Effects Induced by the Temperature and Chemical Environment on the Fluorescence of Water-Soluble Gold Nanoparticles Functionalized with a Perylene-Derivative Dye

Abstract

We developed a fluorescent molecular probe based on gold nanoparticles functionalized with N,N′-bis(2-(1-piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide dihydrochloride, and these probes exhibit potential for applications in microscopic thermometry. The intensity of fluorescence was affected by changes in temperature. Chemical environments, such as different buffers with the same pH, also resulted in different fluorescence intensities. Due to the fluorescence intensity changes exhibited by modified gold nanoparticles, these materials are promising candidates for future technologies involving microscopic temperature measurements.

Full text

Citation: Lindstaedt, A.; Doroszuk, J.;
Machnikowska, A.; Dziadosz, A.;
Barski, P.; Raffa, V.; Witt, D. Effects
Induced by the Temperature and
Chemical Environment on the
Fluorescence of Water-Soluble Gold
Nanoparticles Functionalized with a
Perylene-Derivative Dye. Materials
2024,17, 1097. https://doi.org/
10.3390/ma17051097
Academic Editor: Maria Harja
Received: 1 February 2024
Revised: 16 February 2024
Accepted: 21 February 2024
Published: 28 February 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
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/).
materials
Article
Effects Induced by the Temperature and Chemical Environment
on the Fluorescence of Water-Soluble Gold Nanoparticles
Functionalized with a Perylene-Derivative Dye
Agnieszka Lindstaedt 1, Justyna Doroszuk 1, Aneta Machnikowska 1, Alicja Dziadosz 1, Piotr Barski 1,
Vittoria Raffa 2and Dariusz Witt 3,*
1ProChimia Surfaces Sp. z o.o., Zacisze 2, 81-850 Sopot, Poland; jusdor3101@gmail.com (J.D.);
aneta.machnikowska@gmail.com (A.M.); alicja.dziadosz.ad@gmail.com (A.D.); office@prochimia.com (P.B.)
2Dipartimento di Biologia, Universitàdi Pisa, S.S. 12 Abetone e Brennero, 4 56127 Pisa, Italy;
vittoria.raffa@unipi.it
3Faculty of Chemistry, Gdansk University of Technology, Narutowicza 11/12, 80-233 Gdansk, Poland
*Correspondence: dariusz.witt@pg.edu.pl
Abstract: We developed a fluorescent molecular probe based on gold nanoparticles functionalized
with N,N
′
-bis(2-(1-piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide dihydrochloride,
and these probes exhibit potential for applications in microscopic thermometry. The intensity of
fluorescence was affected by changes in temperature. Chemical environments, such as different
buffers with the same pH, also resulted in different fluorescence intensities. Due to the fluorescence
intensity changes exhibited by modified gold nanoparticles, these materials are promising candidates
for future technologies involving microscopic temperature measurements.
Keywords: gold nanoparticles; gold nanorods; temperature-dependent fluorescence; perylene-
derivative dye
1. Introduction
Temperature is among the most important parameters that reflect the energy of a sys-
tem. By measuring the temperature, we can at least partially characterize some physical or
chemical process in the living or still environment. For living cells, temperature changes are
indicative of disease pathologies associated with disturbed pathways for energy production
and consumption. The methods used to measure temperature at the macroscopic scale are
well known and understood. These methods involve analog thermometers based on liquid
expansion, thermocouples, infrared probes, and thermal imaging cameras. However, it
is challenging and complicated to measure temperature at the microscopic scale, such as
the temperature of objects just a few micrometers in size, such as cells. In the last decade,
several fluorescent molecular probes have been developed to determine the temperature
at the microscopic scale [
1
]. Through these probes, the temperature inside microreactors,
cells, or living organisms can be measured. By determining the temperature with high
special and temporal resolution, we can understand micro reactions and rationally develop
effective therapies at the cellular scale.
Generally, there are five principal methods used to measure temperature-dependent
fluorescence. The first and most common method is based on changes in emission in-
tensity [
2
–
6
]. The second method to measure temperature is based on the ratiometric
fluorescence of a molecular probe [
7
–
16
]. The third approach involves changes in the life-
time of the probe [
17
,
18
]. The fourth method involves measuring the ratio of emission peaks
of the two conjugated fluorophores that exhibit different responses to temperature [
19
,
20
].
The fifth method follows the emission peak shift vs. temperature [
21
–
27
]. Each method
exhibits advantages and disadvantages. Most frequently, photobleaching [
28
], sensitivity
Materials 2024,17, 1097. https://doi.org/10.3390/ma17051097 https://www.mdpi.com/journal/materials

Materials 2024,17, 1097 2 of 13
to ionic strength, pH [
2
,
29
], and concentration dependence are observed. The observation
of changes in emission intensity provides a rapid method for determining the temperature.
