US 11,624,664 B2

9

variable. To train the model, measurement of RATIO2 are
obtained by introducing aqueous solution of known pH
values to pH photothermal spectrometer 200. The measured
RATIO2 values and corresponding pH values are input to
the regression model to calculate a linear relationship
between RATIO2 and pH. To measure the pH of analyte
medium 204 whose pH may not be known prior to mea-
surement, RATIO2 is measured using 200 following which
the RATIO? is used as input variable in calibration model to
solve for pH.

It should be appreciated that pH photothermal spectrom-
eter 200 and proces disclosed herein have numerous
beneficial uses including a small form factor and biocom-
patibility of constituent components. Advantageously, pH
photothermal spectrometer 200 overcomes limitations of
technical deficiencies of conventional devices such as tem-
perature dependence by employing ratiometric measure-
ments. Further, pH photothermal spectrometer 200 does not
require excitation light 208 and 209 to be measured after

interaction with chromophore removing a technical barrier 2

to the use of optical pH sensors where losses in optical
power of excitation light 208 and 209 are a source of
measurement error.

Beneficially, pH photothermal spectrometer 200 does not

use toxic materials such as metals, corrosive salts or toxic ?

dyes. Moreover, pH photothermal spectrometer 200 and
processes herein unexpectedly provide a low drift measure-
ment where the device does not need to be recalibrated over
a period of 2-3 weeks. Conventional devices require recali-
bration daily to provide accurate pH measurement. As such,
pH photothermal spectrometer 200, decontaminated and
calibrated, can be embedded into an analyte, e.g., a micro-
tissue or biomanufacturing vessel, and used over the course
of cell proliferation, differentiation and activity cycle with-
out loss of measurement accuracy. The ability to embed pH
photothermal spectrometer 200 for duration of cell life
eliminates a potential route through which deleterious infec-
tion could be introduced.

The articles and processes herein are illustrated further by
4

the following Examples, which are non-limiting.
EXAMPLES
Example 1. Photothermal Detection of pH
Here, a pH photothermal spectrometer measured a local
pH over a small volume that was contained in an intersection
of a light cone and a light receiver over several days without
recalibration, wherein data is shown in FIG. 6

Example 2. Photothermal Detection of pH

An excitation optical fiber with embedded chromophore
measured changes in the absorption spectra of a chro-

mophore due to changes in pH of the analyte, wherein data 5

is shown in FIG. 7.

Example 3. Photonic pH Sensor Based on
Photothermal Spectroscopy

Three-dimensional tissue analogues and cell-laden con-
structs composed of synthetic or biopolymeric matrices in
combination with cells can be used for in vitro pathophysi-
ology models for drug screening and tissue engineered
constructs for regenerative medicine. As these cell-based
platforms take center stage in testing, development and
manufacturing of pharmaceuticals, quality control tools can

4

5

10

be used to reproducibly generate and maintain these plat-
forms. The fiber-optic based pH sensor is tolerated by cells
and measures local pH change in a physiologically-relevant
range of pH 5-8 with minimal drift in calibration over 21
days.

Three-dimensional microtissues and synthetically grown
constructs are tools used in regenerative medicine and drug
development. These cell-based platforms are used in testing,
development and manufacturing of pharmaceuticals, and a
need exists for quality control tools to test reproducibility of
producing and maintaining these platforms. Sensing in
active biomanufacturing environment challenges conven-
tional sensor technology. Sensors embedded in biological
medium include biocompatible materials to minimize bio-
fouling, leaching of toxic substances from sensor surface,
and to provide non-toxicity of components in contact with
cell media. The sensor matches mechanical properties of the
surrounding tissue environment and are durable enough to
survive sterilization conditions with minimal reliability drift
over many weeks to avoid re-calibration. Re-calibration can
introduce foreign cells or pathogens to the cell construct.
The pH photothermal spectrometer overcomes these disad-
vantages of conventional devices.

Conventional electrical sensors can fail to meet measure-
ment and material science challenges involved with embed-
ded bio-measurement specifications. Photonic sensors pro-
vide biocompatible sensors for physical, mechanical, or
chemical sensing that can include an optical sensor (e.g. a
florescent nanoparticle or molecular dye) or fiber optic-
based sensor for chemical detection. Fiber-optic based sen-
sors have a small probe size (¢.g., 100 um diameter) and
chemical inertness.

Photothermal spectroscopy can use a fiber optic thermom-
eter (¢.g., a fiber Bragg grating (FBG)) in the pH photother-
mal spectrometer. The pH photothermal spectrometer
includes FBGs to monitor a magnitude of thermalization of
free-space propagated light absorbed by a pH sensitive
chromophore (e.g., red cabbage extract). As the pH changes,
light absorption (and heat generation) properties of the
chromophores changes. As a result, the temperature
increases upon light absorption by the chromophore that
corresponds to the chromophore’s UV-visible optical
absorption spectrum. Ratiometric measurement of relative
temperature increases measure pH of the analyte medium,
and the measurement is independent of photodiode and FBG
response sensitivity.

The pH photothermal spectrometer includes a photonic
pH optrode in cell growth media with phenol red (PR) as a
pH indicator dye. Phenol Red was added to growth media as
a visual pH assay. The pH photothermal spectrometer
included an uncoated FBG and PR powder. For calibration,
PR powder added a buffer composition. Buffers can include
citric acid, sodium citrate, acetic acid, sodium acetate, tris,
CHES, and phosphate. Buffer pH was measured with a
conventional pH probe. For cell-based experiments, PR
powder was added to cell wash off collected after 1-3 days
of cell growth. For these experiments the cells were grown.
in clear cell media. Blue (450 nm+5 nm, 75 mW) and green
(520 nm+5 nm, 75 mW) LEDs with FC adapters were used.
UV-Vis spectra were acquired.

