Article

Volume 13, Issue 6, 2023, 547

https://doi.org/10.33263/BRIAC136.547

Structural and Spectroscopic Studies for Chitosan/Fe3O4

Nanocomposites as Glycine Biosensors
Taha M. Tiama 1, Asmaa M. Ismail 2, Hanan Elhaes 3 , Medhat A. Ibrahim 2,*
1 Basic Science Department, October High Institute for Engineering & Technology, 6th October City, Cairo, Egypt
2 Molecular Spectroscopy and Modeling Unit, Spectroscopy Department, National Research Centre, 33 El-Bohouth St.,
12622, Dokki, Giza, Egypt
3 Physics Department, Faculty of Women for Arts, Science, and Education, Ain Shams University, 11757 Cairo, Egypt
* Correspondence: [email protected] (M.A.I.);
Scopus Author ID 8641587100
Received: 3.12.2022; Accepted: 5.01.2023; Published: 24.02.2023

Abstract: Owing to the unique properties of chitosan, composites of chitosan/Fe3O4 (0.25, 0.5, 0.75 wt.
%) were cast, forming films, then subjected to different concentrations of glycine to be applied as a
sensor for the amino acid. Prepared films were characterized using X-ray diffraction (XRD) and ATR-
FTIR spectroscopy. Sensing performance was validated using UV-Vis spectroscopy. XRD data
indicated that there are hydrogen bonds between Fe3O4 and chitosan structure. The interaction between
chitosan and magnetite (Fe3O4) could be described as a composite in the same matrix. ATR-FTIR
spectra demonstrated the broadening of chitosan/Fe3O4 bands in the range of 3360-3041 cm-1, as well
as an increase in the intensity of the bands at 1638, 1564, 1151, and 704 cm-1 with the addition of Fe3O4
associated with a shift of the band at 2987 cm-1 toward lower wavenumber, indicating that Fe3O4 is
embedded and interacted with chitosan. The shift of band positions of functional groups to lower
wavenumber can be interpreted as the strengthening of the chemical bonds due to molecular adsorption.
UV-Vis results indicated that chitosan/ Fe3O4 0.25 wt % ratio had the best sensitivity compared to pure
chitosan and other chitosan/ Fe3O4 ratios. Correlating the data, it can be concluded that chitosan/Fe3O4
could act as a biosensor for the amino acid glycine.

Keywords: chitosan/Fe3O4; glycine; biosensor; XRD; UV-Vis; ATR-FTIR.

© 2023 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Polymer blends incorporated with nanoparticles (NPs) are now widely applied owing
to their enhanced physical, chemical, and biological properties [1-3]. Chitosan and other
biopolymers show unique hydrogen bonding [4, 5]. It was reported that chitosan could be
modified with nanometal oxide to enhance its ability for further applications [6-8]. In order to
investigate the effect of metal oxide on the electronic properties and, subsequently, the
functionality, molecular modeling is consulted. Using molecular modeling calculations,
chitosan was enhanced with graphene and then successfully applied to remove heavy metals
from wastewater [9, 10]. Based on theoretical and experimental results, it was stated earlier
that nano metal oxide such as TiO2 enhanced the ability of nano chitosan blend to act as
biosensors for amino acids [11]. The analyses of modeling data indicated that the presence of
metal oxide is changing the electronic properties by decreasing the value of HOMO/LUMO
band gap energy and increasing the total dipole moment (TDM). Earlier, the reactivity of a
given structure was correlated with physical properties such as HOMO/LUMO and TDM [12,