However, each measurement must be calibrated with a baseline. Moreover, the results
are greatly affected by photobleaching and the variation in the concentration of probes in
each cell [
2
,
3
,
5
,
7
]. From that point of view, the ratiometric self-calibrated method is more
attractive. However, these thermal probes are often polymers and can be affected by ionic
strength or pH [
8
]. Moreover, their large dimensions [
2
,
9
,
12
] can be responsible for the
limited internalization, diffusion, and disturbance of cell functions. The major advantage of
using the lifetime, a ratio of emission peaks, or the emission peak shift as the temperature
indicator is that the results are independent of thermal probe concentration. For the method
based on the lifetime measurement of the probe, photobleaching is not a problem, as the
bleached fluorophores are not measured. Although several molecular probes have been
obtained to determine the temperature, further studies are needed to develop reliable,
widely available, commercial molecular thermometers.
Perylene dyes are an important class of chromophores. Due to the properties of
these dyes, several applications with perylene dyes have been developed thus far. These
dyes have been used as fluorescence standards [
30
–
32
], thin film transistors [
33
–
35
], liq-
uid crystals [
36
–
38
], light emitting diodes [
39
], and photovoltaic devices [
40
–
42
]. The
perylene molecule consists of five benzene rings fused together to provide an extended
π
-conjugated planar structure. A variety of perylene dyes can be readily obtained when
perylene tetracarboxylic acid dianhydride is used as the starting material [
43
–
45
]. Moreover,
water-soluble perylene diimide derivatives were produced to provide potential antitumor
drugs, fluorescence tags, and elements of self-assembled photoactive films [
46
–
51
]. In
this study, to explore a new reversible micro thermometer, gold nanoparticles (AuNPs)
functionalized with N,N
′
-bis(2-(1-piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic acid
diimide dihydrochloride (PZPER) were synthesized, and their reversible optical properties
were investigated.
2. Materials and Methods
Sodium citrate (cat# 71402), EDCI (cat# E7750), sodium N-hydroxysulfosuccinimide
(cat# 56485), AgNO
3
(cat# 209139), perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA)
(cat# P11255), L-ascorbic acid (cat# A92902) benzylhexadecyldimethylammonium chloride
(BDAC) (cat# B4136), hexadecyltrimethylammonium bromide (CTAB) (cat# 52365), 1-
(2-aminoethyl)piperazine (cat# A55209), and an Amicon ultra centrifugal filter (50 kDa,
MWCO) (cat# UFC9050) were purchased from Sigma Aldrich (St. Louis, MO, USA). HAuCl
4
×
H
2
O (cat# 12325) and HSPEG3000COOH (cat# PEG1099) were purchased from Alfa
Aesar (Waltham, MA, USA) and IRIS Biotech (Marktredwitz, Germany), respectively.
2.1. Preparation of N,N
′
-bis(2-(1-Piperazino)ethyl)-3,4,9,10-perylenetetracarboxylic Acid Diimide
Dihydrochloride (PZPER)
PZPER was obtained using a previously described method [
52
]. Perylene-3,4,9,10-
tetracarboxylic dianhydride (PTCDA) was treated with excess 1-(2-aminoethyl)piperazine
and acidified with 2 M HCl. The reaction product was precipitated as the hydrochloride
salt in acetone. The spectra were in agreement with the previously reported data [52].
2.2. Preparation of Gold Nanoparticles and Nanorods
Gold nanoparticles (AuNPs, 12 nm) stabilized with citrate were prepared according to
a previously described method [
53
]. Spherical nanoparticles were obtained by reducing
gold chloride using sodium citrate in hot water.
Gold nanorods (AuNRs, 8.8
×
39.8 nm) stabilized with BDAC and CTAB were ob-
tained, as described previously [
54
]. The gold nanorod AuNRs were produced by seed-
mediated growth method based on a CTAB-capped seed. The silver content of the growth
solution was used to grow NRs to a desired length. The experimental procedure is provided
in the Supporting Information (page S3).

Materials 2024,17, 1097 3 of 13
2.3. Preparation of Functionalized Gold Nanoparticles
A solution of HSPEG3000COOH (2.5
µ
mol) in DI water (2 mL 0.2
µ
m CA filter) was
added to 50
µ
mol of 12 nm Au citrate-stabilized nanoparticles. The mixture was stirred for
1 h at RT. Then, the mixture was transferred to an Amicon filter 50 kDa and centrifuged for
20 min using a bench top centrifuge (2300 rpm). Centrifugation was continued until the
minimum retention volume reached ca. 200–300
µ
L. Mixing by pipetting and washing was
repeated 3 times with DI water using a centrifugal filter (Amicon 50 kDa). This step aims to
remove excess free HSPEG3000COOH. AuNPs were suspended in DI water (10 mL), and
the full characteristics (UV–Vis, DLS, zeta potential) were determined.