‘A configuration for evaluating the temperature-increases
upon illumination of pH-sensitive chromophores is shown in
FIG. 10 to FIG. 13. An LED controller controlled the optical
output of the LED. Optical fiber cable had a 105 ym
diameter and 0.22 NA optogenetic Y-cable that delivered
half of the excitation light from the LED directly to the
analyte medium while the other half impinged on a photo-



U.S. Patent Apr. 11, 2023 Sheet 12 of 13 US 11,624,664 B2

Ratio of temperature rise (Green:Blue excitation)

= 988
= 08.
= i
6 08) 7
= i ”
it *
~ Os. o
ae |
sed ap. a
Sea iv H st
a4 i oa
peed :
SB as.
ew eee
ey °
: Ag
é ge 0b a
E |
ge 0.55 ”
oe ee
inn
8 as. 6
& i
DAS nnn
x
§ 58 § 68 ? 73 8 as

FIG. 12


U.S. Patent Apr. 11, 2023 Sheet 3 of 13 US 11,624,664 B2

Ito
I>
IS

201903, 204
Af |
i

/

FIG. 3


US 11,624,664 B2

11

diode for real-time measurement of LED power output.
‘These readings were used as a correction factor in calibra-
tion.

For pH measurement, a glass tube (4 mm OD, 3 mm ID)
was filled with a solution of PR. The FBG was disposed in
the tube, which was aligned such that the excitation light
overlapped the FBG sensor. The FBG sensor was probed
with laser light that was scanned over several nanometers.
‘The light reflected off of the FBG and measured with a
power meter. Changes in temperature changed the FBG
resonant wavelength, and a temperature increases resulted in
a red shift of the FBG resonant wavelength while tempera-
ture decreases resulted in a blue shift. For each measure-
ment, the FBG was interrogated for several minutes before
turning on the excitation light.

More specifically, with reference to FIG. 11, a small
amount of pH indicator dye (red cabbage extract) was
dissolved in a buffered solution of known pH value. The

solution (0.1 ml to 1 ml) was then filled into the glass tube 2

and an FBG sensor inserted into the tube, A 0.22 NA fiber
carrying excitation light was placed orthogonal to the FBG
such that the light cone exiting the fiber enveloped the FBG.
temperature sensor. At the excitation light source red and

green lights were periodically tured on and off for several 2.

minutes and the FBG spectra was recorded for both the “on’
and “off” state. The relative rise in temperature was mea-
sured by measuring the shift in wavelength center of the
FBG response curve. The ratio of wavelength shift for both
red and green excitation was then calculated. From pH 2.5
to 10, the ratio of wavelength shift (i.e. temperature rise) for
green and red excitation was plotted and a linear regression
was employed to calculate a calibration model. The calibra-
tion model has the form of pH={[AAjq/AAgreen/-b}/S:
where $ and b are calculated using linear regression on
calibration data. To determine the pH of unknown solution,
the Adjc/AAgreen iS measured and the values of S and b
parameter are used to calculate the solution pH.

Results of measurements are shown in FIG. 12 and FIG.
13. As shown in FIG. 12, a ratio of temperature rise at green
and blue excitation light was collected a function of pH.
Here, a small amount of pH indicator dye (phenol red) was
dissolved in a buffered solution of known pH value. 0.1 ml
to 1 mlof the prepared solution was then filled into the glass
tube and an FBG sensor inserted into the tube, A multimode
fiber carrying excitation light was placed orthogonal to the
FBG such that the light cone exiting the fiber enveloped the
FBG temperature sensor. At the excitation light source blue
and green lasers were periodically turned on and off for

several minutes and the FBG spectra was recorded for both 5

the “on” and “off” state. The relative rise in temperature was
measured by measuring the shift in wavelength center of the
FBG response curve. The ratio of wavelength shift for both
red and green excitation was then calculated. Over the pH.

range of 5 to 8, the ratio of wavelength shift (i.e. temperature

rise) for green and red excitation was plotted and a linear
regression was employed to calculate parameters of the
calibration model.

With reference to FIG. 13, repeat measurements of a pH
solution with the same probe were made over a 2-week
period. Here, phenol red was dissolved in pH 6.7 buffered
solution. The pH of the buffered solution was then measured.
using a standard electronic pH probe. A small amount of the
buffered solution with phenol red was placed in the appa-
ratus and ratio metric measurement of wavelength shift
under green and blue excitation were made for 21 consecu-
tive days. These measurements indicate the pH of the

&

&

0

12

solution as measured by ratiometric method does not show
any significant changes over the course of 3 weeks.

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.

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 (c.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-
text. “Or” means “and/or.” 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 (eg., it includes the degree of error associated
with measurement of the particular quantity). The conjune-
tion “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 circum-
stances

What is claimed is:

1. A pH photothermal spectrometer for performing pH
photothermal spectroscopy, the pH photothermal spectrom-
eter comprising:

a container comprising an interior bounded by a wall of

the container, such that container:

receives an analyte medium and a pH-sensitive chro-
mophore, the pH-sensitive chromophore comprises
an optical absorption spectrum that adjusts to a pH of
the analyte medium, the analyte medium comprising
an analyte temperature; and

optionally receives a fiber encapsulation matrix in
which are disposed the analyte medium and the
pH-sensitive chromophore;

an excitation fiber disposed in the container in optical

communication with the pH-sensitive chromophore

and that:

receives a first excitation light comprising a first wave-
length and a second excitation light comprising a
second wavelengt



US 11,624,664 B2

15
depends on the receiver temperature of the light
receiver, the reflected light comprising the probe light
wavelength; and
determining the pH of the analyte medium from a ratio of
the first optical amount to the second optical amount to
perform pH photothermal spectroscopy.
19. The process of claim 18, further comprising:
communicating, from the light receiver, the reflected light
to the temperature detector fiber; and
receiving, by the temperature detector fiber, the reflected
light from the light receiver.
20. The process of claim 18, further comprising: calibrat-
ing the pH photothermal spectrometer so that the second
optical amount provides a pH of the analyte medium

+e eee

16


US 11,624,664 B2

5

or Brillouin), or a florescent particle coupled to temperature
detector fiber 205, e.g., nitrogen vacancy (NV) diamond. It
should be appreciated that light receiver 214 receives probe
light 217 in first optical amount O1 from temperature
detector fiber 205 and reflects probe light 217 as reflected
light 218 in second optical amount 02. Second optical
amount of 2 depends on receiver temperature TR of optical
receiver 214 because resonance wavelength LR at which
light receiver 214 reflect probe light 217 as reflected light
218 changes as a function of receiver temperature TR.
Moreover, light receiver 214 in combination with tempera-
ture detector fiber 205 make up optical thermometer 216
Exemplary optical thermometer 216 is described in U.S.
patent application Ser. No. 16/589,793, which is incorpo-
rated by reference in its entirety.

