polymers Article
Optical pH Sensor Based on Immobilization Anthocyanin
fromDioscorea alataL. onto Polyelectrolyte Complex
Pectin–Chitosan Membrane for a Determination Method of
Salivary pH
Eka Satri
1,
*
, Hani Humaira
1
, Murniana Murniana
1
, Nazaruddin Nazaruddin
1
, Muhammad Iqhrammullah
2
,
Nor Diyana Md Sani
3
, Chakavak Esmaeili
4
, Susilawati Susilawati
1
, Muhammad Mahathir
5
and
Salsabilla Latansa Nazaruddin
5





Citation:Satri, E.; Humaira, H.;
Murniana, M.; Nazaruddin, N.;
Iqhrammullah, M.; Md Sani, N.D.;
Esmaeili, C.; Susilawati, S.; Mahathir,
M.; Latansa Nazaruddin, S. Optical
pH Sensor Based on Immobilization
Anthocyanin fromDioscorea alataL.
onto Polyelectrolyte Complex
Pectin–Chitosan Membrane for a
Determination Method of Salivary
pH.Polymers2021,13, 1276.
doi.org/10.3390/polym13081276
Academic Editor: Akif Kaynak
Received: 15 March 2021
Accepted: 8 April 2021
Published: 14 April 2021
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4.0/).
1
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala,
Banda Aceh 23111, Indonesia; [email protected] (H.H.); [email protected] (M.M.);
[email protected] (N.N.); [email protected] (S.S.)
2
Graduate School of Mathematics and Applied Sciences, Universitas Syiah Kuala,
Banda Aceh 23111, Indonesia; [email protected]
3
Sanichem Resources Sdn. Bhd., No 7/7A, Jalan Timur 6/1A, Mercato Enstek,
Bandar Estek 71060, Negeri Sembilan, Malaysia; [email protected]
4
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran 4176-14411, Iran;
[email protected]
5
Faculty of Dentistry, Universitas Syiah Kuala, Banda Aceh 23111, Indonesia; [email protected] (M.M.);
[email protected] (S.L.N.)
*Correspondence: e.sa[email protected]
Abstract:
A simple optical pH sensor based on immobilization,Dioscorea alataL. anthocyanin
methanol extract, onto a pectin–chitosan polyelectrolyte complex (pectin–chitosan PEC), has been
successfully fabricated. The optical pH sensor was manufactured as a membrane made of pectin–
chitosan PEC and the extracted anthocyanin. This sensor has the highest sensitivity of anthocyanin
content at 0.025 mg/L in phosphate buffer and 0.0375 mg/L in citrate buffer. It also has good
reproducibility with a relative standard deviation (%RSD) of 7.7%, and gives a stable response at time
values greater than 5 min from exposure in a buffer solution, and the sensor can be utilized within ve
days from its synthesis. This optical pH sensor has been employed to determine saliva pH of people
of different ages and showed no signicant difference when compared to a potentiometric method.
Keywords:
optical pH sensor; anthocyanin;Dioscorea alataL.; polyelectrolyte pectin–chitosan; saliva
1. Introduction
Pectin and chitosan are natural polymers that have been widely used in the food
industry, medicine, and the environment [1–6]. Membranes made of the two polymers have
also been applied as a matrix in the development of sensors and biosensors. In regards to
sensor and biosensor development, pectin and chitosan have been used as matrices for the
binding of active substances, which will interact with analytes or other ingredients added,
to improve the performance of sensors or biosensors. In this case, compatibility between the
matrix and the active substance plays an important role in terms of sensitivity and excellent
response time [7]. In many cases, synthetic polymer matrices possess several advantages,
such as excellent mechanical strength, temperature resistance, and plasticity [8]. Frequently,
synthetic polymer matrices are hydrophobic and are very suitable for sensors or biosensors
that require immersion in aqueous media, such as voltammetry and potentiometry methods.
Accordingly, such hydrophobic polymer membranes will not easily leach out from the
matrices into the sample or solution, hence minimizing lost in sensor sensitivity. However,
Polymers2021,13, 1276.

