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Journal ArticleDOI

Performances and Robustness of a Fluorescent Sensor for Nearly Neutral pH Measurements in Healthcare

01 Apr 2020-IEEE Transactions on Instrumentation and Measurement (Institute of Electrical and Electronics Engineers (IEEE))-Vol. 69, Iss: 10, pp 7658-7665
TL;DR: This article theoretically and experimentally investigates the performances and the robustness of a fluorescent sensor that was recently proposed for the in-line and real-time monitoring of blood pH in ECC and demonstrates that the proposed sensor allows achieving significant robustness to unevennesses in the production process of the sensing element.
Abstract: The capability to measure pH is fundamental in many fields ranging from healthcare and agrifood, to chemistry and industrial applications. In medicine, the possibility of in-line and real-time monitoring critical care analytes, such as pH, is recognized to provide relevant information for the diagnosis and treatment of a variety of disorders and would be of considerable support for the management of extracorporeal (blood) circulation (ECC). In this article, we theoretically and experimentally investigate the performances and the robustness of a fluorescent sensor that we recently proposed for the in-line and real-time monitoring of blood pH in ECC. Indeed, being low-cost and biocompatible, the proposed sensor can be advantageously exploited for the in-line and real-time monitoring of the pH of fluids, particularly in applications such as healthcare where safe and low-cost disposables are usually required for all parts which come into contact with the patient. The reported results demonstrate that the proposed sensor allows achieving significant robustness to unevennesses in the production process of the sensing element.

Summary (2 min read)

Introduction

  • Given the interest in blood pH monitoring, many measurement methods and measuring systems have been presented in the literature in the last years i.e. [1], [6]–[14].
  • In such a paper [5], the authors described the proposed measuring instrument and measurement method, they report about a preliminary investigation on biocompatibility and, finally, they reported the results obtained by simulating with bovine blood an about 6 h ECC treatment.
  • As will be demonstrated, such performances and robustness are mainly due to both the measurement method and model and, the developed sensing element.

A. The sensing element

  • The proposed sensing element is based on HPTS (8- Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) an inexpensive and nontoxic ratiometric pH indicator.
  • The fluorophore is fixed to the polymeric substrate by using anion exchange microbeads and a biocompatible polyurethane hydrogel according to the 6-step production process described in their recent paper [5].

B. Measurement model

  • According to the optical properties of HPTS that will be shown in subsection III-B and the following demonstration, 2 Polymeric substrate Anion exchange microbeads ``loaded'' with HPTSPolyurethane hydrogel Fig.
  • The yellows circles represent the anion exchange microbeads “loaded” with HPTS.
  • The sensing element is thus composed of the microbeads “loaded” with HPTS and the polyurethane hydrogel.
  • Therefore, from (7) it is evident that the ratio between the fluorescent powers emitted by the sensing elements depends on both the pH and, the irradinaces I0(λex−1) and I0(λex−2).

C. Sensor characterization

  • Sensor characterization has been aimed at investigating the performances and robustness of the proposed sensor and measurement method to unevennesses in the production of the sensors.
  • PBS has been preferred to blood since blood-pH is known to vary with the blood-pCO2.
  • 1) Substrate absorbances and inter-sensors uniformity: Absorbance spectra have been investigated by using the MPR previously described and modifying the pH of the PBS solution filling each well.
  • Then, unevennesses in the fluorescence signals generated by the various sensors have been investigated similarly to what was done for the analysis of the absorbance spectra.

IV. DISCUSSIONS AND CONCLUSIONS

  • Nowadays no measurement method has been able to provide safe, economical, accurate and reliable estimates of pH in the bloodstream to be used routinely during ECC treatments [5], thus blood pH is generally not monitored during such treatments.
  • To overcome such a limitation, in their recent paper [5] the authors propose a measuring system composed of a disposable fluorescent sensing element and a non-disposable opticalhead aimed at the contactless reading of the sensor.
  • The reported values have been obtained according to (14) and analyzing the two peaks in Fig. Dots (●) and error bars represent the Rmean and the experimental standard deviations of the mean obtained from the Ns = 6 sensors.
  • As a matter of fact, in their previous articles, the authors have benefited from optics in ECC for the inline measurement of blood flow [20], monitoring of the urea clearance [21] and, measurement of the hemolysis [22], [23].
  • Concluding, the proposed sensing element offers the possibility to transform polymeric tubes and containers into sensors that can be read contactless from outside, offering robust inline and real-time monitoring of the pH of the inside fluid.