To 10
µ
mol of Au_12_PEG3000COOH, a solution of EDCI (0.38
µ
mol) in DI water
(1 mL) was added, and then the mixture was stirred for 5 min at RT. Next, a solution of
N-hydroxysulfosuccinimide sodium salt (0.38
µ
mol) in DI water (1 mL) was added, and
the mixture was stirred for 20 min at RT. Then, PZPER hydrochloride (0.19
µ
mol) was
added to the mixture and stirred for 1 h at RT. The mixture was transferred to an Amicon
filter 50 kDa, and the samples were centrifuged for 20 min using a bench top centrifuge
(2300 rpm). Centrifugation was continued until the minimum retention volume reached ca.
200–300
µ
L. Mixing by pipetting and washing was repeated 5 times with DI water using a
centrifugal filter (Amicon 50 kDa). AuNPs were suspended in DI water (2 mL), and the full
characteristics (UV–Vis, DLS, zeta potential) were determined.
2.4. Determination of Size and Zeta Potential
A dynamic light scattering (DLS) Zetasizer Ultra (Malvern Panalytical Ltd., Malvern,
UK) analyzer was used to determine the hydrodynamic particle size and zeta potential
of AuNPs. The detection angle was set at 90
◦
, the temperature was set at 25
◦
C, and the
refractive index was set at 1.33. A helium–neon laser beam and a clear polystyrene cuvette
(3 mL, 10 ×10 ×45 mm) were used for sample analysis.
2.5. Transmission Electron Microscopy (TEM)
The size and morphology of the prepared AuNPs were determined using a JEM 1400
(JEOL Co., Tokyo, Japan, 2008) with an energy-dispersive full range X-ray microanalysis
system (EDS INCA Energy TEM, Oxford Instruments, London, UK), a tomographic holder,
and an 11 Megapixel TEM Camera MORADA G2 (EMSIS GmbH, Münster, Germany)
(JEOL, Japan; Laboratory of Electron Microscopy, the Nencki Institute of Experimental
Biology Polish Academy of Science, Warsaw, Poland). The samples were prepared by
placing them on carbon-coated copper and leaving it to air-dry before imaging.
2.6. UV–Vis and Fluorescence Measurement
UV–Vis spectroscopy was performed using a Lambda 365 spectrophotometer (PerkinElmer,
Waltham, MA, USA). Fluorescence spectra were recorded by a FL 6500 fluorescence spec-
trometer (PerkinElmer, USA).
3. Results and Discussion
The presence of protonated peripheral nitrogen atoms improves the solubility of
PZPER in water and polar organic solvents and potentially provides a chromophore that
can be highly modulable as a function of pH, temperature, and the dielectric constant of
the solution. The optical properties of the dye were first characterized in aqueous solution,
and spectral changes at different temperatures were examined (Figure 1).

Materials 2024,17, 1097 4 of 13
Materials 2024, 17, x FOR PEER REVIEW 4 of 12
the solution. The optical properties of the dye were first characterized in aqueous solution,
and spectral changes at different temperatures were examined (Figure 1).
Figure 1. Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water at 25, 50, 60, 70, 75, and 80
°C.
Although minor changes vs. temperature at 540 nm (UV‒Vis) can be observed (sup-
porting information, page S1, Figure S1), for AuNPs functionalized with PZPER, the
amount of chromophore will likely be insufficient to observe a reliable response vs. tem-
perature. Moreover, the plasmon resonance of gold nanoparticles will overlap the ex-
pected signal from PZPER. The problems with the gold background can be overcome by
employing the fluorescence properties of PZPER. In this case, AuNPs cannot disturb the
response of fluorescence probes to temperature. We examined the fluorescence of an aque-
ous solution of PZPER (Figure 1).
3.1. Effects Induced by the Temperature and Buffers on the PZPER Fluorescence
As presented in Figure 1, the changes in the intensity of emission at 590 nm can be
related to the changes in temperature. However, every fluorescent probe must be tested for
variables such as polarity, pH, viscosity, and molecular interactions before application in
complex media/systems. We tested the fluorescence of PZPER in water and buffers (10 mM,
pH 7.4), PBS, Tris, and MOPS at 25 and 80 °C. The results are summarized in Figure 2a.
(a) (b)
Figure 2. (a) Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water and buffers (10 mM, pH
7.4) at 25 and 80 °C, (b) fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water and MOPS
buffer (1 mM and 10 mM, pH 7.4) at 25 and 80 °C.