Polymers2021,13, 1276 2 of 12
the hydrophobic membrane causes a slow diffusion process between active material and
the analyte [9], resulting in a slow response.
Pectin and chitosan, taken individually, have many weaknesses [10,11]. Pectin is
soluble in water, while chitosan is very soluble in an aqueous environment at low pH [8,12].
Some sensors have been fabricated based on pectin and chitosan membranes. The use
of pectin in the development of optical pH sensors with less volume of the sample has
been previous reported [13,14]. Pectin membrane possesses hydrogel properties, and it
is not appropriate to be used for the immersion of such sensors in samples during anal-
ysis. At the same time, a number of studies on chitosan-based sensors have also been
reported [15–17]. Therefore, efforts to combine both polymers have already started with the
formation of a polyelectrolyte complex (PEC) membrane. In this particular case, two poly-
electrolytes (polymers carrying ionizable functional groups as side-substituents anchored
along their backbones) of opposite charges interact favorably to give an interpolymeric
complex, PEC [18–22]. The modication of pectin and chitosan into a pectin–chitosan PEC
membrane has good mechanical strength. PEC membranes are formed through electro-
static interactions between the carboxylate groups (-COO

) in the pectin and the primary
ammonium groups (-NH3
+) in chitosan. Chitosan acts as a polycation in acidic solutions,
and pectin acts as a polyanion after it is dissolved in water. The formation of a polyelec-
trolyte complex pectin–chitosan may be induced most likely by attractive electrostatic
interactions between the two macromolecular [15].
In this study, a chitosan-pectin PEC membrane has been obtained and used as a matrix
to develop an optical pH sensor by immobilizing the anthocyanin-a pH sensitive compound
extracted from theDioscorea alataL. Anthocyanin is capable of displaying different colors
over a wide pH range and, therefore, is suitable to be employed as an active pH substance
for optical measurement [16,17]. Moreover, all of the materials used in the construction
of the optical pH sensor are non-toxic, hence it is safe to use for in situ analysis. Indeed,
a similar PEC system with immobilized anthocyanin has been employed to construct a
pH indicator [18]. However, in the aforementioned study, the performance was analyzed
based on the change of the color of anthocyanin at different pH values using a colorimeter,
hence the analysis was only qualitative. In contrast, our work performed a qualitative
approach using UV–Vis spectrophotometer to investigate and optimize the sensitivity,
response time, linear range, reproducibility, and response stability of the fabricated sensor.
Additionally, during the construction of our optical pH sensor, the selection of the com-
position ratio was based on the one that could yield a transparent membrane as opposed
to the previous study [18] that was based on the stoichiometric calculation. This sensor
was then utilized to determine the acidity of human saliva and is able to work for a small
amount of sample. Generally, an analytical method, such as determining the pH of saliva
using a pH ion selective electrode (ISE), requires a larger sample volume. Large volumes
of saliva can be difcult to obtain, so analytical techniques that require large numbers of
samples are not suitable for this kind of analysis. The usefulness of the sensor described
herein relies on the fact that the examination of saliva is a routine analysis especially for
dental and oral health assessment [19–21].
2. Chemicals and Apparatus
Chemicals used in this research, such as monopotassium phosphate (KH2PO4) and
dipotassium phosphate (K2HPO4) were acquired from Fluka (Steinhem, Germany); and pectin
and methanol from Sigma (St Louis, MO, USA).
Chitosan was acquired from Tokyo Chemical Industry Co., Ltd., Japan, obtained from
shrimp shell, with a deacetylation degree of 75–85%.Dioscorea alataL. was purchased from
a local market; HCl, NaOH, citrate acid, sodium citrate, pectin, acetic acid from Merck
(Darmstadt, Germany); saliva from volunteers.
Absorbance measurements associated to the response of the biosensor were performed
by spectrophotometer UV–Vis Shimadzu 1800 (Kyoto, Japan); buffer pH was monitored
with a pH-meter Thermo Scientic Orion Star A211 (Waltham, MA, USA). Pectin membrane