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13]. The unique electronic properties of chitosan and other biopolymers enhanced with
nanometal oxide pave the way toward applying metal oxide-enhanced biopolymers as sensors
owing to their biodegradability, easy handling, and low cost [14- 16]. The application of
molecular bioelectronics has allowed the production of highly responsive biosensors to detect
various analytics of interest [17]. Because of their possible applications in biomedical research,
and environmental and biotechnology analyses, biosensors have attracted much attention.
Different factors, such as electrode material and morphology, are involved in applying a
biosensor and its compatibility with biosensing molecules [18]. Chitosan is one of the most
widely used natural polymers to disperse nanomaterials due to its excellent properties, such as
good film formation, biocompatibility, non-toxicity, mechanical strength, and water
permeability. Chitosan can adsorb negatively charged material or bind to a surface with
negative charges when dissolved and introduced with its positively charged NH 3+ group. The
composition of the primary NH2 group helps in grafting the chitosan chain with nanomaterials,
leads to good dispersion of nanomaterials, and forms stable chitosan nanocomposite materials
[19]. Due to their low toxicity and superparamagnetic activity, magnetic NPs such as Fe 3O4
have shown tremendous potential for various applications such as wastewater treatment,
enzyme immobilization, biosensors, and drug delivery applications [20, 21]. This magnetic
material possesses significant advantages, including the high specific surface area, which
enhances its binding performance, low resistance to mass transfer, decreased error and selective
separation under a magnetic field of the immobilized enzymes, and, thus, lower operating costs
[22]. However, Fe3O4 NPs have a high surface area-to-volume ratio of high surface energy and
appear to accumulate, reducing their range of applications [23, 24].
Fe3O4 NPs are dedicated to many applications based on their unique physical, chemical,
and surface properties [25-28]
Preparation of chitosan/Fe3O4 NPs using the casting method provides a simple method
for preparing polymer/nanocomposite films with good properties. In addition, this
polyaminosaccharide has specific chemical properties that allow the formation of positively
charged complexes through its free amino groups, creating reactive sites to attach Fe-based
NPs [29, 30]. Recently, chitosan/Fe3O4 nanocomposite was effectively and safely applied as a
catalyst [31]. Based on its catalytic activity, it was used with sodium alginate and ZnO to
remove pharmaceuticals [32,33] efficiently. A mixture of chitosan with Fe3O4 NPs was
reported to improve the oxidation current in gallic acid (GA) and is used for electrochemical
studies and the determination of (GA) [34]. Chitosan could be coated with Fe3O4 and subjected
to different characterizations [35].
Further enhancement for such nanocomposite is conducted with graphene quantum dots
to act as a magnetic imaging agent [36]. Chitosan-agarose is functionalized with core-shell type
Fe3O4 to act as an anticancer against liver and lung cancer cells [37]. Many researchers reported
that chitosan/iron oxide show application efficient green catalyst [38]. It also could act as an
active photocatalytic removing agent [39]. It also could be applied for the possible removal of
dye and mercury from wastewater [40].
Based on the above considerations, chitosan could be enhanced for application as a
biosensor. Accordingly, the present work is conducted to study the structural and optical
properties of chitosan and chitosan/Fe 3O4 nanocomposites with different weight percentages
using XRD, ATR-FTIR spectroscopy, and UV-Vis spectroscopy to assess their performance
as a biosensor for amino acid glycine and to determine the best ratio for this purpose.

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2. Materials and Methods

2.1. Materials.

Medium molecular weight chitosan was supplied from Sigma-Aldrich. Ferrous sulfate
(FeSO4.7H2O) Extra pure was purchased from Sham Lab, and Ferric chloride (FeCl 3.5H2O)
was supplied from Laboratory Rasayan (LR). Ammonium hydroxide solution (NH4OH) was
purchased from AbcoChemie, England, an assay of 25% NH3 (AR). Acetic acid was purchased
from Merck Company, and glycine was purchased from Adwic Company.

2.2. Preparation of Fe3O4.

Fe3O4 NPs were synthesized using the co-precipitation method. At 70ºC, FeSO4.7H2O
and FeCl3.6H2O (Fe2+ and Fe3+) with a molar ratio 2:1, respectively, were dissolved separately
in distilled water. After that, both solutions were mixed under continuous stirring. The final pH
of the solution was adjusted to 10 using NH4OH. A dark precipitate was formed and finally left
to dry at 50ºC for 24 hrs.

2.3. Preparation of chitosan/Fe3O4 polymer nanocomposite.

An appropriate weight of chitosan was dissolved in 98 ml of DI water and 2 ml of acetic

acid under continuous stirring at 60ºC for 4 hrs, until a homogenous viscous solution was
formed. Different weight percentages of Fe3O4 (0.25, 0.5, 0.75 wt%) were added to the chitosan
solution. The solution is magnetically stirred for 6 hrs, then sonicated using Hielscher UP100H
ultrasonic for 1 hr to prevent agglomeration of the NPs. The nanocomposite solutions were cast
in plastic Petri dishes and dried at 40ºC for 24 hrs. The resulting films were approximately 20
μm in thickness.