Fiber port 212 can be disposed on container 201 for
disposal of optical thermometer 216 or excitation fiber 207
through and in container 201. Fiber port can include a
grommet, sleeve, or pigtail for continuous, non-interrupted

optical communication of light (e.g., first excitation light 2

208, second excitation light 210, probe light 217, reflected
light 218) through excitation fiber 207 or temperature detec-
tor fiber 205.

Fiber encapsulation matrix 203 can be disposed in con-

tainer 201 to encapsulate light receiver 214, excitation fiber 2

207, or a combination thereof to maintain a relative position
in interior 202. Fiber encapsulation matrix 203 can be a gel
such as a hydrogel or other sol-gel composite. It is contem-
plated that fiber encapsulation matrix 203 is permeable to
fluid flow or disposal of analyte medium 204 and pH-
sensitive chromophore 206 therein. Fiber encapsulation
matrix 203 is thermally conductive to equilibrate the tem-
perature of light receiver rapidly thermally 214 to analyte
medium 204 in which is disposed pH-sensitive chromophore
206. Further, fiber encapsulation matrix 203 provides rapid
contact between pH-sensitive chromophore 206 and analyte
medium 204 so that pH-sensitive chromophore 206 is sub-
ject to the pH of analyte medium 204.

Analyte medium 204 is a fluid that a has a pH to which
pH-sensitive chromophore 206 is sensitive so that the
absorption spectrum of pH-sensitive chromophore 206
changes due to the pH of analyte medium 204 as shown in
FIG. 9 and FIG. 10. Analyte medium 204 can provide a pH
that is from 1 to 15, specifically from 2 to 12, and more
specifically from 5 to 8. It is contemplated analyte medium
204 can be single moiety or can include multiple moieties
that can include a solvent, organic compound, inorganic,
carbohydrate, cellular component (¢.g., mitochondria and
the like), a biological sample such as a cell (¢.g., prokaryote

or eukaryote) that can be an animal cell or plant cell, and the

like. In an embodiment, analyte medium 204 is human blood
that includes typical components of human blood, wherein
the human blood can be synthetic or naturally occurring or
from a healthy or diseased human.

In an embodiment, pH-sensitive chromophore 206 can be 5

subject to diffusion in analyte medium 204; disposed in the
fiber encapsulation matrix 203 via entropic trapping, co
lent bond attachment, or electrostatic trapping; and the like.
Exemplary pH-sensitive chromophore 206 includes an acid-
base indicator (e.g., phenol red, cabbage extract, and the
like) that can be compatible with biological samples or
incompatible with biological samples. A color change (i.e.,
a change of the optical absorption spectrum by shifting a
maximum of absorbance in the spectrum) of pH-sensitive
chromophore 206 can occur at a selected pH such as around
an acidic pH, around a neutral pH, or around a basic pH,
wherein the color change can occur over a broad pH range

&

6

(eg., more than 1 pH units such as 2, 3, or 4 pH units) or
narrow pH range (e.g., 0.25 to 1 pH unit)

‘A configuration of optical thermometer 216 relative to
excitation fiber 207 can be selected to accord to a particular
use of pH photothermal spectrometer 200, e.g., as an implant
in biological tissue (e.g., human muscle tissue) to monitor a
biological condition (e.g., acidosis) or presence or amount of
a compound (e.g., a therapeutic drug) is such biological
tissue. In an embodiment, with reference to FIG. 1, optical
thermometer 216 can be disposed at an orthogonal or obtuse
angle to excitation fiber 207 such that optical thermometer
216 terminates in interior 202 of container 201. In an
embodiment, with reference to FIG. 3, optical thermometer
216 can be disposed at an orthogonal or obtuse angle to
excitation fiber 207 such that optical thermometer 216
dually extends into interior 202 of container 201 from two
ports of container 201. In an embodiment, with reference to
FIG. 4, optical thermometer 216 can be disposed parallel to
excitation fiber 207 in container 201. According to an
embodiment, with reference to FIG. 4 and FIG. 5, excitation
fiber 207 can terminate as a straight fiber section proximate
to light receiver 214 as shown in FIG. 4 or can terminate in
optical taper 219 proximate to light receiver 214 as shown
in FIG. 5.

In an embodiment, with reference to FIG. 8, pH photo-
thermal spectrometer 200 includes excitation light source
221 in optical communication with excitation fiber 207,
wherein excitation light source 221 provides first excitation
light 208 and second excitation light 210 to excitation fiber
207. Excitation light source 221 can be a laser or set of lasers
that provides arbitrary wavelengths of light according to
desired wavelengths for first wavelength 209 and second
wavelength 211. Probe light source 222 can be in optical
communication with temperature detector fiber 205 to pro-
vide probe light 217 to temperature detector fiber 205. Probe
light source 222 can be a laser that provides an arbitrary
wavelength of light according to desired wavelength for
probe light 217. Detector 223 can be in optical communi-
cation with temperature detector fiber 205 to receive
reflected light 218 from optical thermometer 216. An exem-
plary detector 223 includ photodiode or power meter.

In an embodiment 200 includes a signal generator (XYZ)
that produces an excitation signal, e.g., a square wave pulse
that may be used to control 221 such as to produce a
sequence of pulses of first excitation light 208 and first
wavelength 209 of time period T, wherein T can be from 1
femtosecond to 1000 seconds, specifically 1 nanoseconds to
300 seconds, and more specifically 1 microsecond to 100
second. An optical splitter, in communication with excita-
tion light source 221 and excitation fiber 207 can deliver first
excitation light 208 and first wavelength 209 to a light
receiver, e.g., a photodiode or power meter, for ratiometric
measurement of optical power of first excitation light 208
and first wavelength 209. In addition, a signal generator
(XYY), in communication with probe light source 222, that
produces a periodic signal such as a sine wave or a triangular
wave, can sweep the probe wavelength of probe light source
222 for measurement of resonance wavelength of light
receiver 214. A lock-in amplifier and laser locking circuit in
communication with detector 223 provides locking of probe
light source 222 to light receiver 214 for automatic selection
of probe light 217. In addition, an optical splitter in com-
munication with temperature detector fiber 205 and probe
light source 222 can deliver probe light 217 to wavelength
reference that can be, e.g., a wavemeter, frequency comb, or
gas wavelength reference cell. An optical circulator in
communication with temperature detector fiber 205 and



U.S. Patent Apr. 11, 2023 Sheet 8 of 13 US 11,624,664 B2

200
223 222
217
218 05
205-1 7
201
207
208
210-4
221

FIG. 8


U.S. Patent Apr. 11, 2023 Sheet 5 of 13 US 11,624,664 B2

203 204

FIG. 5


U.S. Patent Apr. 11, 2023 Sheet 4 of 13 US 11,624,664 B2

ND
iS

203 204

202 201 220 26
\ 213 \
\ f

FIG. 4


US 11,624,664 B2

7
probe light source 222 can separate reflected light 218 from
probe light 217 and communicate reflected light 218 to
detector 223.