Polymers2021,13, 1276 3 of 12
surface morphology was analyzed employing a Zeiss Merlin/Merlin Compact/Supra 55VP
Field Emission Scanning Electron Microscope (FESEM) (Carl Zeiss, Jena, Germany) at an
acceleration voltage of 10kV and a magnication of 4 kx. Investigation on structural and
thermal properties of the obtained systems was performed by IR spectroscopy (FTIR)
Cary 630 Anti Agilent instrument (Santa Clara, CA, USA). Shimadzu DTG-60 thermal
gravimetric analyzer (DTG) and Shimadzu DSC-60 differential scanning calorimetry (DSC)
(Shimadzu, Kyoto, Japan), respectively.
3. Experimental
3.1. Preparation of Anthocyanin/Pectin–Chitosan
Anthocyanin was extracted fromDioscorea alataL. using methanol solvent following
a previously reported procedure [22]. Pectin–Chitosan PEC membrane was prepared by
mixing each pectin solution and chitosan 1% (w/v) in a weight ratio of 3:7. Pectin and
chitosan were dissolved using distilled water and 4 M CH3COOH to the nal volume of
100 mL The mixture was stirred until homogeneous. The pectin–chitosan PEC solution
was mixed with anthocyanin from its stock extract of 0.150 mg/L to obtain PEC solutions
containing anthocyanin at concentrations of 0.0250, 0.0375, and 0.050 mg/L. A sample of
30L PEC-anthocyanin mixture was dripped onto a circle with a diameter of 6 mm onto
a 40.75 cm mica plastic as illustrated in Figure

C for
24 h.
Figure 1.Optical pH sensor design.
3.2. Membrane Characterization
Pectin, chitosan, and PEC membrane functional groups were analyzed using a Fourier-
transform-infrared spectroscopy (FTIR). IR spectra were acquired in the range of 4000–650 cm
1
.
As for the membrane's surface morphology, it was investigated by employing a scanning
electron microscope with the following operating parameters: Accelerating voltage 10 kV
and magnification of 4 kx with the following parameters: Voltage = 10 kV,distance = 25 mm,
current intensity = 200 pA, and vacuum pressure = 10
5
Torr (1.310
3
Pa).
A sample, as much as 1 mg, was placed on an aluminum crucible and heated from
50

C to 600

C at 40

C/min under dynamic nitrogen atmosphere (ow rate: 20 mL/min)
in a Shimadzu DTG-60 Thermal Gravimetric Analyzer. DSC thermograms were acquired
on a DSC instrument under nitrogen atmosphere (ow rate—20 mL/min) and temperature
range of 40

C to 600

C (40

C/min).
3.3. Sensitivity Optical pH Sensor Determination in Citrate and Phosphate Buffer Solutions
The sensitivity of optical pH sensor was determined based on measuring absorbance in
citrate (pH 4–8), and phosphate (pH 4–9) buffer solutions at each wavelength of maximum
absorbance. Thus, volumes of 30L, taken individually, of 0.1 M buffer solution (citrate