Although the largest changes are observed for water, the pH cannot be controlled.
The quenching of the signal was the smallest for the MOPS buffer. Further experiments
were performed in this buffer at different concentrations. We verified the fluorescence of
PZPER at 25 and 80 °C in MOPS (1 mM and 10 mM, pH 7.4). When the concentration of
PZPER (50 µM) is relatively low, a concentration of 1 mM of buffer should be sufficient to
Figure 1. Fluorescence spectra of PZPER (50
µ
M,
λex
= 450 nm) in water at 25, 50, 60, 70, 75, and
80 ◦C.
Although minor changes vs. temperature at 540 nm (UV–Vis) can be observed
(Supporting Information, page S1, Figure S1), for AuNPs functionalized with PZPER,
the amount of chromophore will likely be insufficient to observe a reliable response vs.
temperature. Moreover, the plasmon resonance of gold nanoparticles will overlap the
expected signal from PZPER. The problems with the gold background can be overcome
by employing the fluorescence properties of PZPER. In this case, AuNPs cannot disturb
the response of fluorescence probes to temperature. We examined the fluorescence of an
aqueous solution of PZPER (Figure 1).
3.1. Effects Induced by the Temperature and Buffers on the PZPER Fluorescence
As presented in Figure 1, the changes in the intensity of emission at 590 nm can be
related to the changes in temperature. However, every fluorescent probe must be tested
for variables such as polarity, pH, viscosity, and molecular interactions before application
in complex media/systems. We tested the fluorescence of PZPER in water and buffers
(10 mM, pH 7.4), PBS, Tris, and MOPS at 25 and 80
◦
C. The results are summarized in
Figure 2a.
Materials 2024, 17, x FOR PEER REVIEW 4 of 12
the solution. The optical properties of the dye were first characterized in aqueous solution,
and spectral changes at different temperatures were examined (Figure 1).
Figure 1. Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water at 25, 50, 60, 70, 75, and 80
°C.
Although minor changes vs. temperature at 540 nm (UV‒Vis) can be observed (sup-
porting information, page S1, Figure S1), for AuNPs functionalized with PZPER, the
amount of chromophore will likely be insufficient to observe a reliable response vs. tem-
perature. Moreover, the plasmon resonance of gold nanoparticles will overlap the ex-
pected signal from PZPER. The problems with the gold background can be overcome by
employing the fluorescence properties of PZPER. In this case, AuNPs cannot disturb the
response of fluorescence probes to temperature. We examined the fluorescence of an aque-
ous solution of PZPER (Figure 1).
3.1. Effects Induced by the Temperature and Buffers on the PZPER Fluorescence
As presented in Figure 1, the changes in the intensity of emission at 590 nm can be
related to the changes in temperature. However, every fluorescent probe must be tested for
variables such as polarity, pH, viscosity, and molecular interactions before application in
complex media/systems. We tested the fluorescence of PZPER in water and buffers (10 mM,
pH 7.4), PBS, Tris, and MOPS at 25 and 80 °C. The results are summarized in Figure 2a.
(a) (b)
Figure 2. (a) Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water and buffers (10 mM, pH
7.4) at 25 and 80 °C, (b) fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water and MOPS
buffer (1 mM and 10 mM, pH 7.4) at 25 and 80 °C.
Although the largest changes are observed for water, the pH cannot be controlled.
The quenching of the signal was the smallest for the MOPS buffer. Further experiments
were performed in this buffer at different concentrations. We verified the fluorescence of
PZPER at 25 and 80 °C in MOPS (1 mM and 10 mM, pH 7.4). When the concentration of
PZPER (50 µM) is relatively low, a concentration of 1 mM of buffer should be sufficient to
Figure 2. (a) Fluorescence spectra of PZPER (50
µ
M,
λ
ex = 450 nm) in water and buffers (10 mM,
pH 7.4) at 25 and 80
◦
C, (b) fluorescence spectra of PZPER (50
µ
M,
λ
ex = 450 nm) in water and MOPS
buffer (1 mM and 10 mM, pH 7.4) at 25 and 80 ◦C.
Although the largest changes are observed for water, the pH cannot be controlled.
The quenching of the signal was the smallest for the MOPS buffer. Further experiments
were performed in this buffer at different concentrations. We verified the fluorescence of
PZPER at 25 and 80
◦
C in MOPS (1 mM and 10 mM, pH 7.4). When the concentration of
PZPER (50 µM) is relatively low, a concentration of 1 mM of buffer should be sufficient to
maintain a stable pH. The spectra are presented in Figure 2b (spectra in water are added
for comparison).

