Here, we report the use of highly fluorescent zinc phthalocyanine-conjugated cellulose nanocrystals (ZnPc@CNC) for chemical sensing and imaging applications. Cellulose nanocrystals (CNCs) are crystalline nanorods synthesized through the acid hydrolysis of cellulosic resources like wood pulp, cotton fibers, carded hemp, etc. and lab-synthesized octacarboxylated zinc phthalocyanine molecules are conjugated to these CNCs forming a brightly fluorescent-conjugated molecular aggregate (ZnPc@CNC), which was then used in both liquid suspensions and solution-processed thin films. ZnPc@CNC conjugates showed reproducible and reliable photoluminescence (PL) quenching behavior when exposed to terephthalic acid (TA) of concentration 0.2 mM to 0.8 mM. The PL sensing of TA followed modified Stern-Volmer kinetics with the Stern-Volmer constant (Kapp) determined to be 147.1 M-1. The mechanism of sensing involves the change in the electron density of the π-conjugated phthalocyanine metallocycle core due to the strong electronic interaction with the benzenedicarboxylic acid. This work opens the way to conjugating several other chromophores and fluorophores to CNCs for colorimetric and fluorescence-based chemical sensing using paper-like films and membranes. Likewise, highly emissive ZnPc@CNC nanocomposites were shown to behave as fluorescent staining agents on the surface of TiO2 microrods. This technique can be used to render non-fluorescent micro- and nanomaterials emissive, enabling them to be imaged using fluorescence microscopy

1. Chemical sensing and imaging using fluorophore-
conjugated cellulose nanocrystals
Narendra Chaulagain1
, John Garcia1
, Navneet Kumar1
, Harshitha Rajashekhar1
,
Xiaoyuan Liu2
, Pawan Kumar1
, Alkiviathes Meldrum2
, Kazi M. Alam1
, and Karthik Shankar1,
*
1
Department of Electrical and Computer Engineering, University of Alberta, 9211 - 116 St, Edmonton, AB T6G 1H9, Canada
2
Department of Physics, University of Alberta, 9211-116 St, Edmonton, AB T6G 1H9, Canada
Received: 9 October 2022
Accepted: 21 December 2022
Ó The Author(s), under
exclusive licence to Springer
Science+Business Media, LLC,
part of Springer Nature 2023
ABSTRACT
Here, we report the use of highly fluorescent zinc phthalocyanine-conjugated
cellulose nanocrystals (ZnPc@CNC) for chemical sensing and imaging appli-
cations. Cellulose nanocrystals (CNCs) are crystalline nanorods synthesized
through the acid hydrolysis of cellulosic resources like wood pulp, cotton fibers,
carded hemp, etc. and lab-synthesized octacarboxylated zinc phthalocyanine
molecules are conjugated to these CNCs forming a brightly fluorescent-conju-
gated molecular aggregate (ZnPc@CNC), which was then used in both liquid
suspensions and solution-processed thin films. ZnPc@CNC conjugates showed
reproducible and reliable photoluminescence (PL) quenching behavior when
exposed to terephthalic acid (TA) of concentration 0.2 mM to 0.8 mM. The PL
sensing of TA followed modified Stern-Volmer kinetics with the Stern-Volmer
constant (Kapp) determined to be 147.1 M-1
. The mechanism of sensing involves
the change in the electron density of the p-conjugated phthalocyanine metallo-
cycle core due to the strong electronic interaction with the benzenedicarboxylic
acid. This work opens the way to conjugating several other chromophores and
fluorophores to CNCs for colorimetric and fluorescence-based chemical sensing
using paper-like films and membranes. Likewise, highly emissive ZnPc@CNC
nanocomposites were shown to behave as fluorescent staining agents on the
surface of TiO2 microrods. This technique can be used to render non-fluorescent
micro- and nanomaterials emissive, enabling them to be imaged using fluores-
cence microscopy.