Polymers2021,13, 1276 4 of 12
and phosphate) with a certain pH, were dropped onto the pectin/chitosan/anthocyanin
membrane (optical pH sensor). Afterwards, the measurements were performed, taking
into account the maximum absorbance values exhibited by anthocyanin. The measuring of
absorbance was carried out 10 min after dropping 30L of buffer solution at various pHs.
Dependences between the absorbance and pH values on different pH ranges were plotted
to obtain the corresponding dynamic ranges. The calculated slopes of these linearly tted
dependencies are considered the sensor sensitivity values.
3.4. Determination of Sensitivity of Optical pH Sensors at Various Phosphate Buffer
Concentrations
The buffer solution, which showed the highest sensitivity value, was changed in its
concentration (0.01, 0.03, 0.05, and 0.1 M) to get the maximum sensitivity within a wide
pH range.
3.5. Reproducibility, Response Time, and Lifetime Studies on the Optical pH Sensor
The response time was determined by measuring the response of sensors at 5, 10, 15,
20, 25, and 30 min after dripping 30L samples of various pHs of the buffer solutions
(with optimum concentration) onto the sensor surface. The reproducibility was assessed
based on absorbance measurements of 10 sensors by adding 30L of 0.1 M of PBS at pH 7.
The lifetime was studied by measuring the absorbance of sensor at different days (1, 2, 3, 4,
5, 10, 15, and 20).
3.6. Real Sample Measurement
A real sample study was carried out on the saliva pH testing. About 30L of saliva
from each volunteer was dropped on the surface of the membrane sensor, and absorbance
was measured after 5 min. The absorbance value obtained was then converted to a pH
value using a linear equation obtained from a corresponding calibration curve.
4. Results and Discussion
4.1. Synthesis and FTIR Characterization of the Investigated Systems
As mentioned above, pectin–chitosan PEC membrane is mainly a result of attrac-
tive electrostatic interactions between the two weak polyelectrolytes, one of them being
polyanion (pectin) and the other one (chitosan)-polycation.
During PEC formation, the exibility of each type of macromolecular chain is pro-
gressively reduced and, concomitantly, all the hydrophilic ionizable groups are pointed
towards each other in a pairwise manner (carboxylate groups to primary ammonium ones),
separated from the aqueous environment. As a result of these salt-like bridges established
between the two kinds of oppositely charged groups, pectin–chitosan PEC membrane be-
come practically insoluble in aqueous medium. At the same time, the interaction between
the two polymers pectin and chitosan will be able to improve the mechanical characteristic
of the pectin–chitosan PEC membrane produced [15].
In this study, membranes with a composition expressed as weight ratio between pectin
and chitosan of 3:7 were manufactured. These PEC membranes exhibited a homogeneous
surface and a good transparency. The results of the study show that the composition with
chitosan exceeding this optimum ratio led to a transparent membrane, but rigid and brittle.
Furthermore, pectin–chitosan PEC membranes containing pectin levels are higher than
the specied level yielded pectin–chitosan PEC membranes that are less homogenous and
with a great availability to gel forming in water. That is why the specied composition of
the pectin–chitosan PEC membranes was selected for manufacturing the pH sensor matrix.
Structural characterization of pectin, chitosan, and PEC (3:7) membranes was carried out
by FTIR spectroscopy and the results were graphically collected in Figure.

Polymers2021,13, 1276 5 of 12
Figure 2.
FTIR spectra acquired for membranes of (a) pectin, (b) chitosan, and (c) pectin–chitosan
PEC (3:7).
Based on the FTIR spectroscopy data, the presence of the functional moieties belong-
ing to pectin (Figurea) were revealed by the IR vibrations at 1597, 1716, and 3232 cm
1
assigned to carboxylate (—COO—), carbonyl (C=O) from ester and hydroxyl (intra- and in-
termolecular hydrogen bonding) vibrations, respectively [1,23]. FTIR spectrum of chitosan
(Figureb) shows the presence of C=O stretching at 1642 cm
1
associated to the remaining
acetyl moieties (amide I), a vibration band located at 1564 cm
1
assigned to N—H bending
of amine groups, a vibration band at 1320 cm
1
(amide III), skeletal vibrations involving
C—O stretching around 1000 cm
1
, and a broad band at 3450 cm
1
ascribed to O—H
stretching mixed with N—H stretching vibrations of amide groups [24,25]. The analysis of
IR spectrum of pectin–chitosan PEC membrane moieties show a sharp peak at 1566 cm
1
assigned to an amide II band. On the other hand, in the PEC membrane, it is difcult
to individually identify the presence of carboxylate groups of pectin (involved in the at-
tractive interactions between the two oppositely charged polyelectrolytes) because their
IR vibrations overlap the vibrations of C=O stretching of the remaining acetyl groups of
chitosan. Thus, the band located near 1640 cm
1
in the PEC membrane can be ascribed to
the common contribution of the two types of functionalities just mentioned.
FTIR spectra of anthocyanin extracted fromDioscorea alata,by comparison with those
of the membranes made of pectin–chitosan and anthocyanin pectin–chitosan, are shown in
Figure. The main IR spectral features characteristic to anthocyanin dye consist of a broad
band at around 3500 cm
1
attributed to OH groups responsible for hydrogen bonding,
a band at ca. 1750 cm
1
representing C=O stretching vibrations, a region between 1500 and
1700 cm
1
assigned to C=C stretches of aromatic moieties, and a band located at around
1000 cm
1
associated with C—O—C (ether) stretching vibrations [26]. By comparing IR
proles in Figure, it is obvious the presence of anthocyanin within the pectin–chitosan
PEC membrane (IR spectrum c).