Materials 2024,17, 1097 5 of 13
A lower (1 mM) concentration of MOPS buffer provided a higher intensity of the
PZPER signal. It can be expected that a higher concentration of any salt can quench
fluorescence as well. The optimal buffer was MOPS at a 1 mM concentration. We also
examined this buffer at different pH values (5.35 and 7.4) (Figure 3a). Spectra in water were
added for comparison.
Materials 2024, 17, x FOR PEER REVIEW 5 of 12
maintain a stable pH. The spectra are presented in Figure 2b (spectra in water are added
for comparison).
A lower (1 mM) concentration of MOPS buffer provided a higher intensity of the
PZPER signal. It can be expected that a higher concentration of any salt can quench fluo-
rescence as well. The optimal buffer was MOPS at a 1 mM concentration. We also exam-
ined this buffer at different pH values (5.35 and 7.4) (Figure 3a). Spectra in water were
added for comparison.
(a) (b)
Figure 3. (a) Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water and MOPS buffer (1 mM,
pH 5.35 and 7.4) at 25 and 80 °C, (b) fluorescence spectra of PZPER (50 µM, λex = 450 nm) in MOPS
buffer (1 mM, pH 5.35) at variable temperatures (25 (black), 50 (red), 60 (green), 70 (purple), 75 (or-
ange), and 80 (violet) °C.
Surprisingly, the signal for PZPER in MOPS buffer (1 mM) was the strongest at 80 °C
and was the same as that in water at 25 °C when the buffer pH was 5.35. The comparison
of the spectra at 25 °C, pH 7.4 (green) and pH 5.3 (violet), suggests that changes in the
fluorescence intensity are related to changes in the pH (Figure 3a). Determining the opti-
mal conditions for the detection of PZPER can be helpful in the case of functionalized
AuNPs. We performed fluorescence measurements of PZPER under optimal conditions
(MOPS buffer, 1 mM) at variable temperatures (Figure 3b). The spectra were recorded at
25, 50, 60, 70, 75, and 80 °C and can be used to prepare calibration curves to determine
temperature based on the fluorescence of PZPER solution in MOPS buffer. Based on the
obtained results, PZPER is a promising fluorescence probe for the determination of tem-
perature. The reversibility of the developed conditions for the determination of tempera-
ture is of great interest. The spectra of PZPER solution were recorded at 25 °C, then after
heating to 80 °C, and again at 25 °C after cooling. We performed three cycles, with three
measurements at 25 and 80 °C (Figure 4).
Figure 4. Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in MOPS buffer (1 mM, pH 5.35) at
25 (1 cycle (green), 2 cycle (blue), 3 cycle (black)) and 80 °C (1 cycle (red), 2 cycle (orange), 3 cycle
(purple)) (three cycles).
Figure 3. (a) Fluorescence spectra of PZPER (50
µ
M,
λex
= 450 nm) in water and MOPS buffer
(1 mM, pH 5.35 and 7.4) at 25 and 80
◦
C, (b) fluorescence spectra of PZPER (50
µ
M,
λex
= 450 nm) in
MOPS buffer (1 mM, pH 5.35) at variable temperatures (25 (black), 50 (red), 60 (green), 70 (purple),
75 (orange), and 80 (violet) ◦C.
Surprisingly, the signal for PZPER in MOPS buffer (1 mM) was the strongest at 80
◦
C
and was the same as that in water at 25
◦
C when the buffer pH was 5.35. The comparison
of the spectra at 25
◦
C, pH 7.4 (green) and pH 5.3 (violet), suggests that changes in the
fluorescence intensity are related to changes in the pH (Figure 3a). Determining the optimal
conditions for the detection of PZPER can be helpful in the case of functionalized AuNPs.
We performed fluorescence measurements of PZPER under optimal conditions (MOPS
buffer, 1 mM) at variable temperatures (Figure 3b). The spectra were recorded at 25, 50, 60,
70, 75, and 80
◦
C and can be used to prepare calibration curves to determine temperature
based on the fluorescence of PZPER solution in MOPS buffer. Based on the obtained
results, PZPER is a promising fluorescence probe for the determination of temperature. The
reversibility of the developed conditions for the determination of temperature is of great
interest. The spectra of PZPER solution were recorded at 25
◦
C, then after heating to 80
◦
C,
and again at 25
◦
C after cooling. We performed three cycles, with three measurements at
25 and 80 ◦C (Figure 4).
Materials 2024, 17, x FOR PEER REVIEW 5 of 12
maintain a stable pH. The spectra are presented in Figure 2b (spectra in water are added
for comparison).