Address correspondence to E-mail: kshankar@ualberta.ca
https://doi.org/10.1007/s10854-022-09724-2
J Mater Sci: Mater Electron (2023)34:538 (0123456789().,-volV)
(0123456789().,-volV)

2. 1 Introduction
Cellulose is a carbohydrate polymer which is the
fundamental constituent of paper and one of the most
abundant biopolymers on the surface of the earth.
Cellulose nanocrystals (CNCs) are optically trans-
parent, monocrystalline nanorods of cellulose with a
diameter of 4–20 nm, and a length of 100–500 nm [1].
CNCs also have other interesting properties, such as
the ability to form chiral nematic liquid crystals [2], a
low thermal expansion coefficient, and a large den-
sity of surface hydroxyl groups amenable to func-
tionalization [3, 4]. The biodegradable and renewable
nature of cellulose renders CNCs interesting for a
variety of applications in sustainable technologies.
Previously, CNCs have been primarily viewed as
low-density reinforcing agents in high-strength-to-
weight polymer nanocomposites for structural (load-
bearing) and mechanical applications [5, 6]. How-
ever, in mechanical and structural applications, fur-
ther technological development has been obstructed
by engineering problems related to the aggregation of
CNCs in polymer matrices. Systematic characteriza-
tion methodologies such as scanning and transmis-
sion electron microscopy are not very insightful in
revealing the internal structure of blends due to poor
contrast between CNC and polymer phases [7].
Therefore, new imaging methodologies are needed
[8], and developing fluorescent CNCs [9] is essential
in enabling high-contrast fluorescence microscopy of
CNC-based blends and nanocomposites. Due to the
nanoscale nature of CNCs, advanced fluorescence
microscopic techniques, such as super-resolution
microscopy, multiphoton microscopy, and fluores-
cence lifetime imaging microscopy, will likely pro-
vide the best results.
Unlike mechanical and structural applications, the
optoelectronic and biomedical applications of CNC-
based substrates, platforms, and composites have
received very little attention. In the past few years,
many reports have found that CNCs, due to their
needle-shaped morphology (Fig. 1a & b), can disrupt
or modify the aggregation of p-conjugated organic
semiconductors in CNC-organic semiconductor
nanocomposites [10, 11]. In the case of conjugated
polymers, it has been demonstrated that the polymer
chains tend to wrap themselves around the CNC
nanorods, which serve as a scaffold or template for
the morphological organization of the resulting
polymer film. Such wrapping modifies the photolu-
minescence and charge transport properties of the
resulting CNC-organic semiconductor nanocompos-
ites. Since CNCs are polar and strongly hydrophilic, a
limited number of organic semiconductors that are
either water-soluble or soluble in solvent mixtures of
water and polar aprotic solvents have been used to
form nanocomposites with cellulose nanocrystals
[12]. Therefore, covalent conjugation of CNCs to
render them soluble in organic solvents is the next
logical step in the evolution of CNC-based active
layers and sensing platforms.
Metallophthalocyanines (MPcs) are a family of
organic semiconductors with unique optoelectronic
and sensing properties. Members of this family are
excellent p-type organic semiconductors (hole trans-
porters) with good charge carrier mobilities, while
perfluorination is known to result in n-type com-
pounds as well [13]. MPcs are characterized by the
presence of an extensive macrocyclic p-conjugated
ring system containing 18 p-electrons that are delo-
calized over the entire molecule. These delocalized 18
p-electrons also result in a significant transition
dipole moment which manifests itself in the form of
an extreme optical absorption coefficient in the blue-
ultraviolet (Soret band) and red-near infrared (Q-
band) spectral regions. Selected members of this
family (e.g., ZnPc) have a medium-to-high fluores-
cence quantum yield, which, when combined with
the high extinction, produces a very bright fluo-
rophore. MPcs are also excellent active layer sensing
materials with the ability to detect and measure metal
ions [14], volatile organic compounds (VOCs) [15],
neurotransmitter molecules [16], etc., using electro-
chemical or fluorescence techniques. It was reported
that the zinc complexes of phthalocyanines and por-
phyrins showed high affinities for amines, and the
nickel complexes showed high affinities for aromatic
compounds which were then employed to fabricate
optical amine sensors [17–19]. For these reasons, we
chose to form covalent conjugates of CNCs with ZnPc
(Fig. 1c). ZnPc@CNC conjugates are highly fluores-
cent with Soret band and Q-band emission peaks at
* 400 and 720 nm, respectively. MPc@CNC conju-
gates exhibited unusual packing and aggregation
behavior with a 20–50 nm thick phthalocyanine shell
wrapping the CNCs (Fig. 1d).