It should be appreciated that pH photothermal spectrom-
eter 200 can be made in various ways. In an embodiment, a
process for making pH photothermal spectrometer 200
includes: providing container 201; forming a first port in
wall 220 to receive excitation fiber 207; disposing excitation
fiber 207 in interior 202 through the first port; forming a
second port in wall 220 to receive optical thermometer 216;
disposing optical thermometer 216 in interior 202 through
the second port; disposing fiber encapsi trix 203 in
interior 202 to fixedly maintain a position of light receiver
214 and temperature detector fiber 205 in interior 202 to
make pH photothermal spectrometer 200.

The process for making pH photothermal spectrometer
200 also can include parallel arrangement of temperature
detector fiber 205 and excitation fiber 207 with fiber encap-
sulation matrix that fix the relative position of the two fibers.
Excitation fiber 207 can have an adiabatic taper by the
application of constant tension under a heat source. The
adiabatic tapers allow the excitation light first excitation
light 208 and first wavelength 209 to gradually exit excita-
tion fiber 207 over the tapered area allowing second exci-
tation light 210 and second wavelength 211 to interact with
the chromophore interposed between temperature detector
fiber 205 and excitation fiber 207. The fiber matrix can be
formed using photo polymerization or thermal setting of
polymers such as polyethylene.

It should be appreciated that pH photothermal spectrom-
eter 200 can be made in various ways. In an embodiment, a
process for making pH photothermal spectrometer 200
includes: providing container 201; forming a first port in
wall 220 to receive excitation fiber 207; disposing excitation
fiber 207 in interior 202 through the first port; forming a
second port in wall 220 to receive optical thermometer 216;
disposing optical thermometer 216 in interior 202 through
the second port, disposing fiber encapsulation matrix 203 in
interior 202 to fixedly maintain a position of light receiver
214 and temperature detector fiber 205 in interior 202 to
make pH photothermal spectrometer 200.

The process for making pH photothermal spectrometer
200 also can include parallel arrangement of temperature
detector fiber 205 and excitation fiber 207 with fiber encap-
sulation matrix being used fix the relative position of the two
fibers. Excitation fiber 207 may have an adiabatic taper by
the application of constant tension under a heat source. The
adiabatic tapers allow the excitation light first excitation
light 208 and first wavelength 209 to gradually exit excita-

tion fiber 207 over the tapered area allowing the excitation 5

wavelength second excitation light 210 and second wave-
length 211 to interact with the chromophore interposed
between temperature detector fiber 205 and excitation fiber
207. The fiber matrix may be formed using photo polym-

erization or thermal setting of polymers such as polyethyl-

ene glycol or poly-vinyl alcohol.

Disclosed is a process for performing pH photothermal
spectroscopy with pH photothermal spectrometer 200, the
process including: receiving, by excitation fiber 207, first
excitation light 208 that includes first wavelength 209 and
second excitation light 210 that includes second wavelength
211; contacting pH-sensitive chromophore 206 with analyte
medium 204; adjusting optical absorption spectrum of pH-
sensitive chromophore 206 in response to the pH of analyte
medium 204; communicating first excitation light 208 and
second excitation light 210 from excitation fiber 207 to
pH-sensitive chromophore 206; receiving, by pH-sensitive

20

&

&

8

chromophore 206, first excitation light 208 and second
excitation light 210; absorbing, by pH-sensitive chro-
mophore 206, first amount Al of first excitation light 208
based on the pH of analyte medium 204; absorbing, by
pli-sensitive chromophore 206, second amount A2 of sec-
ond excitation light 210 based on pH of analyte medium
204, adjusting analyte temperature TA of analyte medium
204 in response to pH-sensitive chromophore 206 absorbing
first excitation light 208 and second excitation light 210
through thermal equilibration of analyte medium 204 to the
temperature of pH-sensitive chromophore 206; adjusting
receiver temperature TR of light receiver 214 to match
analyte temperature TA of analyte medium 204 in response
to adjusting analyte temperature TA of analyte medium 204
through heat transfer between analyte medium 204 and light
receiver 214; receiving, by light receiver 214, probe light
217 in first optical amount O1 from temperature detector
fiber 205; producing, by light receiver 214 from probe light
217, reflected light 218 in second optical amount 2 that
depends on receiver temperature TR of light receiver 214,
reflected light 218 including probe light wavelength 224;
and determining the pH of analyte medium 204 from a ratio
of first optical amount O1 to second optical amount 02 to
perform pH photothermal spectroscopy.

Determining the pH of analyte medium 204 from the ratio
of first optical amount OI to second optical amount 2
(RATIO) includes constructing a calibration model of using
linear regression where the RATIO is the dependent variable
and pH the independent variable. To train the model, mea-
surement of RATIO are obtained by introducing aqueous
solution of known pH values to pH photothermal spectrom-
eter 200. The measured RATIO values and corresponding
pH values are used as input to the regression model to
calculate a linear relationship between RATIO and pH. To
measure the pH of analyte medium 204 whose pH may not
be known prior to measurement, RATIO is measured using
pH photothermal spectrometer 200 following which the
RATIO is used as input variable in calibration model to solve
for pH.

In an embodiment, the process for performing pH pho-
tothermal spectroscopy further includes communicating,
from light receiver 214, reflected light 218 to temperature
detector fiber 205; and receiving, by temperature detector
fiber 205, reflected light 218 from the light receiver 214.
Here, detector 223 can receive reflected light 218 to obtain
second optical amount O2 of reflected light 218 from which
the pH of analyte medium 204 can be determined.