A lower (1 mM) concentration of MOPS buffer provided a higher intensity of the
PZPER signal. It can be expected that a higher concentration of any salt can quench fluo-
rescence as well. The optimal buffer was MOPS at a 1 mM concentration. We also exam-
ined this buffer at different pH values (5.35 and 7.4) (Figure 3a). Spectra in water were
added for comparison.
(a) (b)
Figure 3. (a) Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in water and MOPS buffer (1 mM,
pH 5.35 and 7.4) at 25 and 80 °C, (b) fluorescence spectra of PZPER (50 µM, λex = 450 nm) in MOPS
buffer (1 mM, pH 5.35) at variable temperatures (25 (black), 50 (red), 60 (green), 70 (purple), 75 (or-
ange), and 80 (violet) °C.
Surprisingly, the signal for PZPER in MOPS buffer (1 mM) was the strongest at 80 °C
and was the same as that in water at 25 °C when the buffer pH was 5.35. The comparison
of the spectra at 25 °C, pH 7.4 (green) and pH 5.3 (violet), suggests that changes in the
fluorescence intensity are related to changes in the pH (Figure 3a). Determining the opti-
mal conditions for the detection of PZPER can be helpful in the case of functionalized
AuNPs. We performed fluorescence measurements of PZPER under optimal conditions
(MOPS buffer, 1 mM) at variable temperatures (Figure 3b). The spectra were recorded at
25, 50, 60, 70, 75, and 80 °C and can be used to prepare calibration curves to determine
temperature based on the fluorescence of PZPER solution in MOPS buffer. Based on the
obtained results, PZPER is a promising fluorescence probe for the determination of tem-
perature. The reversibility of the developed conditions for the determination of tempera-
ture is of great interest. The spectra of PZPER solution were recorded at 25 °C, then after
heating to 80 °C, and again at 25 °C after cooling. We performed three cycles, with three
measurements at 25 and 80 °C (Figure 4).
Figure 4. Fluorescence spectra of PZPER (50 µM, λex = 450 nm) in MOPS buffer (1 mM, pH 5.35) at
25 (1 cycle (green), 2 cycle (blue), 3 cycle (black)) and 80 °C (1 cycle (red), 2 cycle (orange), 3 cycle
(purple)) (three cycles).
Figure 4. Fluorescence spectra of PZPER (50
µ
M,
λex
= 450 nm) in MOPS buffer (1 mM, pH 5.35) at
25 (1 cycle (green), 2 cycle (blue), 3 cycle (black)) and 80
◦
C (1 cycle (red), 2 cycle (orange), 3 cycle
(purple)) (three cycles).

Materials 2024,17, 1097 6 of 13
The cyclic changes are reliable and reversible. The results also demonstrate that
PZPER is stable at higher temperatures under aqueous conditions, which is important for
the development of AuNPs soluble in water or buffer.
3.2. Functionalization of AuNPs with PZPER
Gold nanoparticles (AuNPs, 12 nm) were stabilized with citric acid and function-
alized with PEG 3000 thiol terminated with carboxylic groups. We decided to use this
thiol to stabilize AuNPs and provide a relatively large distance between the gold surface
and immobilized PZPER. In this way, we attempted to avoid the potential quenching of
fluorescence by gold and obtain a more efficient excitation of the chromophore. Moreover,
PEG units can improve the solubility of functionalized AuNPs in aqueous solution. The
terminal carboxylic groups were activated with EDCI/N-hydroxysulfosuccinimide sodium
salt and treated with PZPER. AuNPs were purified by an Amicon filter 50 kDa to remove
side products and free PZPER. The synthesis of the functionalized AuNPs is presented in
Figure 5.
Materials 2024, 17, x FOR PEER REVIEW 6 of 12
The cyclic changes are reliable and reversible. The results also demonstrate that
PZPER is stable at higher temperatures under aqueous conditions, which is important for
the development of AuNPs soluble in water or buffer.
3.2. Functionalization of AuNPs with PZPER
Gold nanoparticles (AuNPs, 12 nm) were stabilized with citric acid and functional-
ized with PEG 3000 thiol terminated with carboxylic groups. We decided to use this thiol
to stabilize AuNPs and provide a relatively large distance between the gold surface and
immobilized PZPER. In this way, we aempted to avoid the potential quenching of fluo-
rescence by gold and obtain a more efficient excitation of the chromophore. Moreover,
PEG units can improve the solubility of functionalized AuNPs in aqueous solution. The
terminal carboxylic groups were activated with EDCI/N-hydroxysulfosuccinimide so-
dium salt and treated with PZPER. AuNPs were purified by an Amicon filter 50 kDa to
remove side products and free PZPER. The synthesis of the functionalized AuNPs is pre-
sented in Figure 5.
Figure 5. Synthesis of AuNPs functionalized with PZPER.