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1
Performances and robustness of a fluorescent sensor
for nearly-neutral pH measurements in healthcare
Stefano Cattini, Member, IEEE, Stefano Truzzi, Luca Accorsi and, Luigi Rovati, Member, IEEE,
Abstract—The capability to measure pH is fundamental in
many fields ranging from healthcare and agri-food, to chemistry
and industrial applications. In medicine, the possibility of in-line
and real-time monitoring critical care analytes such as pH is
recognized to provide relevant information for the diagnosis and
treatment of a variety of disorders and would b e of considerable
support for the management of extracorporeal (blood) circulation
(ECC). In this paper, we theoretically and experimentally investi-
gate the performances and the robustness of a fluorescent sensor
we recently proposed for the in-line and real-time monitoring
of blood pH in extracorporeal circulation (ECC). Thanks to the
low-cost and biocompatibility, the proposed sensor can be used
for the in-line and real-time monitoring of the pH of fluids
especially in applications such as healthcare where safe and
low-cost disposable are required for all parts in contact with
the patient. The reported results demonstrate that the proposed
sensor allows achieving significant robustness to unevennesses in
the production process of the sensing element.
Index Terms—Ratiometric fluorescent sensor, pH measure-
ment, Biomedical measurement, Healthcare, Extracorporeal cir-
culation (ECC), Hemodialysis, Hemofiltration, Extracorporeal
membrane oxygenation (ECMO).
I. INTRODUCTION
In spite of the long history of pH measurement, many
problems are still open and new measuring systems are
continuously developed to meet measurement needs. Indeed,
pH monitoring is fundamental in many fields ranging from
medicine and agri-food, to chemistry and industrial applica-
tions [1]–[5].
In healthcare, the acid-base status of critically ill patients is
often altered, thus blood pH is one of the most important
parameters to be monitored in the emergency room, recovery
room and intensive care unit. In particular, the possibility
of real-time and in-line monitoring blood pH would be of
significant support for the management of extracorporeal blood
circulation (ECC). ECC includes a set of different medical
procedures in which the patient’s blood flows outside the body
for therapies such as blood purification or heart and lungs
temporary replacement as in cardiac surgery.
Given the interest in blood pH monitoring, many measure-
ment methods and measuring systems have been presented in
the literature in the last years i.e. [1], [6]–[14]. However, as
discussed in more detail in our recent paper [5], nowadays no
S. Cattini and L. Rovati are with the Department of Engineering “Enzo
Ferrari”, University of Modena and Reggio Emilia, Via Vivarelli 10, 41125,
Modena, Italy. All the authors are with the Science & Technology Park for
Medicine, TPM, Democenter Foundation Mirandola, Modena, Italy e-mail:
stefano.cattini@unimore.it
Manuscript received XXX, XXX; revised XXXX.
measurement method has been able to provide safe, econom-
ical, accurate and reliable estimates of pH in the bloodstream
to be used routinely during ECC treatments, thus blood pH i s
generally not monitored during ECC treatments. To overcome
all the above limitations, in our recent paper [5], we proposed
and described a measuring system composed of a disposable
fluorescent sensor and a non-disposable optical-head for the in-
line and real-time monitoring of blood-pH in extracorporeal-
circulation (ECC). In such a paper [5], we described the
proposed measuring instrument and measurement method, we
report about a preliminary investigation on biocompatibility
and, finally, we reported the results obtained by simulating
with bovine blood an about 6 h ECC treatment. In this
paper, we investigate and describe the performances and the
robustness of the proposed sensor to unevennesses in the
production process.
As will be demonstrated, such performances and robustness
are mainly due to both the measurement method and model
and, the developed sensing element. As a result, in the fol-
lowing, section II describes the sensing element, derives the
measurement model and, reports the experimental activities
carried out for the sensor characterization. The obtained results
are r eported in section III and conclusions are drawn in
section IV.
II. MATERIALS AND METHODS
The following subsections, briefly describe the sensing
element (subsection II-A), analyze in detail the measurement
model (subsection II-B), and, give a detailed account of the
experimental activities carried out to characterize the proposed
sensing element (subsection II-C).
A. The sensing element
The proposed sensing element is based on HPTS (8-
Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt) an inex-
pensive and nontoxic ratiometric pH indicator. The fluorophore
is fixed to the polymeric substrate by using anion exchange
microbeads and a biocompatible polyurethane hydrogel ac-
cording to the 6-step production process described in our
recent paper [5]. An “out of scale” cross-section of the sensing
element is shown in Fig. 1.
Excluding the polymeric substrate, the cost of the raw mate-
rials used for a single sensor is about a tenth of a euro.
B. Measurement model
According to the optical properties of HPTS that will be
shown in subsection III-B and the following demonstration,

