 212 can be selected based on
the size of the analytical region and that provides selected
convective or diffusion of the liquid medium between opti-
cal interrogation zones. The microchannel length can be
from 1 micrometer to 1 meter, and specifically from 10
micrometers and 10 centimeters. In an embodiment, the flow
member has a rectangular cross section and has width and

height that are independently 125 micrometers. In an 3

embodiment, the flow member is a glass capillary that has an
internal diameter of 150 micrometers.

In optical flow meter 200, fluid receiver 214 and fluid
transmitter 216 connect the meter to an external source of

fluid or sinks, respectively, or to fluid transmitter or fluid 3

receiver from another flow meter, respectively. The fluid
receiver and fluid transmitter can provide smooth transition
to the flow member and can have similar cross-sectional
dimensions. Moreover, a fluid receiver or fluid transmitter
can include a seal or a mechanical device so that all fluid is
directed down the microchannel. Additionally, fluid trans-
mitter 216 can include valves or bifurcations to control
liquid flow down the channel.

Fluid flows through the microchannel and can be a gas,
liquid, or a combination thereof. It is contemplated that the
fluid includes solid particles, compounds, or small mol-
ecules disposed in a liquid or gas. Exemplary fluids include
water, bodily fluids, and organic solvents such as alcohols
and fuels. The solid particles, compounds, and small mol-
ecules can include chemical compounds that absorb, scatter
or emit light such as fluorescent dyes, inorganic or organic
micro- or nano-particles, biological materials such as pro-
teins and protein aggregates, lipid vesicles, exosomes,
organelles and cells. Moreover, the fluid can include a
combination of a solution of different materials.

Depending on substrate 212, fluid, and dimensions of the
flow member, a pressure of the fluid in the system can be
from 0 Pascals (Pa) to 700 kPa, specifically from 0 Pa to 130
kPa, and more specifically from 0 Pa to 70 kPa. A viscosity
of the fluid can be from 1x10~¢ to 1 Pa's, specifically from
1.0x10-° to 0.1 Pars, and more specifically from 1xi0~* to
0.01 Pa:s. Flow can be unidirectional, and pressure can be
positive. With respect to FIG. 1 through FIG. 7, the device
performs similarly with flow in either direction. In an
embodiment, with reference to FIG. 8, a plurality of flow
readers 252 can be used to determine absolute direction of
the flow.

40

&

3!

0

8

In optical flow meter 200, analyte 222 and pre-analyte 221
can independently include reflective, absorptive, fluores-
cent, reactive, or optically active particles that permit con-
trast in the presence of light. The analyte and pre-analyte
independently can be made, e.g. from fluorescein and
derivatives thereof. Specifically, a derivative could be fluo-
rescein-conjugated dextran or a CMNB-caged carboxyfluo-
rescein, and its derivatives. The analyte or pre-analyte
independently can be selected based on the wavelength of
light to activate, deactivate, or excite the analyte or pre-
analyte, respectively. The analyte or pre-analyte can also be
selected, independently, based on its diffusion coefficient,
absorbance, or emission spectra. Analyte and pre-analyte
excitation, bleaching, and emission wavelengths indepen-

5 dently can be from 240 nm and 1000 nm, and specifically

from 350 nm and 750 nm.

In optical flow meter 200, activation light paths (224, 238,
244) can be disposed in substrate 212 as a microchannel,
e.g., a void in substrate 212. The light paths are in optical
communication with light source (e.g., 236, 240) or photo-
detector 248 by an optical fiber or along light paths as
activation light 226, analytical light 232 or photoanalyte
light 246. The light path can include a bifurcation or optical
element that changes a direction, intensity, or spectrum of

5 light along the light path.

In optical flow meter 200, analytical light source 240 can
include light-emitting diodes or lasers to emit light at a
desired wavelength. The properties of the source light and its
type can be selected based on the packaging of the flow
meter, level of interaction with analyte 222, or transmission
of light by activation light path 238.

In optical flow meter 200, analytical light 232 can be used
to induce fluorescence, scatter, or absorption in photoanalyte
242 in optical interaction region 228. In an embodiment the
photoanalyte 242 is fluorescein. The analytical light may be
selected based on interaction with photoanalyte 242 and can
have a wavelength from 240 nm to 1000 nm, more specifi-
cally from 350 nm to 750 nm. In an embodiment, the
wavelength of the analytical light is from 450 nm to 500 nm.
In an embodiment, the wavelength is from 470 nm to 490
nm. An intensity of the analytical light is selected based on
interrogation of the photoanalyte, nature of the flow, and
desired characteristics of the detected light. The intensity of
the analytical light can be from 1 nanowatt (nW) to 1 W. In

5 an embodiment, the intensity of the analytical light can be

from 1 microwatt to 70 mW.