Polymers2021,13, 1276 6 of 12
Figure 3.
FTIR spectra of (a) anthocyanin, (b) pectin–chitosan, and (c) anthocyanin/pectin–chitosan/
membranes.
4.2. Morphological Properties
The morphological properties of the PEC membrane were revealed by SEM. To a
high electric conductivity in order to avoid any damages under the action of the electron
beam, all the samples were rstly coated by a thin Pt layer. Thus, the obtained morpholo-
gies for PEC (3:7) membranes (with and without anthocyanin) are presented in Figure.
The SEM images show relatively smooth, homogeneous membrane surface, even though
the supercial texture appears to be slightly changed for the membrane with anthocyanin.
Figure 4.Morphologies of (a) pectin–chitosan, and (b) anthocyanin/pectin–chitosan membranes revealed by SEM.
4.3. Thermal Characteristics
Thermal behavior of the manufactured PEC membranes was investigated by thermo-
gravimetric analysis (TGA) performed on a temperature range of 35–600

C (Figurea ).
Initial broad minimum located on derrivative TGA (DTGA) curves at T
peakof 58

C is
attributed to water loss moisture retained due to the membranes' hydrophilicity [27].

Polymers2021,13, 1276 7 of 12
More signicant weight loss occurs within the temperature ranges of about 235–435

C for
chitosan and pectin–chitosan and around 260–373

C for pectin, suggesting a process of
polymer degradation/decomposition. TGA/DTG data are roughly conrmed by those of
differential scanning calorimetry (DSC) obtained on the same samples (Figureb).
Figure 5.
Thermal characteristics of pectin, chitosan, and pectin–chitosan as shown by (a) TGA/DTGA and (b) differential
scanning calorimetry (DSC) thermograms.
In DSC experiments, the loss of water can be observed by a slight endothermic shoul-
der before the temperature reaches 120

C was observed in all samples similar with the TGA
prole. However, a sharper endothermic peak observed at higher temperature (ca. 120

C
peak temperature) in anthocyanin/pectin–chitosan indicates a moisture content strongly
retained/bound to the polymer matrix of the studied systems. Similar to other DSC data
reported elsewhere [28,29] a number of exothermic processes located at peak temperatures
of 281

C, 298

C, 309

C, and 303

C were assigned to the polymer chain decomposi-
tion in pectin–chitosan, anthocyanin/pectin–chitosan, chitosan and pectin membranes,
respectively.
Due to the weight composition of PEC systems richer in chitosan, both DTGA and
DSC traces of the investigated PEC membranes roughly exhibit a resemblance closer to the
chitosan membrane than that of pectin, as expected, and as can be seen in Figure.
4.4. Optical pH Sensor Optimization
Effect of the Anthocyanin Concentration on Sensor Sensitivity
The effect of anthocyanin content as an active and pH-sensitive compound on sensor
sensitivity was studied. The corresponding absorbance values of the sensor membranes
prepared with the three different concentrations of anthocyanin prior to membrane forma-
tion (0.0250, 0.0375, and 0.0500 mg/mL) were measured after the addition of 0.1M buffer
solution (both citrate and phosphate buffer of various pHs) and the results of sensitivity
determination are collected in Table. The best sensitivity for citrate buffer was at concen-
tration of 0.0375 mg/L within pH range 4.0–5.5 and sensitivity value of 0.0795 (R
2
= 0.9985).
The highest sensitivity in phosphate buffer was found at a concentration of 0.025 mg/L
with a pH range of 4.8–9.0 and a sensitivity value of 0.0785 (R
2
= 0.983). The optimum
anthocyanin concentration obtained was used for further characterization.