In an embodiment, the process for performing pH pho-
tothermal spectroscopy further includes calibrating pH pho-
tothermal spectrometer 200 by measuring the ratio of probe
wavelengths 218 before after excitation light 208 and 209
are exposed to the chromophore (call it RATIO2). This
measurement provides the relative increase in temperature
due to absorption of excitation light 208 and 209 and is
equivalent to RATIO. To achieve a higher accuracy, the
RATIO2 maybe multiplied by the ratio of optical power of
excitation light 208 and 209. Taking the ratio of reflected
light 218 before and after excitation with excitation light 208
and 209 removes step of calibrating the temperature to
frequency response of light receiver 214. Scaling this
response factor by the ratio of excitation light 208 and 209
corrects drift in power of the excitation light and removes a
source of error in pH measurement. In this manner, the
scaled RATIO2 pH photothermal spectrometer 200 can be
used to provide the pH of analyte medium 204 by construct-
ing a calibration model of using linear regression where the
RATIO? is the dependent variable and pH the independent



U.S. Patent Apr. 11, 2023 Sheet 7 of 13 US 11,624,664 B2

UV-Vis-based detection

Dye covalently attached to
Visible polymer, composite material pulled Measure
excitation into optical fiber absorption

Optical fiber clad in polymer that
has dye covalently attached.

Visible
excitation

Measure
absorption

FIG. 7


a2) United States Patent
Ahmed et al.

US 11,624,664 B2

(10) Patent No.:

(54) PH PHOTOTHERMAL SPECTROMETER
AND PERFORMING PH PHOTOTHERMAL
SPECTROSCOPY

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

(72) Inventors: Zeeshan Ahmed, Washington, DC
(US); Matthew Robert Hartings,
Gaithersburg, MD (US)

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

(*) Notice: Subject to any disclaimer, the term of this
is extended or adjusted under 35

. 154(b) by 307 days.

(21) Appl. No.: 17/110,996
(22) Filed: Dee. 3, 2020

(65) Prior Publication Data
US 2022/0099503 Al Mar. 31, 2022

Related U.S. Application Data

(60) Provisional application No. 63/084.313, filed on Sep.
28, 2020.

(51) Int. CL

GOIK 1132 (2021.01)
GOIN 21/88 (2006.01)
GOIN 21/80 (2006.01)
AGIB 5/145 (2006.01)
GOIN 21/77 (2006.01)

(45) Date of Patent: Apr. 11, 2023
(52) US. Cl.
cpc . GOIK 11/32 (2013.01); A6IB 5/14539

(2013.01); GOLN 21/7703 (2013.01); GOIN
21/80 (2013.01); GOIN 2021/7786 (2013.01)
(58) Field of Classification Search

CPC... A6IB 5/686; AG1B 5/0075; A6IB 5/01:
A61B 5/14539; GOIK 11/32; GOIN
21/80; GOIN 21/7703; GOIN 2201/08:
(IN 2021/7786

See application file for complete search history.

(56) References Cited
U.S. PATENT DOCUMENTS.

SLISS81L A 5/1992 Hartlaub et al.

5,280,548 A 1/1994 Atwater et al
10,105,080 BL* 10/2018 Kam AGIB 5/14514
2008/0275659 AI* 11/2008 Miller GOIN 21/33
702/84

(Continued)

OTHER PUBLICATIONS,

‘Yang, X.H., etal., “Fluorescence pH probe based on microstructured
polymer optical fiber”, Optics Express, 2007, p. 16478-16483, vol
15 No. 25.

(Continued)

Primary Examiner — Sang H Nguyen
(74) Attorney, Agent, or Firm — Office of Chief Counsel
for National Institute of Standards and Technology

(57) ABSTRACT

A pH photothermal spectrometer includes a container that
receives an analyte medium and pH-sensitive chromophore.
An excitation fiber and optical thermometer are disposed in
the container. The optical thermometer include a light
receiver disposed on a temperature detector fiber.

20 Claims, 13 Drawing Sheets



U.S. Patent Apr. 11, 2023 Sheet 10 of 13 US 11,624,664 B2

ene 88 $B mime sat BAP owner gt BE venga EP neem gb ME

ee

pS

Absorbance (au)

Wavelength (nm)
FIG. 10


U.S. Patent Apr. 11, 2023 Sheet 9 of 13 US 11,624,664 B2

Optical Absorption Spectrum of pH-Sensitive Chromophore

Analyte pH ———_____»

A Shift in optical absorption
as analyte pH changes
|
c=]
g
=
$
A
2
<
3
2
Ss
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Wavelength

FIG. 9


U.S. Patent Apr. 11, 2023 Sheet 6 of 13 US 11,624,664 B2

Photo-thermal detection of pH

Visible
excitation

Infrared
probe light

>

Le Dye covalently attached to polymer

coating FBG

Dye covalently attached to surface
of polymer coating FBG

FIG. 6


US 11,624,664 B2

13

communicates the first excitation light and the second

excitation light to the pH-sensitive chromophore,

such that the pH-sensitive chromophore:

receives the first excitation light and the second
excitation light; and

absorbs a first amount of the first excitation light and
a second amount of the second excitation light
based on the pH of the analyte medium such that
absorption of the first excitation light or the sec-
ond excitation light increases the analyte tempera-
ture of the analyte medium: and

an optical thermometer disposed in the container and

comprising:
a temperature detector fiber that
receives a probe light comprising a probe light
wavelength in a first optical amount; and
receives a reflected light comprising the probe light
wavelength from a light receiver; and
the light receiver:
disposed on a terminus of the temperature detector
fiber in optical communication with the tempera-
ture detector fiber and in thermal communication
with the analyte medium; and
comprising:
a receiver temperature that adjusts to match the
analyte temperature; and
an optical resonance at a resonance wavelength
that changes in response to a change in the
receiver temperature,
such that the light receiver:
receives the probe light from the temperature
detector fiber;
produces, from the probe light, a reflected light in
a second optical amount and comprising the
probe light wavelength, such that the second
optical amount of the reflected light depends on.
the receiver temperature of the light receiver, so
that a ratio of the first optical amount to the
second optical amount provides the pH of the
analyte medium; and
communicates the reflected light to the tempera-
ture detector fiber; and

the temperature detector fiber receives the reflected light

from the light receiver.