Poly (ethylene terephthalate) (PET) and phthalate
esters (PAEs) are extensively used as consumer
plastics and industrial plasticizers, where
538 Page 2 of 12 J Mater Sci: Mater Electron (2023)34:538

3. terephthalic acid (TA) is the primary building block
and hydrolysis product of PET and PAEs. TA directs
the degree of degradation of PET and PAEs. Due to
the absence of viable screening approaches to detect
terephthalic acid, the potential of directed evolution
of enzymatic degradation reactions for plastics has
not yet been completely exploited. High-performance
liquid chromatography, which is bulky, expensive,
and frequently onerous, has been used to detect TA
leaching into edible oils from plastic bottles [20]. A
fluorometric assay is an excellent tool for product
detection, but terephthalic acid is not fluorescent in
nature. Another option consists of using metal oxide-
based chemiresistive gas sensors [21]. The primary
disadvantage of oxide-based gas sensors is that their
application in gas sensing is constrained by the
microfabrication processes required to fabricate the
sensor. Metal oxide gas sensors also tend to have
lower selectivity than fluorescence sensors. Jiawei
et al. employed the transcription factor XylS to build
a biosensor for the fluorometric detection of tereph-
thalic acid [22].
Fluorescence sensing is inherently more selective
than competing techniques such as chemiresistive
and electrochemical sensing which is why it has
become the gold standard for a variety of sensing and
diagnostic applications [23]. It was previously
reported that the zinc phthalocyanine-conjugated
cellulose nanocrystals were able to detect nitroaro-
matic compounds [24]. Here, we report the successful
detection of terephthalic acid which is basically a
benzendicarboxylic acid using the ZnPc@CNC
nanocomposite. This helps to broaden the selectivity
of ZnPc@CNC nanocomposite toward different
functional group containing molecules. Long-lived
charge separation upon light illumination was also
observed for ZnPc@CNC and CoPc@CNC conjugates
[25, 26].
Due to the insufficient difference between CNC
and the polymer lattice, it is challenging to examine
the internal topology of CNC–polymer nanocom-
posites using electron microscopy techniques (a
consequence of deprived secondary electron contrast
between the CNC and polymer). In this regard,
photoluminescence (PL)-based methods can circum-
vent difficulties in exposing the internal structure of
nanocomposites. In this study, the laboratory-made
ZnPc@CNC was tested for sensing capability by
employing terephthalic acid as a fluorescence
quencher to observe photoluminescence quenching.
Further, ZnPc@CNC was blended with microwave-
synthesized anatase titanium dioxide microrods
(TMR), and fluorescence microscopy was used to
evaluate if the surface coating of fluorescent
Fig. 1 a Glycopolymer network constituting the CNCs, b Oblate
spheroidal or rod-like morphology of cellulose nanocrystals
(CNCs), c Molecular structure of octacarboxylated zinc
phthalocyanine, d CNC forming ester linkage to covalently
functionalize with octacarboxylated zinc phthalocyanine to
produce ZnPc@CNC conjugates
J Mater Sci: Mater Electron (2023)34:538 Page 3 of 12 538

4. ZnPc@CNC on TMR allows them to be imaged
readily.
2 Experimental section
2.1 Materials
Cellulose nanocrystals were supplied by Alberta
Innovates and FPInnovations. Terephthalic acid (TA)
(99%) was procured from Acros. Dimethylsulfoxide
(DMSO) (99.9%) was supplied by Sigma Aldrich.
Fresh deionized (DI) water was used for all the
experiments.