In optical flow meter 200, analytical light path 238
transmits light from light source 240 to photo interaction
region 230. Moreover, the light path can include a filter or
bifurcation to reduce the intensity of light or direct part of
the light to a light intensity meter. In an embodiment,
analytical light path 238 includes a waveguide filled with
optical material that permits total internal reflection of light.
In an embodiment, the optical material is a photocurable
adhesive that has a higher index of refraction than the
substrate. In an embodiment, the analytical light path has a
curved end to shape the light in the optical interaction region
230. In an embodiment, the light is approximately uniform
intensity across the optical interaction region as shown in
FIG, 24.

In optical flow meter 200, photo interaction region 230 is
where analytical light 232 interacts with the analyte 222 or
photoanalyte 242 to induce fluorescence, bleaching, scatter,
or absorption by the analyte 222 or photoanalyte 242,

5 independently. In an embodiment, the analytical light

induces the photoanalyte to emit fluorescence. In an embodi-
ment, the analytical light causes some of photoanalyte 242


US 11,035,707 B2

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Jun. 15, 2021

U.S. Patent

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US 11,035,707 B2
Page 2

(51) Int. CL

GOLF 1/704 (2006.01)
€23C 16/00 (2006.01)
GOIP 5/26 (2006.01)
GOSD 7/00 (2006.01)

(58) Field of Classification Search
CPC... BOIL 3/502776; BOIL 3/502715; BOIL
2200/0636

See application file for complete search history.

(56) References Cited

OTHER PUBLICATIONS

M. Ahrens, et al., “Design and uncertainty assessment of a setup for
calibration of microfluidic devices down to SnL min-1”, Measure-
ment and Science Technology, 2014, p. 1-9, 25.

M. Ahrens, et al., “An experimental setup for traceable measure-
ment and calibration of liquid flow rates down to 5 nL/min”,
Biomed. Eng.-Biomed. Tech., 2015, p. 337-345, 60(4).

H. Hosseinkhannazer, et al., ““Two-species microparticle detection
in optofluidic biochips with polymeric waveguides”, Proceedings of
SPIE: Photonics North 2008, 2008, vol. 7099

V. Lien, et al., “Microfluidic flow rate detection cased on integrated
optical fiber canitlever”, The Royal Society of Chemistry: Lab on a
Chip, 2007, p. 1352-1356, 7

E. Pedrol, et al., “Optoftuidic device for the quantification of
circulating tumor cells in breast cancer”, Nature: Scientific Reports,
2017

* cited by examiner


U.S. Patent Jun. 15, 2021 Sheet 32 of 32 US 11,035,707 B2

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US 11,035,707 B2

17

mines simultaneously optical properties of the analyte or
photoanalyte using alternate photoanalyte light paths (e.g.
244B, 244C). Beneficially, the changing the distance
between the optical activation region and the photo analyti-
cal zone can change the range of flow velocities and fluid
(e.g., viscosity) or analyte properties (e.g., diffusion con-
stant) where the flow meter performs and sets the precision
for time-of-flight measurements of convection or diffusion
of analyte or photoanalyte between the activation and mea-
surement points.

Optical flow meter 200 advantageously and unexpectedly
tracks changes in the flow rate with the activation laser on
continuously, e.g. not pulsed (see FIG. 10). Unexpectedly,
optical flow meter 200 has a precision of scaling the flow
rate with laser power that is not a strong function of the
shape of the microchannel or the optical or photo interaction
regions. Beneficially, the optical flowmeter does not require
a microscope to achieve measurements of low flow rates or
low uncertainties.

The articles and processes herein are illustrated further by
the following Examples, which are non-limiting

EXAMPLES

Example 1. Optical Flow Meter with a Waveguide
Using Photobleaching

The optical flow meter measures convection or diffusion
of an activated compound, e.g., a fluorescent marker, at a

measurement point in a liquid flowing through a microchan- 3

nel. FIG. 22, FIG. 23, FIG. 24, and FIG. 25 show optical
flow meters in which fluorescent molecules (fluorescein) are
stimulated with analytical (excitation) light, and emit pho-
toanalyte light (fluorescence) that is collected by a photoana-

lyte light path (optical waveguide) and measured on a 3

photodetector. At relatively high excitation laser power or a
relatively low flow rate, the fluorescent molecules are pho-
tobleached, that is, they are destroyed by the light and no
longer emit fluorescence. Thus, the flow meter provides
measurement of the flow rate based on the fraction of how
much fluorescence remains (relative to maximum, saturated,
or calibrated emission at the highest or calibrated flow rate)
for a particular flow rate and laser power. Laser power
adjustments allow the sensitivity of flow rate to shift to
different speeds, e.g. higher laser power enables measure-
ment of faster flows, and lower laser power enables mea-
surement of lower flows (for a given signal-to-noise ratio of
fractional fluorescence).