2. The pH photothermal spectrometer of claim 1, further
comprising a fiber port disposed on the container through
which is disposed the optical thermometer.

3. The pH photothermal spectrometer of claim 1, further
comprising a fiber port disposed on the container through
which is disposed the excitation fiber.

4. The pH photothermal spectrometer of claim 1, com-
prises the fiber encapsulation matrix that encapsulates the
light receiver, the excitation fiber, or a combination com-
prising at least one of the light receiver and the excitation
fiber.

5. The pH photothermal spectrometer of claim 1, wherein
the fiber encapsulation matrix maintains a position of the
excitation fiber and the light receiver in the container.

6. The pH photothermal spectrometer of claim 1, wherein
the fiber encapsulation matrix comprises a gel.

7. The pH photothermal spectrometer of claim 1, wherein
the pH-sensitive chromophore is subject to diffusion in the
analyte medium.

8. The pH photothermal spectrometer of claim 1, wherein
the pH-sensitive chromophore is disposed in the fiber encap-
sulation matrix via entropic trapping, covalent bond attach-
ment, or electrostatic trapping,

20

3

4

0

&

35

0

14

9. The pH photothermal spectrometer of claim 1, wherein
the light receiver comprises a fiber Bragg grating, an on-chip
thermometer, a Raman fiber optic thermometer, a Brillouin
fiber optic thermometer), or a fluorescent particle.
10. The pH photothermal spectrometer of claim 1,
wherein the light receiver is the fluorescent particle that
comprises a nitrogen vacancy diamond.
11. The pH photothermal spectrometer of claim 1,
wherein the analyte medium comprises a cell medium.
12. The pH photothermal spectrometer of claim 1, further
comprising an excitation light source in optical communi-
cation with the excitation fiber and that provides the first
excitation light to the excitation fiber.
13. The pH photothermal spectrometer of claim 1, further
comprising a probe light source in optical communication
with the temperature detector fiber and that provides the
probe light to the temperature detector fiber.
14. The pH photothermal spectrometer of claim 1, further
comprising a detector in optical communication with the
temperature detector fiber and that receives the reflected
light from the optical thermometer.
15. The pH photothermal spectrometer of claim 1,
wherein the temperature detector fiber extends into and out
of the interior of the container in a plurality of locations of
the wall of the container.
16. The pH photothermal spectrometer of claim 1,
wherein the excitation fiber is disposed orthogonal to the
optical thermometer in the container, parallel to the opti
thermometer in the container, or at an oblique angle with
respect to the optical thermometer.
17. The pH photothermal spectrometer of claim 1,
wherein the excitation fiber comprises an optical taper
disposed at a terminus of the excitation fiber proximate to
the light receiver.
18. A process for performing pH photothermal spectros-
copy with the pH photothermal spectrometer of claim 1, the
process comprising:
receiving, by the excitation fiber, the first excitation light
that comprises the first wavelength and the second
excitation light that comprises the second wavelength;

contacting the pH-sensitive chromophore with the analyte
medium;

adjusting the optical absorption spectrum of the pH-

sensitive chromophore in response to the pH of the
analyte medium;

communicating the first excitation light and the second

excitation light from the excitation fiber to the pH-
sensitive chromophore:
receiving, by the pH-sensitive chromophore, the first
excitation light and the second excitation light;

absorbing, by the pH-sensitive chromophore, the first
amount of the first excitation light based on the pH of
the analyte medium;

absorbing, by the pH-sensitive chromophore, the second

amount of the second excitation light based on pH of
the analyte medium;
adjusting the analyte temperature of the analyte medium
in response to the pH-sensitive chromophore absorbing
the first excitation light and the second excitation light;

adjusting the receiver temperature of the light receiver to
match the analyte temperature of the analyte medium in
response to adjusting the analyte temperature of the
analyte medium:

receiving, by the light receiver, the probe light in the first

optical amount from the temperature detector fiber;
producing, by the light receiver from the probe light, the
reflected light in the second optical amount that



U.S. Patent Apr. 11, 2023 Sheet 2 of 13 US 11,624,664 B2

FIG, 2



US 11,624,664 B2

Sheet 13 of 13

Apr. 11, 2023

U.S. Patent

(9013S 9IDKY ONG Uses) SS1s Sane, jo OneY

Day 12 Day 2t

Day2 Day 3 Day6

Day 4

FIG. 13


US 11,624,664 B2

3

It has been discovered that a pH photothermal spectrom-
eter probes a pH-dependent change in absorbance of a
pH-sensitive chromophore in response to irradiation thereof
by different wavelengths of light to determine a difference in
temperature rise due to differences in light absorbance of the
pH-sensitive chromophore at the different wavelengths of
light. An absorption profile of the pH-dependent absorption
of light of the pH-sensitive chromophore governs an amount
of light absorbed at a particular wavelength. Light absorbed
by the pH-sensitive chromophore is thermalized in an ana-
lyte matrix that includes the pH-sensitive chromophore so
that as a temperature of the analyte matrix changes, the
temperature change of the analyte matrix is detected by an
optical thermometer disposed in the analyte matrix.

Here, the pH photothermal spectrometer measures pH and
can be used, e.g., in bio-industrial processing, tissue engi-
neering industry, and the like that involve a small footprint
probe that is compatible with a biological environment,
wherein the pH photothermal spectrometer does not inter-

fere with cell growth, operates in a high ionic strength 2

solution environment, can be embedded in a substrate to
measure pH via access to an optically dark area, is dispos-
able, or is stable over a lengthy period without weekly
re-calibration.