2.2 Characterization
The Zeiss Sigma FESEM equipment with a 5 kV
accelerating voltage was used to evaluate the surface
structure of all samples. Using a 532 nm excitation
laser and a Renishaw inVia Qontor Confocal Raman
Microscope, the Raman spectra of all materials were
obtained, spanning the Raman shift range of
70–1800 cm-1
. The transmission spectra in liquid
suspensions were obtained using a Perkin Elmer
Lambda-1050 UV–Vis–NIR spectrophotometer. Dif-
fuse reflectance spectroscopy (DRS) mode UV Vis
spectrophotometer (Hitachi U-3900H) was also used
to collect absorption spectra for thin films. A Stel-
larNet spectrometer (SILVER-NOVA) fitted with a
UV-enhanced CCD detector was used to capture
steady-state photoluminescence spectra. As an exci-
tation source, a 405 nm LED was used. Fluorescence
microscopy was performed using a Zeiss LSM710
confocal fluorescence microscope. A 3 mW diode
laser was used to produce a 405 nm excitation
wavelength, which was used to excite the fluo-
rophore. The microscope is equipped with three
photomultiplier tubes (PMTs) used to detect emitted
photons according to specified wavelength ranges.
Fluorescence lifetime imaging microscopy (FLIM)
was performed using a Leica ST8 stimulated emis-
sion depletion (STED) microscope equipped with a
FLIM module. The FLIM module uses a time-corre-
lated single-photon counting (TCSPC) method to
measure fluorescence lifetime. A filtered white light
pulsed laser source was used to produce an excitation
wavelength of 470 nm. A pulse frequency of 20 and
10 MHz was used, corresponding to a pulse interval
of 50 and 100 ns, respectively. A Leica HyD hybrid
photodetector was used to detect emitted photons
based on a given emission wavelength range. For the
time-resolved photoluminescence, the samples were
excited with a 405-nm Picopower LD-405 H laser
(Alphalas, GmbH) with nominally 25 ps pulses. PL
spectra were initially collected using an optical fiber
interfaced through a 425-nm long-pass filter to an
intensity-calibrated USB-2000 ? Ocean Optics
miniature spectrometer. Then the fiber was discon-
nected from the spectrometer and directed to an
HPM100-50 hybrid single-photon detector from
Becker-Hickl. The detector was interfaced to an
SPC130-EMN single-photon counter, and data were
collected using the SPCM software package (all from
Becker-Hickl).
2.3 Synthesis of titania microrods,
ZnPc@CNC-based nanocomposites,
and sample preparation for FM imaging
TiO2 microrods were synthesized utilizing a micro-
wave synthesis approach [27]. In brief, 1.5 ml of
titanium (IV) butoxide was mixed with 1 g of PVP
that had previously been dissolved in EG (50 ml). The
combination was then put into a clean glass beaker
and exposed to a 385 W maximum power (frequency
2.45 GHz) microwave sample preparation equipment
(Panasonic NN-SG626W) for 10 min. The action
produced white flocculi, showing that titanium gly-
colate complexes were being formed because of the
transformation of titanium (IV) alkoxide. Then, for an
additional 10 min, the microwave radiation was
applied at the same power. The resulting suspension
of titanium glycolate microrods was centrifuged at
6000 rpm, cleaned three times with distillated water
and methanol, and dried at 80 °C. Eventually, titania
microrods were synthesized by microwave irradia-
tion (385 W) of titanium glycolate microrods held in
water suspension for 20 min at atmospheric pressure.
The resulting titania microrods were additionally
calcined at 400 °C in the air for one hour, and their
SEM images are shown in Fig. 2.
For fluorescence microscopic imaging, 2 mg of
ZnPc@CNC, and 3 mg of TMR were mixed into 400
lL of DI water in an ultrasonic bath. After 15 min of
sonication, a wet mounting technique was used in
which a suspension of the sample was pipetted onto
a glass slide, and a coverslip (thickness No.1,
* 150 lm) was gently lowered overtop. A 63x oil
immersion objective was used for imaging, and Type
538 Page 4 of 12 J Mater Sci: Mater Electron (2023)34:538

5. A immersion oil was used as the medium. For fluo-
rescence lifetime imaging microscopy too, wet
mounting was used for sample preparation identical
to the method used for fluorescence microscopy. For
imaging, a 40x water immersion objective was used
with water as the immersion medium.