2.4. Sensing experiment.

A 1M glycine solution was prepared and then diluted into 10 −1, 10−2, 10−3, and 10−4 M
solutions. Next, each nanocomposite film was cut into stripes (0.5 x 3 cm), and stripes were
dipped into the four different concentrations of glycine solution for 60 seconds to choose the
optimum ratio that could be used as a biosensor for glycine.

2.5. Characterization techniques.

X-ray diffraction (XRD) in the 2θ range of (5°–80°) is performed using

PANalyticalX'Pert Pro target Cu-Kα with secondary monochromator Holland radiation with
tube operating at 45 kV and wavelength of 0.1540 nm.
Attenuated total reflection infrared (ATR-FTIR) spectra were obtained in the mid-
infrared spectral range of 4000-400 cm-1 with a resolution of 4 cm-1, using VERTEX 80 FTIR
spectrometer from Bruker Optik GmbH, Germany, equipped with platinum diamond ATR
crystal system, which consists of a diamond disc as an internal reflection element.
Ultraviolet-visible absorption spectra were obtained in the 200-1000 nm wavelength
range using a V-630 UV-Vis spectrophotometer (Jasco).

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3. Results and Discussion

3.1. X-ray diffraction (XRD).

Figure 1 shows XRD patterns of chitosan and Fe 3O4. XRD pattern of chitosan shown
in Figure 1-a demonstrated weak diffraction peaks at 2 = 9 and 12 which are assigned to
chitosan's crystal form , and a strong, broad peak at 2 = 20 which is assigned to chitosan's
crystal form , and is attributed to the amorphous nature of the chitosan. The crystalline forms
are due to the nature of the crystallinity of chitosan [34]. Because of hydrogen bonds between
the amino and hydroxyl groups, and the regularity of the chitosan structure, no impurities were
observed, indicating the high purity of chitosan [41-44]. Figure 1-b shows the crystalline
diffraction peaks for Fe3O4 of 2 values at 18.4, 30.19, 35.55, 43.03, 57.17, 62.74 and
74.3 which agree the results previously reported in the literature [45].The average crystal size
of Fe3O4 can be calculated using the Scherrer equation [46]:
𝐷 = 𝐾𝜆⁄𝛽𝑐𝑜𝑠𝜃 (1)
where D is the size of the crystal, k is a constant (0.915), λ is the wavelength of the X-ray
(0.154 nm), β is the full width at half maximum (FWHM) of the diffraction peak (in radian),
and θ (in degrees) is the Bragg's diffraction angle of the maximum peak. The average crystal
size of Fe3O4 was found to be 5 nm. Hence, we found that the surface-to-volume ratios are
high, which results in increased surface reaction activity, catalytic efficiency, and strong
absorption.

Cs Fe3O4
Intensity (a. u. )

Intensity (a. u. )

10 20 30 40 50 60 70 80 90
10 20 30 40 50 60 70 80 90
2 (Degree)
2 (Degree)
(a) (b)
Figure 1. XRD patterns of (a) Chitosan and (b) Fe3O4.

Figure 2 shows XRD patterns for chitosan and chitosan/Fe 3O4 with different weight
percentages of Fe3O4. As demonstrated in the Figure, the intensities of the diffraction peaks of
2 values at 9 and 12 increased with the addition of Fe 3O4 but became less broad when
compared to pure chitosan. This finding indicates the change in the regularity of chitosan chains
resulting from the interaction of chitosan with Fe 3O4. In addition, the diffraction peak at 2 of
20became broader and shifted toward a higher 2 degree, while the characteristic peaks of
Fe3O4 disappeared. These results confirm the interaction between chitosan and Fe3O4 and their
complex formation. The results show that the proposed preparation method resulted in chitosan
incorporated with Fe3O4 as a composite in the same matrix, which agrees with what was
previously reported in the literature [47].