The pH photothermal spectrometer can include optical
waveguides to deliver light and detect temperature change
so that the pH photothermal spectrometer can be disposed in
a biocompatible matrix, e.g., in a region that is in i
to optical microscopy. Furthermore, the pH photothermal
spectrometer can include soft, biocompatible materials that
match a mechanical or chemical environment for cellular or
tissue growth without biofouling the pH photothermal spec-
trometer. In the pH photothermal spectrometer, the pH-
sensitive chromophore can be covalently attached to a
proton-permeable waveguide for optical or photothermal
spectroscopy for measurement of pH. Advantageously,
photo-thermal spectroscopy to measure pH, as compared
with a volumetric change, in a hydrogel that is probed by
changes in a bandgap resonance wavelength can be per-
formed in an ionic solution. In this manner, the pH photo-
thermal spectrometer overcomes technical limitations of
conventional polymeric waveguide sensors that operate at
low ionic strengths but not high ionic strength because such
conventional waveguide sensor infer pH from effects of
‘osmotic pressure-induced swelling of a hydrogel. Benefi-
cially, the pH photothermal spectrometer operates without
undergoing a significant hysteresis upon dehydration to
eliminate device failure that occurs in some conventional
electrical pH devices.

It should be appreciated that pH photothermal spectrom- 5

eter 200 performs pH photothermal spectroscopy to deter-
mine pH of analyte medium 204 via a thermal response of
optical thermometer 216 to light absorption of pH-sensitive
chromophore 206 disposed among analyte medium 204. In

an embodiment, with reference to FIG. 1, FIG. 2, FIG. 3, s

FIG. 4, and FIG. 5, pH photothermal spectrometer 200

ner 201 including interior 202 bounded by
wall 220 of container 201, such that container 201 receives
analyte medium 204 and pH-sensitive chromophore 206,
pH-sensitive chromophore 206 includes optical absorption
spectrum (e.g., as shown in FIG. 9 or FIG. 10) that adjusts
to a pH of analyte medium 204, analyte medium 204
including analyte temperature TA; and optionally receives
fiber encapsulation matrix 203 in which are disposed analyte
medium 204 and pH-sensitive chromophore 206; excitation
fiber 207 disposed in container 201 in optical communica-
tion with pH-sensitive chromophore 206 and that receives

&

&

4

first excitation light 208 including first wavelength 209 and
second excitation light 210 including second wavelength
211; and communicates first excitation light 208 and second
excitation light 210 to pH-sensitive chromophore 206, such
that pH-sensitive chromophore 206 receives first excitation
light 208 and second excitation light 210; and absorbs first
amount A1 of first excitation light 208 and second amount
A2 of second excitation light 210 based on the pH of analyte
medium 204 such that absorption of first excitation light 208
or second excitation light 210 increases analyte temperature
TA of analyte medium 204; and optical thermometer 216
disposed in container 201 and including temperature detec-
tor fiber 205 that receives probe light 217 including probe
light wavelength 224 in first optical amount Ol; and
receives reflected light 218 including probe light wavelength
224 from light receiver 214; and light receiver 214 disposed
‘on terminus of temperature detector fiber 205 in optical
communication with temperature detector fiber 205 and in
thermal communication with analyte medium 204; and
including receiver temperature TR that adjusts to match
analyte temperature TA; and optical resonance OR at reso-
nance wavelength LR that changes in response to a change
in receiver temperature TR, such that light receiver 214
receives probe light 217 from temperature detector fiber

5 205; produces, from probe light 217, reflected light 218 in

second optical amount ©2 and including probe light wave-
length 224, such that second optical amount O2 of reflected
light 218 depends on receiver temperature TR of light
receiver 214, so that a ratio of first optical amount O1 to
second optical amount O2 provides the pH of analyte
medium 204; and communicates reflected light 218 to
temperature detector fiber 205; and temperature detector
fiber 205 receives reflected light 218 from light receiver 214.

Container 201 can be a plate well or other structure that
can contain fluids such as cell media or a composition with
arange of viscosity from very fluidic to highly viscous. Plate
wells are typically arranged in an array format for disposal
of multiple different samples for independent testing. A
volume of container 201 is suflicient to hold fiber encapsu-
lation matrix 203, analyte medium 204, pH-sensitive chro-
mophore 206, light receiver 214, temperature detector fiber
205, and excitation fiber 207 in interior 202 that is bounded
by wall 220. Accordingly, the volume of container 201 can
be from 10 nanoliters to 10° liters, specifically from 100
nanoliters to 10 centiliters, and more specifically from 1
microliter to 100 microliters.

Temperature detector fiber 205 and excitation fiber 207
independently are optical fibers that communicate light (e.g.,
208, 210, 217, 218) into or out of excitation fiber 207,
particularly sensing volume 215, wherein first excitation
light 208 and second excitation light 210 communicate from
excitation fiber 207 and spread into light cone 213 and
interact with analyte medium 204 proximate to light receiver
214. Excitation fiber 207 can be an optical fiber that propa-
gates ultraviolet or visible light as first excitation light 208
or second excitation light 210 that can have respectively first
wavelength 209 and md wavelength 211, e.g., from 200
nanometers (nm) to 1200 nm, specifically from 300 nm to
1000 nm, and more specifically from 400 nm to 900 nm.
Temperature detector fiber 205 can be an optical fiber that
propagates infrared light as probe light 217 or reflected light
218 that can have probe light wavelength 224, e.g., from
1000 nanometers (nm) to 6000 nm, specifically from 1100
‘nm to 4000 nm, and more specifically from 1100 millimeters
2500 nm.

Light receiver 214 can be a fiber Bragg grating (FBG),
on-chip thermometer, fiber optic thermometer (e.g., Raman,



U.S. Patent Apr. 11, 2023 Sheet 1 of 13 US 11,624,664 B2

FIG. |


US 11,624,664 B2

Page 2
(56) References Cited
U.S. PATENT DOCUMENTS.

2012/0075619 AL* 3/2012 Nieman ...........0. GOIN 21/474
356/73
2015/0297086 Al* 10/2015 Hong GOIN 21/6456
600/431
2018/0274988 Al* 9/2018 Sabri ... GOLK 11/20
2019/0128894 A1* 5/2019 Jain .. €23C 16/403
2019/0223795 Al* 7/2019 Patolsky .... AGIB 5/14532,

OTHER PUBLICATIONS,

Netto, E.J., et al., “A fiber-optic broad-range pH sensor system for
gastric measurements”, Sensors and Actuators B, 1995, p. 157-163,
vol. 29.

Gaur, 8.S., et al., “Synthesis and Analysis of Planar Optical Wave-
guides as pH Sensors”, Recent Innovations in Chemical Engineer-
ing, 2018, p. 40-44, vol. 11.