2
Polymeric substrate
Anion exchange microbeads
``loaded'' with HPTS
Polyurethane hydrogel
Fig. 1. Out of scale cross-section of the proposed sensor. The yellows
circles represent the anion exchange microbeads “loaded” with HPTS. The
microbeads are fixed to the polymeric substrate by the polyurethane hydrogel.
The sensing element is thus composed of the microbeads “loaded” with HPTS
and the polyurethane hydrogel.
the pH value can be estimated from the ratio of the fluorescent
optical powers emitted by the s ensor in s mall wavelength band
around λ
em
520 nm while exciting the sensor at wavelengths
λ
ex1
405 nm and λ
ex2
475 nm, respectively.
The fluorescent radiance generated by the sensor is pro-
portional to the excitation power that is absorbed by the
fluorophore [15], [16]. Since the sensing element is sub-
stantially composed of a monolayer of anion exchange mi-
crobeads loaded with HPTS (see Fig. 3), according to Fig. 2
and assuming the sensing element to be illuminated by a
monochromatic infinite plane wave with irradiance I
0
(x, y)
impinging ort hogonally to the surface of the sensor, the overall
irradiance absorbed by a small portion of the sensing element
having thickness d(x, y) and infinitesimal area dσ can be
estimated according to the Beer-Lambert’s law as:
I
0
(x, y)I
T
(x, y) = I
0
(x, y)[1 10
A(x,y)
] , (1)
where I
T
is the irradiance of the beam after crossing the sensor
and, A is the (overall) absorbance [17]:
A(λ, x, y)
def
= log
10
I
0
(λ, x, y)
I
T
(λ, x, y)
=
1
2.303
ln
I
0
(λ, x, y)
I
T
(λ, x, y)
=
N
i=1
ǫ
i
(λ)[C
i
]d DP F +G +A
sub
(λ)
=A
sens
(λ, x, y)+A
sub
(λ, x, y),
(2)
where λ is the wavelength, N = 3 is the number of chro-
mophores light-absorbing species composing the sensing
element (HPTS, anion exchange microbeads and, polyurethane
hydrogel), [C
i
](x, y)and ǫ
i
(λ)are t he concentration and the
molar absorption coefficient of the i
th
chromophore compos-
ing the sensor, DP F (λ, x, y) is the differential pathlength
factor that takes into account potentially extended pathlength
due to scattering, G(λ, x, y) represents potential losses due
to scattering and, A
sub
(λ, x, y) and A
sens
(λ, x, y) represent
the absorbances of the polymeric substrate and the sensing
element, respectively. In (2) the chromophores concentrations
[C
i
](x, y)have been supposed to be uniform throughout the
thickness d(x, y).
It i s important to notice that the fluorescent intensity is not
due to the overall absorbance of the sensing element A
sens
,
but it is proportional to the excitation power that is absorbed
by the fluorophore only:
A
HP
(λ, x, y)= ǫ
HP
(λ)[C
HP
](x, y)d(x, y)DP F (x, y ),
(3)
where ǫ
HP
and [C
HP
] are the molar absorption coefficient
and the molarity of HPTS, respectively.
I (x,y)
0
I (x,y)
T
x y
z
Fig. 2. The fluorescent intensity generated by the sensor is proportional to
the excitation power that is absorbed by the fluorophore. I
0
and I
T
are the
irradiances impinging on the polymeric substrate and after crossing the sensor.
Hence, the overall optical power P
F
relative to the fluo-
rescence generated by the sensor in a small bandwidth around
the λ
em
wavelength once it is illuminated (excited) by a beam
having a small bandwidth around the wavelength λ
ex
and
irradiance I
0
is:
P
F
= Φ
E
k
x,y
I
0
(x, y)1e
2.303A
HP
(x,y)
dxdy , (4)
where Φ
E
(λ
ex
, λ
em
, pH) is the fluorescence quantum yield
the ratio of photons emitted at λ
em
through fluorescence
to photons absorbed at λ
ex
, k(λ
ex
, λ
em
)is a constant that
takes into account the different energies of photons at λ
ex
and
λ
em
. Expanding in series the exponential in (4) and keeping
only the zero and first-order terms, (4) becomes:
P
F
Φ
E
k
x,y
I
0
(x, y)2.303 A
HP
(x, y)dxdy . (5)
Note that the approximation of (5) is valid for A
HP
< 1.
Indeed, as will be shown in Fig. 3, the proposed sensing
element is very thin, thus the absorption is little as it will
be shown in subsection III-A (note that, according to (3) and
(8) the absorbance of the fluorophore A
HP
is only a fraction
of the overall absorbances showed in Fig. 5).
Supposing uniform illumination I
0
(x, y)= I
0
from (3)
and (5) the overall fluorescence power P
F
is:
P
F
Φ
E
k 2.303ǫ
HP
I
0
x,y
[C
HP
]dDP F dxdy . (6)
As a result, the ratio between the overall optical powers P
F 2
and P
F 1
emitted at the emission wavelength λ
em
once the
sensor is respectively excited at the wavelengths λ
ex2
and
λ
ex1
is substantially insensitive to changes in sensor thickness
and fluorophore concentration:
P
F 2
P
F 1
=
Φ
E2
k
2
ǫ
HP
(λ
ex2
)I
0
(λ
ex2
)
Φ
E1
k
1
ǫ
HP
(λ
ex1
)I
0
(λ
ex1
)
=
Φ
E2
k
2
ǫ
HP
(λ
ex2
)P
sen
(λ
ex2
)
Φ
E1
k
1
ǫ
HP
(λ
ex1
)P
sen
(λ
ex1
)
,
(7)
where Φ
E2
= Φ
E
(λ
ex2
, λ
em
), Φ
E1
= Φ
E
(λ
ex1
, λ
em
),
k
2
= k(λ
ex2
, λ
em
), k
1
= k(λ
ex1
, λ
em
)and, P
sen
is the over-
all excitation optical power equal to the product between the I
0
irradiance and the sensor area in the (x, y)plane. Equation (7)
has been obtained assuming that the scattering coefficient
varies little between λ
ex1
and λ
ex2
. Indeed, the DP F is
due by both the concentration and the scattering coefficient of
the scatterers. Since the scatterers are the same both for λ
ex1
and λ
ex2
and, the two wavelengths are not very far apart, we

3
approximated DP F (λ
ex1
, x, y) DP F (λ
ex2
, x, y).
For pH-sensitive ratiometric fluorophores such as HPTS,
Φ
E1
, Φ
E2
, ǫ
HP
(λ
ex1
)and, ǫ
HP
(λ
ex2
)are known to vary
as a function of the pH. Therefore, from (7) it is evident
that the ratio between the fluorescent powers emitted by the
sensing elements depends on both the pH and, the irradinaces
I
0
(λ
ex1
)and I
0
(λ
ex2
). As a result, it is important to mon-
itor and compensate potential drift of the excitation powers.
C. Sensor characterization
Sensor characterization has been aimed at investigating
the performances and robustness of the proposed sensor and
measurement method to unevennesses in the production of
the sensors. The properties of the fluorescent sensing element
have been investigated by analyzing and comparing the perfor-
mances of N
s
= 6 sensors produced following the procedure
previously described in subsection II-A and using a 6-wells
multi-well plate (model 657160 by Greiner) as substrate. A
picture of the sensors is shown in Fig. 3.
Fig. 3. Picture of the N
s
= 6 sensors produced following the procedure
described in subsection II-A and using a 6-wells multi-well plate (model
657160 by Greiner) as substrate. The zoom shows the anion exchange
microbeads “loaded” with HPTS and fixed to the substrate by the polyurethane
hydrogel (microbeads sizes declared by the manufacturer: [200, 400] mesh).
Sensors have been investigated by analyzing the absorbance
and fluorescence spectra as a function of the pH of the medium
the measurand. To modify the pH, wells were filled with
5 ml phosphate-buffered saline (PBS) solutions at different
pH. The pH of the PBS solutions was estimated by using a pH
electrode (model HI9125, Hanna). PBS has been preferred to
blood since blood-pH is known to vary with the blood-pCO2.
Hence, blood-pH would have varied over time due to exposure
to air, giving rise to a relevant uncertainty on the pH value of
the sample.
Then, spectral absorbance and fluorescence were analyzed
by using a Multimode Plate Reader (MPR, model EnSpire
2300 by PerkinElmer). In particular, as shown in Fig. 4, the
absorbance has been investigated by measuring the attenuation
of the light beam traversing the layered structure realized by
the substrate, the sensor and, the PBS. On the other hand, the
fluorescence has been investigated by exciting and measuring
from the bottom of the well.
To guarantee repeatability conditions, for each pH value the
absorbance and fluorescence spectra were measured thermo-
stating the sensors at 27
C using the MPR and, waiting a
warm-up ti me of 5 minutes after the sensors were inserted into
the MPR during the warm-up, sensors were gently shaken
by the MPR. Indeed, both the pH of PBS and the fluorescence
quantum yield Φ
E
are known to vary as a function of the
temperature and, preliminary tests revealed that in our test
conditions (thickness of the sensors and temperatures of the
ambient, the PBS and, the measuring chamber of the MPR)
5 minutes were sufficient to guarantee that the sensing element
reached the steady-state condition both in terms of temperature
and H
+
ions concentration. Then, both the absorbance and the
fluorescent spectra were recorded by setting the MPR to an
intensity equal to 10 flashes.
PBS
I
0
PBS
I
0
I
T
I
fl
Absorbance
Fluorescence
Fig. 4. The absorbance has been investigated by measuring the attenuation of
the light beam traversing the the three-layer structure realized by the substrate,
the sensor and the PBS. I
0
and I
T
are irradiances impinging and transmitted
after traversing the three layers. On the other hand, fluorescence has been
investigated by exciting the sensor and measuring the fluorescence from the
bottom of the well. I
f l
is the fluorescence irradiance. d
s
and d
P BS
are the
(mean) thicknesses of the sensor and the PBS layer, respectively.
1) Substrate absorbances and inter-sensors uniformity:
Absorbance spectra have been investigated by using the MPR
previously described and modifying the pH of the PBS solu-
tion filling each well.
It i s important to notice that absorbance spectra allow to both
investigate the optical properties of the fluorescent sensor and,
to investigate the inter-sensors uniformity. Indeed, according
to (2), the overall absorbance A of a monochromatic and
collimated light beam of irradiance I
0
traversing the three-
layer structure realized by the substrate, the sensor and the
PBS can be estimated according to the Beer-Lambert’s law
as:
A = A
sub
+A
sens
+A
P BS
, (8)
where, A
sub
, A
sens
and A
P BS
are the absorbances of the sub-
strate, the sensor and the PBS. According to subsection II-A,
the sensing element is composed of 3 chromophores, namely
the HPTS, the hydrogel and, anion exchange microbeads.
Since the molar absorption coefficient of HPTS ǫ
HP
is known
to vary as a function of the pH, while the others quantities
basically do not, it is possible to define the following quantity:
γ(λ, pH
1
, pH
2
)= A(λ, pH
2
)A(λ, pH
1
)
[C
HP
]d
s
DP F ǫ
HP
(λ, pH
1
, pH
2
),
(9)
where [C
HP
]is the (mean) concentration of the fluorophore
(HPTS), d
s
is the (mean) thickness of the sensing element,
DP F is the (mean) differential pathlength factor that takes
into account potential scattering, and
ǫ
HP
(λ, pH
1
, pH
2
)= ǫ
HP
(λ, pH
2
)ǫ
HP
(λ, pH
1
). (10)