3 Results and discussions
Due to their ability to modify the emission of com-
posites, metal-organic frameworks with d-metal
centers [28] potentially exhibit luminous characteris-
tics. ZnPc@CNC can be recognized as a workable
compound with promising application performance
in fluorescence sensing because of its outstanding
water stability, thermal stability, and fluorescence
features.
In the UV/blue and red spectral ranges, the ZnPc
exhibits the well-known Soret and Q-bands of met-
allophthalocyanines (Fig. 3a). The electronic absorp-
tion spectrum of MPcs exhibits a band in the
620–650 nm range when aggregation takes place in
the course of the intramolecular interactions between
the phthalocyanine units [29] in conjugated systems.
Here, the Q-band of ZnPc is a doublet, with the
higher energy peak (640 nm) attributed to p-stacked
columnar H-type aggregates and the lower energy
peak (695 nm) owing to the S0?S1 electronic transi-
tion [30]. The Q-band is hypersensitive to environ-
mental disturbances and significantly distorted after
strong interaction between the carboxy group present
in terephthalic acid and zinc phthalocyanine while
the Soret band or B-band is the result of mobile
electrons moving from the ground state (S0) to a
higher energy state (S2) and is responsible for the
electronic bandgap of the material.
DMSO is a potent facilitating solvent and aggre-
gation is the causative factor of the band at roughly
705 nm. However, aggregation of several MPc
derivatives [31] has already been seen to occur in
DMSO. The aggregates in this instance have been
identified as the outstanding J aggregates, which are
scarce in aqueous conditions [31]. Figure 3b shows
Fig. 2 a and b SEM images of anatase TiO2 microrods; c Raman Spectra, d DRS UV Vis spectra of microwave-synthesized anatase
titania
J Mater Sci: Mater Electron (2023)34:538 Page 5 of 12 538

6. the absorption and fluorescence emission spectra for
ZnPc@CNC in DI. Fluorescence emission peaks were
observed at 710 nm for ZnPc@CNC in aqueous
media. These emission peaks are also observed for
the ZnPc@CNC-TMR nanocomposite, but the Soret
band maximum is red shifted as shown in Figure S1
(Supporting Information).
3.1 Test for photoluminescence quenching
In recent years, fluorescence quenching has been
employed and researched in various facets of ana-
lytical chemistry, biomedicine, and biochemistry [32].
In complicated macromolecular structures, fluores-
cence quenching techniques have been found to be
helpful in learning more about the conformation and
dynamic changes of molecules.
The chemical sensing ability of laboratory-pro-
duced conjugated ZnPc@CNC was evaluated by
monitoring the photoluminescence quenching
employing terephthalic acid as a fluorescence
quencher. The conjugation of cellulose nanocrystals
with the metal phthalocyanine aids charge transfer
by enabling a more delocalized excited state in the
molecular aggregates. Charge transfer occurs
between the fluorophore ZnPc (and its conjugate with
CNC) and terephthalic acid, causing the former’s
fluorescence to be quenched.
For ZnPc@CNC dispersed in a deionized water
(DI) suspension, the quenching activity of tereph-
thalic acid has been examined. In both aqueous and
organic media, terephthalic acid is weakly soluble.
The best solubility of terephthalic acid was found in
DMSO compared to all other investigated solvents.
As a result, different concentrations of terephthalic
acid were made in DMSO, and 10 lL of each con-
centration was added to the test samples. According
to Fig. 4a, when terephthalic acid concentration
increases from 0.2 to 0.8 mM, the luminescence
intensity of the suspension is noticeably lowered.