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Intensity (a. u. )
0.75 wt%, Fe3O4

0.50 wt%, Fe3O4

0.25 wt%, Fe3O4

Cs

10 20 30 40 50 60 70 80 90
2 (Degree)
Figure 2. XRD patterns of chitosan and chitosan/Fe3O4 with a different weight percentage of Fe3O4.

3.1.1. ATR-FTIR spectroscopy.

Figure 3 shows the ATR-FTIR spectra of chitosan, Fe3O4, and chitosan/Fe3O4

nanocomposites with the different weight percentages of Fe 3O4 (0.25, 0.5, and 0.75 wt%). As
shown in Figure 3-a, chitosan has two bands at 3337 cm-1 and 3257 cm-1, characteristic of the
stretching vibrations of O-H and N-H, respectively.

Cs Fe3O4
Absorbance ( a. u.)
Absorbance ( a. u.)

4000 3600 3200 2800 2400 2000 1600 1200 800 400
4000 3600 3200 2800 2400 2000 1600 1200 800 400 -1
-1 Wavenumber (cm )
Wavenumber (cm )
(a) (b)

0.75 wt%, Fe3O4

Absorbance ( a. u.)

0.50 wt%, Fe3O4

0.25 wt%, Fe3O4

Cs

Fe3O4

4000 3600 3200 2800 2400 2000 1600 1200 800 400
-1
Wavenumber (cm )

(c)
Figure 3. ATR-FTIR spectra of (a) Chitosan, (b) Fe3O4, and (c) Chitosan/Fe3O4 with a different weight
percentage of Fe3O4.

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The symmetric and asymmetric vibrations of C-H stretching vibration are observed at
2987 cm-1 and 2883 cm-1, respectively. The presence of bands affirms the residual of N-acetyl
groups at 1638 cm-1, which is assigned to C=O stretching vibration of amide I, 1564 cm -1,
which is attributed to bending vibration of N-H group (amide II) and 1321 cm-1 that related to
stretching vibration of C-N group (amide II). The bending vibration of CH2 and symmetrical
deformation of CH3 are noted at 1402 cm-1 and 1377 cm-1, respectively. The band at 1151 cm-
1
corresponds to the asymmetric stretching vibration of the C-O-C bridge [48]. The stretching
vibrations of the C-O group of β (1→4) glycosidic bonds are observed at 1064 cm-1, 1026 cm-
1
, and 897 cm-1. The band at 650 cm-1 is related to the bending vibration of the O-H group [49,
50]. From Figure 3-b, Fe3O4 shows bands at 3193 cm-1 and 1630 cm-1 corresponding to the
stretching and bending vibration of the O-H group of moisture. The strong band at 546 cm-1
corresponds to Fe-O vibration [51].
Figure 3-c presents the ATR-FTIR of chitosan, Fe3O4, and chitosan/Fe3O4 with different
weight percentages of Fe3O4. From the Figure, the bands in the range of 3360-3041 cm-1
become broader. A new band appeared at 593 cm-1, and the intensities of the bands at 1638 cm-
1
, 1564 cm-1, 1151 cm-1, and 704 cm-1 increased and shifted to lower frequencies with the
addition of Fe3O4. In addition, the band at 2987 cm-1 shifted toward a lower frequency. These
results confirm that Fe3O4 is embedded in and interacts with chitosan.

3.1.2. Optical properties.

UV-Vis spectra of chitosan and chitosan/Fe 3O4 with different weight percentages of
Fe3O4 are shown in Figure 4. Chitosan shows a peak at 217 nm and a broad hump at 341 nm
which are attributed to л-л* and n-л* inter-band transitions, respectively [52]. After the
addition of Fe3O4 with different weight percentages, the peak at 217 nm is shifted to a lower
wavelength, 213 nm (blue shift), and the hump at 341 nm decreased in intensity and became
faint with the addition of Fe3O4, and this indicates the complexation and interaction between
chitosan and Fe3O4.

Figure 4. UV-Vis spectra of chitosan and chitosan/Fe3O4 with a different weight percentage of Fe3O4 (0.25, 0.5,
and 0.75 wt%).