4
In (9) the variation in PBS thickness has been neglected as
the thickness of the sensing element is negligible compared to
that of PBS.
Since ǫ
HP
is a property of HPTS, thus the same for all
the 6 sensors, differences between the γ values obtained from
the absorbances of the 6 sensors reveal differences in the
HPTS concentrations [C
HP
], thicknesses d
s
and/or scattering
(DP F ) between the sensors. Thus, inter-sensors uniformity
has been investigated analyzing
γ(λ, pH
1
, pH
2
)=
γ(λ, pH
1
, pH
2
)γ
mean
(λ, pH
1
, pH
2
)
γ
mean
(λ, pH
1
, pH
2
)
,
(11)
and
s
γ
=
1
N
s
1
N
s
i=1
γ(i)γ
mean
2
γ
mean

1
, (12)
where s
γ
(λ, pH
1
, pH
2
) is the relative experimental standard
deviation, γ(i)is the γ value respective to the i
th
sensor and,
γ
mean
(λ, pH
1
, pH
2
)is the mean γ recorded from the N
s
= 6
sensors. Equation (11) has been divided by γ
mean
so that to
remove the contribution of ǫ
HP
, thus providing an estimate
of how much the current sensor differs in percentage from
the “average sensor” in terms of fluorophore concentration,
thickness and, scatterers.
2) Substrate fluorescence and calibration curve: As pre-
viously shown in Fig. 4, for each sensor the fluorescence
has been investigated by using the MPR, and exciting and
measuring the sensor from the bottom of the well.
It is important to notice that the spectra provided by spec-
trofluorimeters are automatically rescaled by the excitation
power used by the spectrofluorimeter to obtain the fluores-
cence signal. Thus, the fluorescence spectra provided by the
Multimode Plate Reader at λ
ex1
and λ
ex2
are corrected with
respect to variation of P
sen
in (7). Then, unevennesses in
the fluorescence signals generated by the various sensors have
been investigated similarly to what was done for the analysis
of the absorbance spectra. Hence, for each of the N
s
= 6
sensors we estimated:
P (i, λ
em
, λ
ex
, pH)=
P (i)P
mean
P
mean
, (13)
and,
s
P
=
1
N
s
1
N
s
i=1
P (i)P
mean
2
(P
mean
)
1
, (14)
where λ
em
and λ
ex
are the emission and excitation wave-
lengths, P (i, λ
em
, λ
ex
, pH)is the fluorescence optical power
recorded from the i
th
sensor and, P
mean
(λ
em
, λ
ex
, pH)and
s
P
(λ
em
, λ
ex
, pH)are the mean and the relative experimental
standard deviation of the P recorded from the N
s
= 6 sensors.
Moreover, according to subsection II-B, from the analysis
of the fluorescence spectrum, it is possible to estimate the pH
of the solution. Thus, in accordance with (7), for each of the
6 sensors we estimate the ratios R:
R(i, pH)=
P (i, λ
em
, λ
ex2
, pH)
P (i, λ
em
, λ
ex1
, pH)
, (15)
and the sigmoid obtained as a function of the pH. Then, the
calibration function pH
est
(R)has been estimated by reversing
such sigmoid function as described in subsection III-B.
3) Sensitivity and pK
a
: The pK
a
of the fluorophore defines
the sensitivity and the measuring interval of the sensor [18].
Thus, it is important to know and match the pK
a
of the
indicator to the pH of the measurand [18]. pK
a
of HPTS
is known in the literature e.g. pK
a
7.30 in 0.066 M
phosphate buffers at 22
C [18]. However, pK
a
depends
on several quantities such as the ionic strength, and the
dielectric constant of the surrounding medium [19]. Hence, it
is important to determine the pK
a
of HPTS once incorporated
in the polyurethane hydrogel and anion exchange microbeads.
The pK
a
of the sensor in PBS has been estimated as the pH
value for which t he second-order derivative of the R values
as a function of the pH of the medium is equal to zero the
inflection point [19]:
pK
a
= pH
2
R
2
pH
= 0 . (16)
Then, the sensitivity η of the sensor has been investigated as
the derivative of the R values as a function of the pH of the
medium:
η =
R
pH
. (17)
III. RESULTS
A. Substrate absorbances and inter-sensors uniformity
As an example, Fig. 5 shows the absorbance spectra ob-
tained from one of the N
s
= 6 sensors realized using the
multi-well plate as described in subsection II-A.
According to subsection II-C, inter-sensors uniformity has
been investigated analyzing the γ parameter defined in (9).
Fig. 6 shows the γ values obtained from the N
s
= 6 sensors
considering pH
1
= 4.11 pH and pH
2
= 9.50 pH. As shown
in Fig. 6, the γ obtained by the “handmade” sensors re-
vealed significant differences. Thus, according to (11), sensors
have significant differences in terms of HPTS concentrations
[C
HP
], thicknesses d
s
and/or scattering (DP F ). Indeed,
according to (12) we obtained s
γ
40% for λ = 408 nm
and s
γ
38% for λ = 464 nm (the two peak wavelengths in
the absorption spectra). Nevertheless, thanks to the use of a
ratiometric fluorophore, the sensors have almost the same ratio
R as it will be shown in subsection III-B.
B. Substrate fluorescence and calibration curve
As an example, Fig. 7 shows the fluorescence spectra
obtained from one of the N
s
= 6 sensors realized using the
multi-well plate as described in subsection II-A.
Fig. 8 and 9 show the P and s
P
values obtained by analyzing
the N
s
= 6 sensors according to (13) and (14). According
to the results obtained by analyzing the absorbance spectra
and previously shown in Fig. 6, also the results relative to
fluorescence spectra shown in Fig. 8 and 9 reveal significant
inter-sensors differences.
Despite the significant inter-sensors differences shown in
Fig. 6, 8 and, 9, the ratiometric analysis described in (15)