After purification, we collected UV‒Vis and fluorescence spectra of the functional-
ized AuNPs in water (Figure 6). Moreover, we performed filtration of functionalized
AuNPs on Amicon filters, and the filtrate was taken for measurement of fluorescence. The
filtrate did not show any fluorescence, demonstrating that the AuNP solution did not con-
tain free PZPER. The free PZPER in solution is approximately 100 times more fluorescent
than PZPR connected with AuNPs (Figure 6).
(a) (b)
Figure 6. UV‒Vis (a) and fluorescence (b) spectra of functionalized AuNPs (red, λex = 450 nm) and
PZPER (black, 50 µM) in water at 23 °C.
The spectra of PZPER were added for comparison (Figure 6 black line). For the UV‒
Vis spectrum, PZPER cannot be detected for functionalized AuNPs. The main problem is
the strong SPR signal from gold nanoparticles (520 nm) and the low concentration of the
Figure 5. Synthesis of AuNPs functionalized with PZPER.
After purification, we collected UV–Vis and fluorescence spectra of the functionalized
AuNPs in water (Figure 6). Moreover, we performed filtration of functionalized AuNPs
on Amicon filters, and the filtrate was taken for measurement of fluorescence. The filtrate
did not show any fluorescence, demonstrating that the AuNP solution did not contain free
PZPER. The free PZPER in solution is approximately 100 times more fluorescent than PZPR
connected with AuNPs (Figure 6).
Materials 2024, 17, x FOR PEER REVIEW 6 of 12
The cyclic changes are reliable and reversible. The results also demonstrate that
PZPER is stable at higher temperatures under aqueous conditions, which is important for
the development of AuNPs soluble in water or buffer.
3.2. Functionalization of AuNPs with PZPER
Gold nanoparticles (AuNPs, 12 nm) were stabilized with citric acid and functional-
ized with PEG 3000 thiol terminated with carboxylic groups. We decided to use this thiol
to stabilize AuNPs and provide a relatively large distance between the gold surface and
immobilized PZPER. In this way, we aempted to avoid the potential quenching of fluo-
rescence by gold and obtain a more efficient excitation of the chromophore. Moreover,
PEG units can improve the solubility of functionalized AuNPs in aqueous solution. The
terminal carboxylic groups were activated with EDCI/N-hydroxysulfosuccinimide so-
dium salt and treated with PZPER. AuNPs were purified by an Amicon filter 50 kDa to
remove side products and free PZPER. The synthesis of the functionalized AuNPs is pre-
sented in Figure 5.
Figure 5. Synthesis of AuNPs functionalized with PZPER.
After purification, we collected UV‒Vis and fluorescence spectra of the functional-
ized AuNPs in water (Figure 6). Moreover, we performed filtration of functionalized
AuNPs on Amicon filters, and the filtrate was taken for measurement of fluorescence. The
filtrate did not show any fluorescence, demonstrating that the AuNP solution did not con-
tain free PZPER. The free PZPER in solution is approximately 100 times more fluorescent
than PZPR connected with AuNPs (Figure 6).
(a) (b)
Figure 6. UV‒Vis (a) and fluorescence (b) spectra of functionalized AuNPs (red, λex = 450 nm) and
PZPER (black, 50 µM) in water at 23 °C.
The spectra of PZPER were added for comparison (Figure 6 black line). For the UV‒
Vis spectrum, PZPER cannot be detected for functionalized AuNPs. The main problem is
the strong SPR signal from gold nanoparticles (520 nm) and the low concentration of the
Figure 6. UV–Vis (a) and fluorescence (b) spectra of functionalized AuNPs (red,
λex
= 450 nm) and
PZPER (black, 50 µM) in water at 23 ◦C.

Materials 2024,17, 1097 7 of 13
The spectra of PZPER were added for comparison (Figure 6black line). For the UV–
Vis spectrum, PZPER cannot be detected for functionalized AuNPs. The main problem is
the strong SPR signal from gold nanoparticles (520 nm) and the low concentration of the
attached PZPER. However, the fluorescence spectrum showed a weak signal at 590 nm,
which can be detected due to the fluorescence lacking gold. The weak signal resulted from
the quenching of fluorescence or the insufficient excitation of the chromophore limited by
gold nanoparticles (Figure 6b).
The hydrodynamic size and zeta potential of functionalized AuNPs were determined
to be 23
±
3 nm and
−
29
±
4 mV, respectively, by dynamic light scattering (DLS) (Figure 7a).
The negative zeta potential indicates that only a minor number of carboxylic groups
were consumed for the attachment of PZPER, and most of the groups were present in an
anionic form.