Here, the same quencher is quenching the fluo-
rophore through complex construction as well as
collisions. In these situations, the Stern-Volmer plots
are distinguished by an upward curve that is concave
toward the y-axis (Fig. 4b). The modified form of the
Stern-Volmer equation is second order in [M], which
accounts for the upward curvature observed when
both static and dynamic quenching (association con-
stants KS and KD) occur for the same fluorophore,
and is given as: I0/I = 1 ? Kapp [M], where Kapp= (KD
? KS) ? KDKS[M] = I0/I-1. Here, [M] is the molar
concentration of the terephthalic acid, (I0) is the initial
fluorescence intensity, (I) is the fluorescence intensity
of the ZnPc@CNC suspensions varying with different
concentrations of terephthalic acid, and (Kapp) is the
quenching constant [M-1
], and this is the crucial
parameter that indicates the sensing capability of a
fluorescent sensor device. In ZnPc@CNC nanocom-
posite, the fluorescence of the overall system is gen-
erated by ZnPc which is conjugated on the surface of
cellulose nanocrystals. Thus, we only take zinc
phthalocyanine into account for its fluorescence and
quenching behavior. Here, quencher constant (KD)-
= Kq 9 r, and (KS)= [I-M]/[I][M], where Kq is the
bimolecular constant and s is the average lifetime of
the fluorophore molecule before quenching. For the
Fig. 3 a UV-VIS spectra of ZnPc@CNC in different solvent, b Absorption and emission spectra of ZnPc@CNC in DI. Excitation
wavelength was set to 405 nm
538 Page 6 of 12 J Mater Sci: Mater Electron (2023)34:538

7. zinc phthalocyanine molecules, the bimolecular
quenching constant is in the order of 1010
M-1
s-1
[33].
The value of KD and KS was obtained as 12.7 M-1
and
1.44 9 103
M-1
respectively. It is evident that in the
quenching regime, ZnPc@CNC follows the modified
Stern-Volmer kinetics, where the Stern-Volmer con-
stant (Kapp ) was calculated to be 147.1 M-1
. As seen
in Fig. 4b, ZnPc@CNC showed a nonlinear quench-
ing behavior, where the I0/I vs. quencher plot is
bending upward. An upward bending curve nor-
mally denotes the combined quenching mechanism
for the detection of terephthalic acid.
3.2 Fluorescence microscopy imaging
of ZnPc@CNC decorated titania
nanostructure
Recent findings on mesoporous titania materials have
shown their viability for use in numerous applica-
tions, including dye-sensitized photovoltaic cells [34],
photoelectrochemical water splitting [35], and
degradation of organics [36], because of their high
specific surface area and distinctive semiconducting
properties. On the other hand, mesoporous titania
composites have only been used in a small number of
biomedical applications. These biocompatible TiO2
mesoporous structures are simple to functionalize
with fluorophores for intercellular bioimaging and to
fill with chemotherapeutic medicines for intercellular
drug administration [37]. Such a titania structure
could serve as a potentially beneficial intracellular
nanovehicle for biomedical applications. We describe
employing ZnPc@CNC fluorophore molecule to
decorate the TMR structure’s surface and imaging the
composites using fluorescence microscopy (Fig. 5).
The Raman spectra of pristine anatase TiO2
microrods shown in Fig. 2c confirm its synthesis. The
anatase crystal phase is characterized by the bands
Eg(1), Eg(2), B1g, A1g, and Eg(3) (3) which is consistent
with reference [38]. Eg(1), which is typically observed
at about 144 cm-1
, is recorded between 140 and
148 cm-1
. The microrods have the highest absorption
capacity at UV region (350 nm) as shown in Fig. 2d.
The fluorescence properties of the octacarboxylated
ZnPc molecule are equally shared by the ZnPc-con-
jugated CNCs. An excitation-dependent shift of the
Soret band emission peak, where the redshift appears
with increased excitation, was also produced by the
conjugation of ZnPc with CNCs [24].