5
400 450 500 550
(nm)
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Absorbance (OD)
Fig. 5. Absorbance spectra as a function of the pH of the PBS solution
obtained from one of the N
s
= 6 sensors realized using the multi-well plate
(PBS solution 1X, at 27
C). pH = 4.11 (
), pH = 5.11 (), pH = 6.13
(
), pH = 7.10 (), pH = 7.50 (), pH = 8.00 (), pH = 8.58 () and,
pH = 9.50 (
). As expected, spectra at low pH values () and ()
substantially overlap.
1 2 3 4 5 6
Sensor
-60
-40
-20
0
20
40
60
80
(%)
Fig. 6. γ values obtained from the N
s
= 6 sensors considering pH
1
=
4.11 pH and pH
2
= 9.50 pH (PBS solution 1X, at 27
C). The reported
values have been obtained according to (11) and analyzing the two peaks in
Fig. 5. In particular, (
) have been obtained by analyzing λ = 408 nm and
(
) have been obtained by analyzing λ = 464 nm.
allows to greatly limit the negative effects due to such inter-
sensors differences. Fig. 10 shows the mean R values and
the respective experimental standard deviations of the mean
s
Rmean
= s
R
N
s
obtained from the sensors changing the
pH of the PBS solutions filling the wells:
R
mean
(pH)=
1
N
s
N
s
i=1
R(i, pH),
s
R
(pH)=
1
N
s
1
N
s
i=1
R(i)R
mean
2
,
(18)
where R(i, pH) is the R value obtained from the
i
th
sensor. In particular, according to (15), each R(i, pH)has
been obtained exciting the sensing element at λ
ex1
= 405 nm
and λ
ex2
= 475 nm and, recording the fluorescence power P
emitted in the band λ
em
= [515, 525] nm. Fig. 12 shows t he
relative experimental standard deviations (s
R
R
mean
) of the
R values obtained from the N
s
= 6 sensors.
As previously described in subsection II-C, the ratio R allows
estimating the pH of the solution. The function obtained by
reversing the sigmoid shown in Fig. 10 is:
pH = α
3
log
10
α
2
α
1
R α
1
1α
1
4
, (19)
where α
1
= 6.5 10
3
, α
2
= 1.867, α
3
= 6.920 and, α
4
=
0.635. Note that (19) has real solutions only for α
1
< R < α
2
.
Therefore, given the limited sensitivity that would be obtained
for pH values at the ends of the sigmoid and the interest in
nearly-neutral pH, we have defined the calibration function as
follows:
pH
est
=f(R)
=α
3
log
10
α
2
α
1
R α
1
1α
1
4
R
min
< R < R
max
=10 R R
max
=4 R R
min
.
(20)
where R
min
= 32.17 10
3
and R
max
= 1.8469. By applying
(20) to the experimental R values it is possible to estimate
both the relative errors
ǫ(i, pH)=
pH pH
est
(i, pH)
pH
estmean
(pH)
, (21)
and, the relative experimental standard deviations s
pHest
of
the pH
est
values:
s
2
pHest
(pH)=
N
s
i=1
pH
est
(i, pH)pH
estmean
(pH)
2
(N
s
1)[pH
estmean
(pH)]
2
(22)
where pH
est
(i, pH) is the estimate obtained from the
i
th
sensor at pH and pH
estmean
(pH) is the mean. The
obtained results are shown in Fig. 11 and 12.
Note that, since Fig. 6 gives an idea of how much the 6 sensors
differ from the “average sensor” in terms of fluorophore
concentration, thickness and, scatterers (see subsection II-C),
while Fig. 11 and 12 show the percentage errors and the
relative experimental standard deviations obtained by using the
same calibration function for all the 6 sensors, by comparing
Fig. 6, 11 and, 12, it is possible to obtain a visual estimate of
the “robustness” of the ratiometric method to unevenness in
the production process.
C. Sensitivity and pK
a
Fig. 13 and 14 show the η and
2
R
2
pH values obtained
from the sigmoid function shown in Fig. 10. As expected,
the pK
a
obtained in Fig. 14 is equal to the α
3
obtained in
(19). As shown in Fig. 10, 13 and 14, the measuring interval
is about [4, 10] pH and the maximum sensitivity is achieved
for nearly-neutral pH values where η 0.7 pH
1
. Thus, the
maximum sensitivity in PBS at 27
C is achieved for a pH
value slightly different from the physiological pH value of the
blood [7.35, 7.45]pH.