Hartings, M.R., et al., “A photonic pH sensor based on photothermal
spectroscopy”, Sensors and Actuators B: Chemical, 2019, p. 127076,
vol. 301

Rovati, L., et al., “Plastic Optical Fiber pH Sensor Using a Sol-Gel
Sensing Matrix”, Fiber Optic Sensors, 2012, p. 415-438, DOL

10.5772/26517.

* cited by examiner


U.S. Patent Apr. 11, 2023 Sheet 11 of 13 US 11,624,664 B2

infrared Probe

Ba oe

See pt

Visible Light

33S nm

isa it

FIG. 11


US 11,624,664 B2

1
PH PHOTOTHERMAL SPECTROMETER
AND PERFORMING PH PHOTOTHERMAL
SPECTROSCOPY

CROSS REFERENCE TO RELATED,
APPLICATIONS

The application claims priority to U.S. Provisional Patent
Application Ser. No. 63/084,313 filed Sep. 28, 2020, the
disclosure of which is incorporated herein by reference in its
entirety.

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-
nology Partnerships Office, NIST, Gaithersburg, Md.,
20899; voice (301) 301-975-2573; email tpo@nist. gov; ref-
erence NIST Docket Number 19-051USI.

BRIEF DESCRIPTION

Disclosed is a pH photothermal spectrometer for perform-
ing pH photothermal spectroscopy, the pH photothermal
spectrometer comprising a container comprising an interior
bounded by a wall of the container, such that the container
receives an analyte medium and a pH-sensitive chro-
mophore, the pH-sensitive chromophore comprises an opti-
cal absorption spectrum that adjusts to a pH of the analyte
medium, the analyte medium comprising a analyte tempera-
ture TA; and optionally receives a fiber encapsulation matrix
in which are disposed the analyte medium and the pH-
sensitive chromophore; an excitation fiber disposed in the
container in optical communication with the pH-sensitive
chromophore and that receives a first excitation light com-
prising a first wavelength and a second excitation light
comprising a second wavelength; and communicates the first
excitation light and the second excitation light to the pH-
sensitive chromophore, such that the pH-sensitive chro-
mophore receives the first excitation light and the second
excitation light; and absorbs a first amount Al of the first
excitation light and a second amount A2 of the second
excitation light based on the pH of the analyte medium such
that absorption of the first excitation light or the second
excitation light increases the analyte temperature TA of the
analyte medium; and an optical thermometer disposed in the
container and comprising a temperature detector fiber that
receives a probe light comprising a probe light wavelength
in a first optical amount O1; and receives a reflected light
comprising the probe light wavelength from a light receiver;

and a light receiver disposed on a terminus of the tempera-

ture detector fiber in optical communication with the tem-
perature detector fiber and in thermal communication with
the analyte medium; and comprising a receiver temperature
TR that adjusts to match the analyte temperature TA; and an
optical resonance OR at a resonance wavelength LR that
changes in response to a change in the receiver temperature
TR, such that the light receiver receives the probe light from
the temperature detector fiber; produces, from the probe
light, a reflected light in a second optical amount O2 and
comprising the probe light wavelength, such that the second
optical amount 2 of the reflected light depends on the
receiver temperature TR of the light receiver, so that a ratio

20

&

&

50

2

of the first optical amount O1 to the second optical amount
O2 provides a pH of the analyte medium; and communicates
the reflected light to the temperature detector fiber; and the
temperature detector fiber receives the reflected light from
the light receiver.

Disclosed is a process for performing pH photothermal
spectroscopy with a pH photothermal spectrometer, the
process comprising receiving, by the excitation fiber, the
first excitation light that comprises the first wavelength and
the second excitation light that comprises the second wave-
length; contacting the pH-sensitive chromophore with the
analyte medium; adjusting the optical absorption spectrum.
of the pH-sensitive chromophore in response to the pH of the
analyte medium; communicating the first excitation light
and the second excitation light from the excitation fiber to
the pH-sensitive chromophore; receiving, by the pH-sensi-
tive chromophore, the first excitation light and the second
excitation light; absorbing, by the pH-sensitive chro-
mophore, the first amount Al of the first excitation light
based on the pH of the analyte medium; absorbing, by the
pH-sensitive chromophore, the second amount A2 of the
second excitation light based on pH of the analyte medium:
adjusting the analyte temperature TA of the analyte medium
in response to the pH-sensitive chromophore absorbing the
first excitation light and the second excitation light; adjust-

5 ing the receiver temperature TR of the light receiver to

match the analyte temperature TA of the analyte medium in
response to adjusting the analyte temperature TA of the
analyte medium; receiving, by the light receiver, the probe
light in the first optical amount O1 from the temperature
detector fiber; producing, by the light receiver from the
probe light, the reflected light in the second optical amount
2 that depends on the receiver temperature TR of the light
receiver, the reflected light comprising the probe light wave-
length; and determining the pH of the analyte medium from
a ratio of the first optical amount O1 to the second optical
amount O2 to perform pH photothermal spectroscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

The following description should not be considered lim-
iting in any way. With reference to the accompanying
drawings, like elements are numbered alike.

FIG. 1 shows a pH photothermal spectrometer;

FIG. 2 shows a cross-section along line A-A of the pH
photothermal spectrometer shown in FIG. 1;

FIG. 3 shows a pH photothermal spectromet

FIG. 4 shows a pH photothermal spectrometer;

FIG. 5 shows a pH photothermal spectrometer;

FIG. 6 shows pH photothermal spectroscopy;

FIG. 7 shows pH photothermal spectroscopy:

FIG. 8 shows a pH photothermal spectromet

FIG. 9 shows an optical absorption spectrum of a pH-
sensitive chromophore as a graph of optical absorption
versus wavelength:

FIG. 10 shows a graph of absorbance versus wavelength
for UV-visible absorbance of red cabbage extract for pH of
2.5, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0,
9.5, and 10.0;

FIG. 11 shows pH photothermal spectroscopy;

FIG. 12 shows a graph of normalized signal versus pH for
phenol red; and

FIG. 13 shows a graph of normalized signal versus time
for phenol red.

DETAILED DESCRIPTION,

A detailed description of one or more embodiments is
presented herein by way of exemplification and not limita-
tion.