Polymers2021,13, 1276 8 of 12
Table 1.Sensitivity of the PEC sensor against various anthocyanin concentrations in 0.1M citrate and phosphate buffers.
Anthocyanin
Concentration (mg/L)
Citrate Buffer Phosphate Buffer
pH Range
Sensitivity
(AU/pH Unit)
R
2
pH Range
Sensitivity
(AU/pH Unit)
R
2
0.025 4.0–6.0 0.0423 0.003 0.933 4.8–9.0 0.0785 0.001 0.9830
0.0375 4.0–5.5 0.0795 0.004 0.999 4.8–9.0 0.0744 0.001 0.9679
0.05 5.0–7.5 0.0128 0.001 0.969 4.8–7.5 0.0730 0.003 0.9711
Table
solution compared to that in phosphate buffer solution. This is because the pKa values
of the conjugate acid-base pairs associated to phosphoric acid (involved in phosphate
buffer) cover a broader buffer range than in the case of citric acid (involved in citrate
buffer). A higher acidity of the environment (like in citrate buffer) causes the color of
anthocyanin to fade, leading to low absorbance and, as a consequence, low sensitivity
of the sensor. Even though citrate buffer yielded slightly higher sensitivity at a pH of
4.0–5.5 and anthocyanin concentration of 0.0375 mg/L, the applicability might be limited
by its narrow pH range. Therefore, PEC sensor in phosphate buffer with anthocyanin
concentration of 0.025 mg/L was selected for further studies.
Table
pH optical sensor. The results indicate that 0.1M phosphate buffer solution led to the best
results with a sensitivity value of 0.0786 and a coefcient of determination (R
2
) of 0.9838.
Table 2.Sensitivity of optical pH sensor in phosphate buffer solution of different concentrations.
Phosphate Buffer
Concentration (M) pH Range Sensitivity (AU/pH Unit) R
2
0.01 7.0–9.5 0.0588 0.0145 0.9760
0.03 7.0–9.5 0.0623 0.0070 0.9616
0.05 6.5–9.5 0.0682 0.009 0.9614
0.075 4.8–9.5 0.056 0.02 0.9746
0.1 4.8–9.5 0.0786 0.001 0.9838
4.5. Characterization of Optical pH Sensor
4.5.1. Response Time
Response time was determined at 0, 5, 10, 15 20, 25, and 30 min in 0.1M phosphate
buffer solution of pH 5 and pH 8, and the data are plotted in Figure.
Figure 6.Response time prole of the selected (most sensitive) optical pH sensor.

Polymers2021,13, 1276 9 of 12
The increase in absorbance during the rst 5 min of measurement is caused by the
reaction time required by anthocyanin and the buffer solution. Then, absorbance values
tend to make a plateau due to the stability of the anthocyanin color after the reaction
time [23]. It is worth mentioning that the reaction may take less than 5 min. However,
the stable response was only obtained afterward. Indeed, paper-based universal pH
indicator may yield faster response due to its hydrophilic properties. However, such pH
indicator has been reported to produce poor quantitative data [30].
4.5.2. Reproducibility of Optical pH Sensors
The reproducibility of the sensor was studied to evaluate the performance of the
sensor by measuring the absorbance of ten different sensors of the same type under the
same experimental conditions. The results obtained are shown in Table.
Table 3.Reproducibility test for the optical pH sensor.No. Optical pH Sensor Absorbance (AU)
1 Sensor A 0.461
2 Sensor B 0.450
3 Sensor C 0.401
4 Sensor D 0.413
5 Sensor E 0.460
6 Sensor F 0.463
7 Sensor G 0.513
8 Sensor H 0.471
9 Sensor I 0.436
10 Sensor J 0.516
Average 0.458
SD 0.0352
RSD (%) 7.687
SD = standard deviation; RSD = relative standard deviation.
Table
with a standard deviation of 0.035 and a percentage relative standard deviation (% RSD) of
around 7.69%. The results indicate that this sensor meets the criteria for reproducibility in
terms of good standard deviation and percentage RSD.
4.5.3. Lifetime Prole of Optical Sensor pH
The sensitivity of the sensor can decrease after prolonged storage and use. Determina-
tion of the lifetime of the pH optical sensor was evaluated by measuring the absorbance of
the sensor on different days and carried out for 20 days. Results are shown in Figure.
The lifetime study was carried out over a time period of 20 days, with absorbance
measurements performed after 1, 2, 3, 5, 10, 15, and 20 days of sensor storage. According
to the test results, the pH optical sensor gives a good performance from day 1 to day 3
based on the increase in absorbance. However, the measurements conducted on the 5th to
20th days showed reduced sensor responses as observed by decreasing absorbance values.
Essentially, by comparison to the signal recorded on the rst day, the percentage decrease
in sensor signal is of 2.9% on the 10th day, 11.4% on the 15th day, and 14% on the 20th day.