Citations
More filters
Proceedings ArticleDOI
05 Mar 2021
TL;DR: The obtained results demonstrate that the robustness of such a measuring system to variations of blood parameters such as blood flow and hematocrit is investigated, thus complying with the metrological requirements for in-line and real-time monitoring of blood-pH during ECC.
Abstract: The possibility to monitor blood-pH has long been acknowledged to provide significant information for the diagnosis, management and treatment of a variety of diseases and it would be of considerable support for the administration of several treatments such as, for example, extracorporeal (blood) circulation (ECC). During ECC, the patient’s blood flows outside the body in disposable bloodlines and devices for treatments such as blood purification or circulation/ventilation/oxygenation support. Although blood-pH can be measured since the early twentieth century by using ion-selective electrodes (ISEs) and, more recently, also by using point-of-care testing (POCT) instruments, nowadays no measurement method has fully succeeded in providing a cost-effective, reliable and accurate estimate of the blood-pH to be routinely used for its real-time monitoring. In a recent paper, we have proposed and demonstrated a measuring instrument for the in-line and real-time monitoring of blood-pH during ECC. Such a measuring system consists of a low-cost fluorescent disposable sensor that can be integrated into the bloodline and, of a non-disposable reading system that interrogates the sensor without contacting the patient’s blood. In this paper, we investigated the robustness of such a measuring system to variations of blood parameters such as blood flow and hematocrit. The obtained results demonstrate that, although during the tests the pH, flow, and hematocrit values were significantly varied — pH from ≈ 6.8 pH, to ≈ 7.4 pH; hematocrit from 32%, to 40%; flow from 250 ml/min, to 400 ml/min, — the measuring system continued to guarantee a measurement error inferior to ±0.04 pH, thus complying with the metrological requirements for in-line and real-time monitoring of blood-pH during ECC

3 citations


Cites background from "Performances and Robustness of a Fl..."

  • ...In a recent paper, Cattini et al.(13) investigated the robustness of the measuring instrument to unevennesses potentially occurring in the production process of the sensing element....

    [...]

  • ...As previously introduced and discussed in more detail by Cattini et al.,12 the measuring system consists of a fluorescent disposable sensor inserted in the disposable bloodline and of a non-disposable optical head that excites the sensor and measures the resulting fluorescence intensities....

    [...]

  • ...To Send correspondence to Luigi Rovati (e-mail luigi.rovati@unimore.it) Optical Diagnostics and Sensing XXI: Toward Point-of-Care Diagnostics, edited by Gerard L. Coté, Proc. of SPIE Vol. 11651, 116510W · © 2021 SPIE CCC code: 1605-7422/21/$21 · doi: 10.1117/12.2576609 Proc. of SPIE Vol. 11651 116510W-1 respond to such a clinical need, we have recently proposed and demonstrated a measuring system that could overcome current limitations, thus enabling the in-line and real-time monitoring of blood-pH in extracorporeal (blood) circulation (ECC).12 Such a measuring system is based on the use of optical radiations to interrogate a disposable sensor inserted in the disposable ECC-bloodline.12 The sensor is equipped with a fluorescent substrate whose emission signal is analyzed through a non-disposable optical head.12 In a recent paper, Cattini et al.13 investigated the robustness of the measuring instrument to unevennesses potentially occurring in the production process of the sensing element....

    [...]

Journal ArticleDOI
TL;DR: A measuring instrument consisting of a low-cost and disposable fluorescent pco-sensor that can be inserted in series with the bloodline, and a nondisposable optical head that contactless reads the sensor is proposed and demonstrated.
Abstract: Since the late 1950s, blood $p$ CO2 can be measured by using electrodes and, more recently, also by using point-of-care instruments such as blood gas and chemistry analyzers Nevertheless, nowadays no measurement method has succeeded in providing an economic, reliable, and accurate estimate of the blood- $p$ CO2 to be routinely used for the monitoring of extracorporeal (blood) circulation (ECC) treatments Indeed, blood is a very complex biological matrix composed of several different constituents, thus apart from a limited maximum-admitted measurement error (about some mmHg) and a quite wide measuring interval (about [20, 100] mmHg), the measuring systems have to provide significant selectivity taking hemocompatibility and safety for the patient as a primary concern In this article, we propose and demonstrate a measuring instrument consisting of a low-cost and disposable fluorescent pco-sensor that can be inserted in series with the bloodline, and a nondisposable optical head that contactless reads the sensor The proposed measuring system is intrinsically safe for the patient, has adequate metrological performance, avoids blood sampling and the related issues, and may allow to comply with cost requirements Preliminary tests, conducted simulating a 6-h ECC treatment by using bovine blood, revealed deviations from the reference measuring instrument of less than ±4 mmHg, while the blood- $p$ CO2 was varied in the range [20, 100] mmHg

3 citations


Cites background from "Performances and Robustness of a Fl..."

  • ...Moreover, the performed ratiometric pH-measurement has been demonstrated to provide great robustness against variation in the geometry of the sensor as well as in the concentration of the fluorophore [24]....

    [...]

  • ...ratiometric fluorophores are recognized to provide a more robust measurement method [23], [24]....

    [...]