The photocatalytic capabilities of TiO2 nanoparti-
cles make them promising candidates for photosen-
sitizer therapy of cancer and tumors [39]. In
interaction with semiconductor surfaces, the ZnPc
fluorophore (and its CNC conjugate) can transfer
absorbed photon energy, resulting in the formation
of electron–hole pairs engaged in photocatalytic
processes. When compared to TiO2, ZnPc absorbs
light at longer wavelengths, resulting in higher
overall light absorption and far more electron–hole
pairs being produced with the ZnPc@CNC-TMR
system. After coating the titania structure with
ZnPc@CNC, their effective imaging by epifluores-
cence microscopy was validated.
To establish a simple, inexpensive process to fluo-
rescently label the metal oxide nanostructure, we
modified the surface of the TiO2 microrods with
Fig. 4 a Quenching of luminescence intensity of ZnPc@CNC fluorophore solution in the presence of a different concentration of
terephthalic acid, b PL intensity ratios of ZnPc@CNC vs. quencher concentration
J Mater Sci: Mater Electron (2023)34:538 Page 7 of 12 538

8. ZnPc@CNC. The creation of hydrophilic surfaces
with the surface - OH groups increase the adsorp-
tion sites for the organic molecules, which creates an
opportunity for hydrogen bonding [40]. Similarly, the
covalent attachment of ZnPc with CNC, performed
through ester bond formation, involves the - OH
groups from CNC surface and - COOH groups from
octacarboxylated ZnPc [1, 24]. The hydroxyl groups
of cellulose nanocrystals are significant in the binding
to both octacarboxylated zinc phthalocyanine and
TiO2. Also, the presence of carboxylated functional
group in ZnPc@CNC facilitates the adsorption of
ZnPc@CNC on the surface of TiO2 microrods. The
photoluminescence (PL) could be excited at relatively
long wavelengths (blue photons). Thus, the
ZnPc@CNC-modified TiO2 microrods are fluores-
cent, emitting in the 550–600 and 600–700 nm spectral
channels for the excitation wavelength of 405 nm, as
shown in Fig. 6. Anatase TiO2 is weakly fluorescent
with a blue PL emission [41] that interferes with the
blue fluorescence of ZnPc@CNCs on the surface of
the TiO2 microrods and results in the poor contrast
seen in Fig. 6a. Poor contrast is also observed in the
yellow channel fluorescence microscope image in
Fig. 6c due to the weak emission of ZnPc@CNCs in
this spectral window. Therefore, sharp and distinct
images of the TiO2 microrods with good contrast are
only observed in the green and red channels (Fig. 6b
and d, respectively) wherein the background fluo-
rescence is low and the ZnPc@CNCs exhibit strong
PL emission.
Fluorescence microscopy is the most commonly
used method to characterize proteins, cells, and
therapeutic agents. While microrods and nanorods of
TiO2 are widely used in photodynamic therapy, bone
implants and drug eluting coatings, the imaging of
such TiO2-based therapeutic agents is severely lim-
ited by the extremely weak fluorescence (quantum
yield 0.1%) of both the anatase and rutile phases of
titania. When ZnPc@CNC was attached to TiO2
microrods, the average lifetime for the material
decreased from 3.23 to 1.86 ns for 470 nm excitation
wavelength and 650–700 nm emission region,
respectively (Figures S2 and S3 in Supporting Infor-
mation). The decrease in emission lifetime indicates
that while some charge transfer occurred from the
ZnPc@CNC, this was not sufficient to quench the
fluorescence emission from TMR coated with
ZnPc@CNC (Fig. 6). Time-resolved photolumines-
cence spectroscopy was used to record the radiative
relaxation from excited states and examine charge
carrier dynamics. The TRPL spectra of the various
materials are displayed in Figures S2 and S3 in Sup-
porting Information. The biexponential decay func-
tion used to model these TRPL graphs is defined as:
I t
ð Þ ¼ A1et=s1
þ A2et=s2
ð1Þ
Here, A1 and A2 represent the normalized per-
centages of the individual decay components, while
s1 and s2 stand for the lifetimes of individual decay
components. The average lifetime has been calculated
using the following equation:
savg ¼ A1s2
1 þ A2s2
2
= A1s1 þ A2s2
ð Þ ð2Þ
The average lifetime for ZnPc@CNC and
ZnPc@CNC-TMR samples was 7.51 ns and 2.12 ns
using 470 nm as the excitation wavelength. The
average lifetime was recorded as 1.27 ns and 1.05 ns
using 405 nm excitation wavelength. Turquoise
Fig. 5 Schematic representation of fluorophore-TiO2 nanocomposite
538 Page 8 of 12 J Mater Sci: Mater Electron (2023)34:538

9. photoexcitation is preferable to violet excitation for
maximizing the fluorescence imaging signal from
TMR labeled with ZnPc@CNC.