The flow meter was made by first creating topographic
features on silicon wafers using photolithography. For the
optical flow meter, photolithography was conducted at the
Center of Nanoscale Technology (CNST) at NIST. A nega-
tive photoresist made of a polymer (here, SU8) was used as
a photosensitive polymer to produce topographic patterns on.
a silicon wafer. The features on the SU8 were approximately
125 micrometers tall. The features on the SU8 were treated
with a silane for removal of substrates after casting. Devices
were made of poly(dimethylsiloxane) (PDMS) after casting
and curing against the topographic features. Two PDMS
layers were bonded together to enclose a device containing
open-ended microchannels and light paths. The light paths
were filled with a UV-curable optical adhesive (commer-
cially available as Norland Optical Adhesive 88) that had a
higher index of refraction compared to PDMS. Cleaved
optical fibers connected to light sources and power meters
were inserted into light paths and then cured with UV light.
Analytical light was supplied by an Omicron 488 nm laser

3

40

g

18
with up to 60 mW output power. The power meter was a dual
channel meter from Newport Corporation fitted with pho-
todiode sensors having sensitivity in the 400 nm to 1100 nm
wavelength range.

‘An analyte (e.g., fluorescein) in a flow was excited when
the analyte crossed an optical path that communicated
analytical light absorbed by the analyte or that communi-
cated fluorescence from the analyte. To determine changes
in absorption (or obscuration) of analytical light, light was
collected from the opposite side of the microchannel from
the analytical light (as shown in FIG. 22). To determine
changes in emitted or detected light. the light was collected
at some angle relative to the analytical light on the same side
of the microchannel as the analytical light (as shown in
FIGS. 23 and 24). In addition, the amount of light scattered

S from the analyte can be collected in a waveguide on the

opposite side of the microchannel as the analytical light but
at a slight angle (as shown in FIG. 23). Light was collected
in the optical paths and was delivered to a detector that was
a power meter.

The waveguide collecting fluorescence emission was at a
selected angle and selected distance from the source wave-
guide to maximize emitted light collection while reducing an
amount of excitation light communicated to the detector. In
this respect, with reference to FIG. 24, a microscope imaged
488 nm light impinging on a stream of fluorescein, which
represents the area of measurement through which the
activated molecule (fluorescein) passed. Fluorescein flowed
down the central microfluidic channel and lit up when it
crossed the excitation path, which was collected by the
microscope as shown in the inset of FIGS. 22 and 24 and by
the power meter(s). Fluorescence emission from the fluo-
rescein can be collected via the emission path, and the
excitation light can be excluded using optical filters. In this
example, the optical filter was a bandpass filter than passed
greater than 95% of light between 500 nm and 540 nm,
which blocking greater than 99.99% of light outside of that
range from reaching the power meter measuring fluores-
cence emission. This wavelength range was chosen because
it maximizes transmission of the emitted fluorescence light
from fluorescein while excluding the analytical light.

Advantageously, the optical flow meter measures the
change in fluorescence emission with respect to a maximum,
saturated, or calibrated fluorescence intensity as a function
of flow rate (or in reference to a calibrated flow rate), as
shown in FIG, 25. In FIG, 25, the calibrated flow rate was
a commercial flow meter specified to have accuracy to
within 350 nl/min (5% of full scale) over the range shown.
Loss of fluorescence intensity (e.g. photobleaching) as a
function of flow rate is demonstrated in the microscope
images the top of FIG. 25. The upper panel of FIG. 25 shows
a microscope view of the amount of fluorescence being
emitted for a particular analytical light power as a function
of flow rate. At slower flows, the photoanalyte spends more
time in the interaction region and tends to be photobleached
and no longer emits light. At very fast flow rates, there is no
bleaching of the analyte and the full region of analytical light
interacting with the analyte is visible. Fluorescent molecules
on the sides of the channels are most likely to bleach first due
to the no-slip boundary condition (e.g. zero velocity at the
channel walls) in pressure driven flows. The lower panel
shows a plot of measured photoanalyte light as a function of
flow rate.

Example 2. Optical Flow Meter with Activation
Light for Time-of-Flight Measurement of Flow
Rate

FIGS. 26 and 27 show optical flowmeters that use an
activation light source and an analytical light source to


U.S. Patent Jun. 15, 2021 Sheet 27 of 32 US 11,035,707 B2

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US 11,035,707 B2

Sheet 11 of 32

Jun. 15, 2021

U.S. Patent

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US 11,035,707 B2

Sheet 14 of 32

Jun. 15, 2021

U.S. Patent

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