Proceedings ArticleDOI
16 May 2022
TL;DR: In this paper , the performance of a real-time, non-invasive pH measuring sysem for extracorporeal circulation (ECC) is analyzed, focusing on the analysis of the effects that temperature of the measurand may have on the error in estimating blood pH.
Abstract: Under physiological conditions, the body maintains blood pH within the very narrow range [7.36, 7.44] pH. Small deviations from this range can reveal the onset of pathological states. In this work the performances of a real-time, non-invasive pH measuring sysem for extracorporeal circulation (ECC) are analyzed. In particular, this study focuses on the analysis of the effects that temperature of the measurand may have on the error in estimating blood pH. Indeed, the sensor is based on the analysis of the fluorescence produced by HPTS, which is known to vary with temperature. The extent of such a variation, however, depends on various factors, including the chemical environment. Blood temperature in ECC is often thermostated at 37 °C. Nevertheless, there are treatments in which the blood temperature is varied by a few Celsius degrees, generally reduced, from the physiological temperature of 37 °C. Therefore, the first objective of this study was to evaluate whether a modest reduction in temperature, that is a few Celsius degrees, introduce an error such as the measuring system no longer conforms to the maximum permissible measurement error of ±0.04 pH. Once verified that the temperature-induced error could exceed the limit of ±0.04 pH, a correction factor for temperature compensation was investigated and its robustness to unevenness in the sensor production was explored. The results obtained from this preliminary study performed using Phosphate Buffer Saline (PBS) showed how the addition to the measuring system of a temperature sensor can effectively allow to maintain the measurement error within the ±0.04 pH range, even when the temperature of the measurand decreases by a few degrees from the physiological temperature of 37 °C.

1 citations

Proceedings ArticleDOI
16 May 2022
TL;DR: In this paper , the performance of a real-time, non-invasive pH measuring sysem for extracorporeal circulation (ECC) is analyzed, focusing on the analysis of the effects that temperature of the measurand may have on the error in estimating blood pH.
Abstract: Under physiological conditions, the body maintains blood pH within the very narrow range [7.36, 7.44] pH. Small deviations from this range can reveal the onset of pathological states. In this work the performances of a real-time, non-invasive pH measuring sysem for extracorporeal circulation (ECC) are analyzed. In particular, this study focuses on the analysis of the effects that temperature of the measurand may have on the error in estimating blood pH. Indeed, the sensor is based on the analysis of the fluorescence produced by HPTS, which is known to vary with temperature. The extent of such a variation, however, depends on various factors, including the chemical environment. Blood temperature in ECC is often thermostated at 37 °C. Nevertheless, there are treatments in which the blood temperature is varied by a few Celsius degrees, generally reduced, from the physiological temperature of 37 °C. Therefore, the first objective of this study was to evaluate whether a modest reduction in temperature, that is a few Celsius degrees, introduce an error such as the measuring system no longer conforms to the maximum permissible measurement error of ±0.04 pH. Once verified that the temperature-induced error could exceed the limit of ±0.04 pH, a correction factor for temperature compensation was investigated and its robustness to unevenness in the sensor production was explored. The results obtained from this preliminary study performed using Phosphate Buffer Saline (PBS) showed how the addition to the measuring system of a temperature sensor can effectively allow to maintain the measurement error within the ±0.04 pH range, even when the temperature of the measurand decreases by a few degrees from the physiological temperature of 37 °C.

1 citations

Journal ArticleDOI
TL;DR: In this paper , a linear correction factor for temperature compensation was proposed to compensate for the measurement error caused by temperature variation in extracorporeal circulation (ECC) measurements. But the results showed that the temperature-induced error could exceed the maximum permissible measurement error of ± 0.04 pH.
Abstract: Under physiological conditions, the human body maintains blood pH within [7.36, 7.44] pH. Small deviations from this range can reveal the onset of pathological states and worsen the patient’s condition. This paper reports the performance analysis of a real-time, non-invasive pH measuring system for extracorporeal circulation (ECC). In particular, this study focuses on the analysis of the effects that the measurand temperature may have on the error in estimating blood pH. Even if the blood temperature in ECC is often thermostated at 37 °C, there are treatments in which the blood temperature is varied by a few Celsius degrees, and the exploited measurement principle - fluorescence - is known to be affected by temperature. First, we verified that the temperature-induced error could exceed the maximum permissible measurement error of ±0.04 pH. Hence a linear correction factor for temperature compensation was proposed. The results obtained showed how the simple addition to the measuring system of a temperature sensor and the use of a linear correction factor can effectively allow maintaining the measurement error within the ±0.04 pH range, even when the fluid - phosphate buffer saline (PBS) and blood - temperature is varied in the range [30, 39] °C.
References
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TL;DR: The acid dissociation constant (pKa) is among the most frequently used physicochemical parameters, and its determination is of interest to a wide range of research fields as discussed by the authors, however, it is difficult to determine its application in modern chemistry.
Abstract: The acid dissociation constant (pKa) is among the most frequently used physicochemical parameters, and its determination is of interest to a wide range of research fields. We present a brief introduction on the conceptual development of pKa as a physical parameter and its relationship to the concept of the pH of a solution. This is followed by a general summary of the historical development and current state of the techniques of pKa determination and an attempt to develop insight into future developments. Fourteen methods of determining the acid dissociation constant are placed in context and are critically evaluated to make a fair comparison and to determine their applications in modern chemistry. Additionally, we have studied these techniques in light of present trends in science and technology and attempt to determine how these trends might affect future developments in the field.

262 citations

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TL;DR: In this paper, a wide range of methods and materials used for in vivo measurement of pH levels, such as using the optical fibers, pH-sensitive polymers, ion-sensitive field effect transistors, near infrared spectroscopy, nuclear magnetic resonance, and fluorescent pH indicators.
Abstract: Advances in semiconductor sensor technology, medical diagnostics, and health care needs a rapid boost in research into novel miniaturized pH sensors, which can be used in vivo for continuous patient monitoring. Requirements for the in vivo sensor are materials biocompatibility, high measurement precision, a response time of an order of less than seconds, and the possibility of continuous 24-h monitoring. Monitoring of the pH values is important in the study of tissue metabolism, in neurophysiology, cancer diagnostics, and so forth. Muscle pH can be used to triage and help treat trauma victims as well as to indicate poor peripheral blood flow in diabetic patients. Clearly, to avoid infection and spread of diseases, all in vivo monitoring devices should be single-use/disposable, which puts strict requirement on their price. This paper reviews the wide range of methods and materials used for in vivo measurement of pH levels, such as using the optical fibers, pH-sensitive polymers, ion-sensitive field effect transistors, near infrared spectroscopy, nuclear magnetic resonance, and fluorescent pH indicators.