Polymers2021,13, 1276 10 of 12
Figure 7.Lifetime prole of optical pH sensor.
5. Salivary pH Determination
The optimized sensor was then tested on human saliva samples. The saliva samples
were categorized according to age and the before and after meals in the span of 1 h. The pH
values of saliva were determined based on the absorbance measurements, which were then
converted to pHs according to a calibration curve. The results of testing saliva samples are
shown in Table.
Table 4.Salivary pH determination from three peoples.
No.
Age of People from
Whom Saliva Samples
Were Tested before
Meals (Years)
pH Determined
by Optical
pH Sensor
pH Determined
by Ion Selective
Electrode (ISE H
+
)
T
valueT
table
1. 6 7.23 7.3 0.835
2.922. 22 6.83 7.15 2.29
3. 56 6.50 6.8 1.42
Determination of saliva pH was also carried out on pregnant women before and after
meals within 1 h. Saliva samples were taken every 15 min after each meal. The results of
before and after meal saliva tests are collected in Table.
Table 5.pH determination on saliva samples from pregnant women before and after meal.
No. Time after Meal (Min) Optical pH Sensor ISE H
+
T
value T
table
1. Before meal 6.83 7.15 2.29
2.92
2. 15 6.76 6.73 1.48
3. 30 6.62 6.63 0.539
4. 45 6.85 6.81 1.09
5. 60 6.96 6.91 1.80
The data from Tables
not signicantly different from pH measured by ISE H
+
.

Polymers2021,13, 1276 11 of 12
Based ont-test calculations, the obtained t-values were lower than that of t-table.
It means the two methods for measuring salivary pH were not signicantly different. In ad-
dition, normal pH in the mouth is 7. Salivary acidity (pH) is one of the important factors
that favor dental caries, periodontal abnormalities, and other diseases of the oral cavity.
At the same time, bacterial growth on teeth is optimum at salivary pH from 6.5–7.5. On the
other hand, low salivary pH (4.5–5.5) will facilitate the growth of acidogenic germs such
asStreptococcus mutantsandLactobacillus[20,31,32]. All of these aspects, abovementioned,
show the importance of easy detection of intraoral pH by using very small saliva samples
and employing an appropriate sensor.
6. Conclusions
Anthocyanin from the tuber ofDioscorea alataL. has been used as a pH-sensitive
active component, and it has been successfully immobilized onto a pectin–chitosan PEC
membrane for designing an optical pH sensor. The formation of PEC from polyanionic
pectin and polycationic chitosan was successfully carried out and conrmed by FTIR
spectroscopy. Thermal of the anthocyanin/pectin–chitosan reveals a good stability of the
optical pH sensor in over a large range of temperature. Analytical performance and its
capacity to give a reliable response on small sample volumes within a wide range of pHs
make the optical pH sensor very useful in measuring human saliva pH.
Author Contributions:
Conceptualization, E.S.; Data curation, H.H.; Formal analysis, M.M. (Muhammad
Mahathir); Funding acquisition, E.S.; Investigation, E.S. and M.I.; Methodology, E.S., S.L.N. and M.M.
(Muhammad Mahathir); Project administration, S.S.; Resources, N.N.; Software, M.I.; Supervision,
E.S. and M.M. (Murniana Murniana); Validation, C.E. and N.N.; Writing—original draft, E.S., N.D.M.S.
and M.I.; Writing—review & editing, E.S. and M.I. All authors have read and agreed to the published
version of the manuscript.
Funding:
We acknowledge nancial support from Universitas Syiah Kuala for the experiment via
grants Lektor Kepala (Contract Number 266/UN11/SPK/PNBP/2020).
Institutional Review Board Statement:
Ethical review and approval were waived for this study,
due to the nature of this study which is observational and without involving any intervention on
human subjects.
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conicts of Interest:The authors declare no conict of interest.
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