One of the fundamental parameters that depend on the wavelength of the incident light
and gives detailed data on the attenuation of the intensity of the incident light by the studied
samples is the optical absorption coefficient (). This coefficient can be determined from the
absorbance (A) and thickness of the sample (T) using the Swapole formula [53]:
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2.303
𝛼(λ) = 𝐴 (2)
𝑇
Figure 5 shows the relation between the calculated  and the photon energy (hʋ) for all
the studied samples. The absorption edge values can be estimated by extrapolating the linear
portions of the α curve to zero absorption values.

Figure 5. Relation between  and hʋ for chitosan and chitosan/Fe3O4 with a different weight percentage of
Fe3O4 (0.25, 0.5, and 0.75 wt%).

Table 1 presents the calculated values of absorption edge, band tail energy, direct
energy gap, and indirect energy gap (Egi) for chitosan and chitosan/Fe 3O4 nanocomposites with
different weight percentages Fe3O4 which were obtained from optical absorption coefficient.
As seen in table 1, the absorption edge values are shifted to lower photon energy. This means
there is a change in the band structure of chitosan, and there is a structural disorder resulting
from adding Fe3O4.
The structure of polymeric materials can be investigated using Urbach energy by
detecting the defect level and measuring the degree of disordering in the forbidden band gap
[54]. The width of the tail was estimated through the following relation:
ℎʋ
𝛼 = 𝛼0 exp ( ) (3)
𝐸𝑡
where α0 is a constant and Et is band tail energy.
Figure 6 shows the relation between ln and hʋ. The reciprocal of the slope of the curve
gives the value of band tail energy. Values of band tail energy are increased by adding Fe 3O4.
As seen in table 1, band tail energy values are increased by adding Fe3O4, which
indicates the high density of the localized tail states and causes the tail-to-tail transitions.
Additionally, the characteristic change in the optical properties causes disorder in the polymer
composite system due to the intermolecular interaction between the blend components and
Fe3O4.
Measuring band gap energy is an important parameter for determining the optical
transition in any material. The nature of electronic transition (μ) in any material is classified
according to band gap into direct and indirect band gaps. In a direct band gap, μ=2 when the
maximum of the valance band and the minimum of the conduction band coexist at the same
momentum point of the zero crystal (k (wave vector) = 0).

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Figure 6. Relation between ln and hʋ for chitosan and chitosan/Fe3O4 with a different weight percentage of
Fe3O4 (0.25, 0.5, and 0.75 wt%).

For indirect band gap, μ=1/2 when the valence band's maximum and the conduction
band's minimum do not lie at the same wave vector. The absorption of the phonon energy will
often be correlated with the right crystal momentum magnitude of the electron transfer from
the valence band to the conduction band. The optical band gap energy is calculated using Mott
and Davis method [55]:
(αhʋ) = B (hʋ− Eg)1/μ (4)
where B is the probability of transition.
Figure 7 shows the relation between (αhʋ)2 and (αhʋ)1/2 against hʋ for chitosan and
chitosan/Fe3O4 with 0.25, 0.5, and 0.75 wt% of Fe3O4. The values of direct and indirect band
gap energy are determined from the extrapolation of the linear portions of (αhʋ)2 and (αhʋ)1/2
curves to zero absorption values. E.g., the value demonstrated the electronic transition between
the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO).

Table 1. Values of absorption edge as (eV), band tail energy, direct energy gap (Egd) as (eV), and indirect
energy gap (Egi) as (eV) for chitosan and chitosan/Fe3O4 with a different weight percentage of Fe3O4.
Absorption Band tail energy Energy gap (eV)
Samples
edge(eV) (eV) Egd Egi
Chitosan 4.55 1.25 3.71 2.21
Chitosan/0.25 wt% Fe3O4 4.29 1.44 3.29 1.84
Chitosan/0. 5 wt% Fe3O4 4.04 1.59 3.01 0.84
Chitosan/0.75 wt% Fe3O4 4.17 2.06 3.17 1.81

(a) (b)
Figure 7. Relation between (hʋ)0.5 and (hʋ)2 versus hʋ for chitosan and chitosan doped with 0.25, 0.5, and
0.75 wt% magnetite Fe3O4.