110 citations


"Performances and Robustness of a Fl..." refers background in this paper

  • ...Indeed, pH monitoring is fundamental in many fields ranging from medicine and agri-food, to chemistry and industrial applications [1]–[5]....

    [...]

Journal ArticleDOI
TL;DR: In this paper, a pH sensor based on polymethylmethacrylate (PMMA) optical fibers has been developed for on-body monitoring in biological fluids, which relies on evanescent wave absorption in a thin film deposited on the fiber core.
Abstract: A pH sensor based on polymethylmethacrylate (PMMA) optical fibers has been developed for on-body monitoring in biological fluids. Detection relies on evanescent wave absorption in a thin film deposited on the fiber core. The sensitive film was prepared using sol–gel technology from a mixture of tetraethyl orthosilicate (TEOS) and methyltriethoxysilane (MTES) or phenyltriethoxysilane (PTES), forming a porous hybrid organic–inorganic layer upon drying. The so-called ORMOSILs (organically modified silicate) were doped with bromophenol blue (BB) and the formulation was optimized to suppress dye leaching by varying the fraction of organic precursor, the sol aging time and the drying conditions. The molecular structure of the films was examined by Fourier-transformed infrared (FTIR) spectroscopy, and their optical responses characterized with UV–VIS absorption spectroscopy. Homogeneous crack-free films were obtained with an optimal MTES content of 50%. Leaching from these films was greatly reduced, with more than 80% of dyes remaining after 2 months in phosphate buffered saline (PBS) buffer. The entrapped indicator showed pronounced increase of apparent pKa and a broader response range compared to the free form in solution. The sensor demonstrated good sensitivity from pH 3 to 9, with an optical signal variation of 3.5 dB. It showed sensitivity to ionic strength variations, but not to temperature in the range between 20 and 50 °C. Reversible pH monitoring between pH 5 and 8 with a precision of 0.2 pH unit was demonstrated in human serum as a model biological fluid, without degradation of the sensing fiber over 24 h. This simple and low cost device will be particularly valuable to support diagnosis and the evaluation of therapies for wound healing.

79 citations


"Performances and Robustness of a Fl..." refers background in this paper

  • ...10, 13 and 14, the measuring interval is about [4,10] pH and the maximum sensitivity is achieved for nearly-neutral pH values where η ≈ 0....

    [...]

Journal ArticleDOI
TL;DR: Approaches and challenges in developing chemical sensor-based methods to accurately and continuously monitor levels of key analytes in blood related directly to the status of critically ill hospitalized patients are reviewed.
Abstract: We review approaches and challenges in developing chemical sensor-based methods to accurately and continuously monitor levels of key analytes in blood related directly to the status of critically ill hospitalized patients. Electrochemical and optical sensor-based technologies have been pursued to measure important critical care species in blood [i.e., oxygen, carbon dioxide, pH, electrolytes (K(+), Na(+), Cl(-), etc.), glucose, and lactate] in real-time or near real-time. The two main configurations examined to date for achieving this goal have been intravascular catheter sensors and patient attached ex vivo sensors with intermittent blood sampling via an attached indwelling catheter. We discuss the status of these configurations and the main issues affecting the accuracy of the measurements, including cell adhesion and thrombus formation on the surface of the sensors, sensor drift, sensor selectivity, etc. Recent approaches to mitigate these nagging performance issues that have prevented these technologies from clinical use are also discussed.

49 citations

Journal ArticleDOI
01 Dec 2018-Small
TL;DR: The fiber-based ratiometric optical pH sensor for use in real-time and continuous in vivo pH monitoring in human tissue outperforms the state-of-the-art reported in the current literature and exhibits promising performance in in vitro whole blood samples.
Abstract: This article reports on a fiber-based ratiometric optical pH sensor for use in real-time and continuous in vivo pH monitoring in human tissue. Stable hybrid sol-gel-based pH sensing material is deposited on a highly flexible plastic optical fiber tip and integrated with excitation and detection electronics. The sensor is extensively tested in a laboratory environment before it is applied in vivo in a human model. The pH sensor performance in the laboratory environment outperforms the state-of-the-art reported in the current literature. It exhibits the highest sensitivity in the physiological pH range, resolution of 0.0013 pH units, excellent sensor to sensor reproducibility, long-term stability, short response time of <2 min, and drift of 0.003 pH units per 22 h. The sensor also exhibits promising performance in in vitro whole blood samples. In addition, human evaluations conducted under this project demonstrate successful short-term deployment of this sensor in vivo.

42 citations

Frequently Asked Questions (2)
Q1. What are the contributions mentioned in the paper "Performances and robustness of a fluorescent sensor for nearly-neutral ph measurements in healthcare" ?

In medicine, the possibility of in-line and real-time monitoring critical care analytes such as pH is recognized to provide relevant information for the diagnosis and treatment of a variety of disorders and would be of considerable support for the management of extracorporeal ( blood ) circulation ( ECC ). In this paper, the authors theoretically and experimentally investigate the performances and the robustness of a fluorescent sensor they recently proposed for the in-line and real-time monitoring of blood pH in extracorporeal circulation ( ECC ). The reported results demonstrate that the proposed sensor allows achieving significant robustness to unevennesses in the production process of the sensing element. 

As an example, as previously introduced, the possibility of in-line and real-time monitoring blood pH would be of considerable support for the management of ECC. Concluding, the proposed sensing element offers the possibility to transform polymeric tubes and containers into sensors that can be read contactless from outside, offering robust inline and real-time monitoring of the pH of the inside fluid. Thanks also to the low-cost and biocompatibility [ 5 ], the proposed sensor can be used for the monitoring of the pH of fluids especially in applications such as healthcare where safe and low-cost disposable are required for all parts in contact with the patient.