Developing nanocomposites with biodegradable
properties using functional nanomaterials cellulosic
materials have a significant potential [42, 43]. The
high biocompatibility of CNCs has led to their
employment in several biomedical applications. It is a
non-toxic chemical with no substantial health haz-
ards when employed in the human body. Modified
CNCs using fluorescent materials may be helpful for
bioimaging and medication delivery systems [44].
TiO2 particles also offer a lot of promise for biological
applications because of their ultra-small-scale and
high specific surface area [45]. However, detect-
ing them within cells has proven to be complicated.
As a result, adding fluorescence characteristics to
titania nanoparticles by controlled functionalization
with an organic fluorophore is a novel and effective
way to make them visible under the microscope. It
was observed that the rough surface of TiO2 micro-
rods allows fluorophore material to adhere to it.
4 Conclusion
This work demonstrates two practical applications of
fluorescent cellulose nanocrystals, in the photolumi-
nescence sensing of small molecules and as a fluo-
rescent stain for non-emissive surfaces. Brightly
luminescent octacarboxylated zinc phthalocyanine-
conjugated cellulose nanocrystals (CNCs) exhibited
encouraging results in PL sensing experiments with
the ability to detect sub-millimolar concentrations of
terephthalic acid. The CNCs provide a robust
mechanical and chemically resistant scaffold for the
fluorophore and prevents it from being leached away
in liquid environments. The underlying principle of
sensing electron density changes in the phthalocya-
nine macrocycle can be applied toward the detection
of other small molecules and polymers containing
Fig. 6 Epifluorescence
micrographs of
ZnPc@CNC_TMR system
using 405 nm excitation
wavelength in (a) Blue,
(b) Green, (c) Yellow, and
(d) Red emission channels
(Color figure online)
J Mater Sci: Mater Electron (2023)34:538 Page 9 of 12 538

10. aromatic rings. The photoluminescence quenching of
fluorophore-conjugated CNCs using terephthalic
acid as a quencher can be applied in chemical sensing
applications. Non-fluorescent TiO2 microrods were
successfully imaged via fluorescence microscopy
using ZnPc@CNC as a fluorescent label which
spontaneously adsorbed on the surface of TiO2.
ZnPc@CNC is an intriguing candidate for bioimaging
applications since phthalocyanines and cellulose
nanocrystals are low toxicity, low-cost, earth-abun-
dant materials.
Author contributions
NC synthesized and characterized the materials,
performed the experiments, and prepared the
manuscript. JG performed select characterization and
interpreted the collected data. NK was responsible
for collecting and analyzing optical characterization
data. HR and XL assisted with data collection and
analysis. PK assisted with fluorophore synthesis,
fluorophore conjugation, and synthesis optimization.
AM supervised the research and provided instru-
mentation for time-resolved photoluminescence
characterization. KMA assisted in writing the
manuscript. KS supervised the research and edited
the manuscript.
Funding
Authors would like to acknowledge direct and
indirect support from the Future Energy Systems
(FES), FP Innovations, Alberta Innovates, NSERC,
and National Research Council Canada (NRC). NC
thanks Alberta Innovates for scholarship support.
Data availability
Research data will be made available upon reason-
able request.
Declarations
Competing interests The authors have no relevant
financial or non-financial interests to disclose.
Supplementary Information: The online version
contains supplementary material available at http
s://doi.org/10.1007/s10854-022-09724-2.
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