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As seen in Table 1, the band gap energy values decreased with adding Fe3O4. This
confirmed that adding Fe3O4 creates localized states, causes defects in chitosan structure, and
increases the degree of disorder of the system.
UV–Vis spectroscopy was also used to monitor the studied nanocomposite samples'
performance as a glycine sensor. Figure 8 shows the absorbance of 10-1, 10-2, 10-3, and 10-4 M
glycine solutions after dipping stripes of chitosan and chitosan/Fe3O4 with different
concentrations of Fe3O4 for 60 seconds. As seen from the Figure, the absorbance of the studied
samples is increased with increasing glycine concentration and shifted toward a longer
wavelength. This may be attributed to the increased pH values of the prepared solutions. In
addition, there are noticeable changes in the absorbance of all samples with different glycine
concentrations. These changes in absorbance are attributed to the interaction between the
carboxylic group of glycine and functional groups of chitosan (N-H and O-H) and Fe3O4, as
indicated earlier [11].

(a) (b)

(c) (d)
Figure 8. UV–Vis absorption spectra for the glycine after exposure to (a) chitosan and chitosan/Fe3O4 with
different weight percentages of Fe3O4 (b) 0.25 wt% Fe3O4, (c) 0.5 wt% Fe3O4 and (d) 0.75 wt% Fe3O4 for
insertion time 60 sec.

Figure 9 shows the UV–Vis absorption spectra for glycine concentration 10-4 M after
exposure to chitosan and chitosan/Fe3O4 with different weight percentages of Fe 3O4. As seen
in Figure 9, chitosan/Fe3O4 nanocomposite containing 0.25 wt% of Fe3O4 showed better
sensitivity than pure chitosan and chitosan/Fe3O4 with other wt%, and this concentration can
be used as a biosensor for glycine. In addition, shifts of band peak positions of the molecular
functional groups to higher wavelength values were noticed and can be interpreted as
strengthening the chemical bonds of these functional groups due to molecular adsorption.
These results suggest that these features could enable chitosan with a nanometal oxide like
Fe3O4 to be used in new signal transmission techniques in biosensors for rapid in vivo analysis.
As the reported literature shows, this is because the dynamic interface properties are based on
surface charge and effective area, roughness, porosity, functional groups, energy and
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valence/conduction states, and the hygroscopic nature of nano metal oxide [56]. The
association of biomolecules with nanometal oxide can be achieved through physical adsorption
or chemical bonding. This interconnection between biomolecules depends on the availability
of functional groups, which can be formed through appropriate chemical reactions. In contrast,
the physical adsorption of the biomolecules arises mainly from weak interactions (for example.
Decomposition, Van Der Waals, electricity) [57].

Figure 9. UV–Vis absorption spectra for the glycine concentration 10−4 M after exposure to chitosan and
chitosan/Fe3O4 with a different weight percentage of Fe3O4 (0.25, 0.5, and 0.75 wt%) for insertion time 60 sec.

4. Conclusions

The casting technique prepared a nanocomposite of chitosan/Fe3O4 (0.25, 0.5, 0.75

wt%). Samples were subjected to 10-1, 10-2, 10-3, and 10-4 M glycine solutions for an insertion
time 60 seconds to identify use the optimal ratio that gives the best sensitivity to be used as a
biosensor for glycine. XRD, ATR-FTIR, and UV-Vis spectroscopy were used to investigate
the effect of adding different concentrations of Fe3O4 to chitosan. XRD and ATR-FTIR
techniques confirmed the complexity and interaction between chitosan and Fe 3O4. The size of
Fe3O4 was found to be 5 nm using the Scherrer equation. UV-Vis absorption spectra
demonstrated a blue shift of the peak at 217 nm, confirming the interaction between chitosan
and Fe3O4. Values of absorption edge, band tails, and direct and indirect band gaps confirmed
the creation of localized states between electronic transitions, thus causing defects in chitosan
structure and increasing the degree of disorder. It was found that chitosan with 0.25 wt% of
Fe3O4 showed the best sensitivity to glycine solutions compared to pure chitosan and other
samples. This ratio between chitosan and Fe 3O4 can be used as a biosensor for glycine amino
acids.

Funding

We receive no funding for this work.

Acknowledgments

We would like to express our deep thanks to the members of the Molecular Spectroscopy and
Modeling Unit at Spectroscopy Department, National Research Centre, Egypt, for their kind
support during this work.

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Conflicts of Interest

The authors declare no conflict of interest.

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