Materials 2024, 17, x FOR PEER REVIEW 7 of 12
aached PZPER. However, the fluorescence spectrum showed a weak signal at 590 nm,
which can be detected due to the fluorescence lacking gold. The weak signal resulted from
the quenching of fluorescence or the insufficient excitation of the chromophore limited by
gold nanoparticles (Figure 6b).
The hydrodynamic size and zeta potential of functionalized AuNPs were determined to
be 23 ± 3 nm and −29 ± 4 mV, respectively, by dynamic light scattering (DLS) (Figure 7a). The
negative zeta potential indicates that only a minor number of carboxylic groups were con-
sumed for the attachment of PZPER, and most of the groups were present in an anionic form.
(a) (b)
Figure 7. (a) DLS measurement of functionalized AuNPs, (b) TEM image of functionalized AuNPs.
The TEM images showed a uniform metallic core size of 12 nm with a calculated
dispersity of ±1 nm (Figure 7b). The PEG shell cannot be detected by this technique.
We also recorded fluorescence spectra for AuNPs functionalized with PZPR in MOPS
buffer (1 mM, pH 5.35) at variable temperatures (Figure 8a).
(a) (b)
Figure 8. (a) Fluorescence spectra of AuNPs (1 × 1012 NP/mL) functionalized with PZPER (λex = 532
nm) in MOPS buffer (1 mM, pH 5.35) at variable temperatures, (b) the correlation between the tem-
perature and fluorescence of AuNPs functionalized with PZPER.
The intensity of the fluorescence at 590 nm can be used to determine the temperature
based on the linear calibration curve (Figure 8b). We were able to determine the amount
of PZPER aached to AuNPs. The solution of AuNPs was treated at pH 1 with a HCl so-
lution for 30 min. Then, the precipitated gold was separated, and the solution was taken
for fluorescence measurement. Based on the calibration curve at pH 1, the concentration
of PZPER released from AuNPs was 5 mM, which corresponds to 16% of all carboxylic
groups functionalized with PZPER (see supporting information, page S2).
We a lso verified the reversibility of the developed fluorescence probes. The fluores-
cence of AuNPs functionalized with PZPER was measured in MOPS buffer in three cycles
between 25 and 80 °C (Figure 9).
Figure 7. (a) DLS measurement of functionalized AuNPs, (b) TEM image of functionalized AuNPs.
The TEM images showed a uniform metallic core size of 12 nm with a calculated
dispersity of ±1 nm (Figure 7b). The PEG shell cannot be detected by this technique.
We also recorded fluorescence spectra for AuNPs functionalized with PZPR in MOPS
buffer (1 mM, pH 5.35) at variable temperatures (Figure 8a).
Materials 2024, 17, x FOR PEER REVIEW 7 of 12
aached PZPER. However, the fluorescence spectrum showed a weak signal at 590 nm,
which can be detected due to the fluorescence lacking gold. The weak signal resulted from
the quenching of fluorescence or the insufficient excitation of the chromophore limited by
gold nanoparticles (Figure 6b).
The hydrodynamic size and zeta potential of functionalized AuNPs were determined to
be 23 ± 3 nm and −29 ± 4 mV, respectively, by dynamic light scattering (DLS) (Figure 7a). The
negative zeta potential indicates that only a minor number of carboxylic groups were con-
sumed for the attachment of PZPER, and most of the groups were present in an anionic form.
(a) (b)
Figure 7. (a) DLS measurement of functionalized AuNPs, (b) TEM image of functionalized AuNPs.
The TEM images showed a uniform metallic core size of 12 nm with a calculated
dispersity of ±1 nm (Figure 7b). The PEG shell cannot be detected by this technique.
We also recorded fluorescence spectra for AuNPs functionalized with PZPR in MOPS
buffer (1 mM, pH 5.35) at variable temperatures (Figure 8a).
(a) (b)
Figure 8. (a) Fluorescence spectra of AuNPs (1 × 1012 NP/mL) functionalized with PZPER (λex = 532
nm) in MOPS buffer (1 mM, pH 5.35) at variable temperatures, (b) the correlation between the tem-
perature and fluorescence of AuNPs functionalized with PZPER.
The intensity of the fluorescence at 590 nm can be used to determine the temperature
based on the linear calibration curve (Figure 8b). We were able to determine the amount
of PZPER aached to AuNPs. The solution of AuNPs was treated at pH 1 with a HCl so-
lution for 30 min. Then, the precipitated gold was separated, and the solution was taken
for fluorescence measurement. Based on the calibration curve at pH 1, the concentration
of PZPER released from AuNPs was 5 mM, which corresponds to 16% of all carboxylic
groups functionalized with PZPER (see supporting information, page S2).
We a lso verified the reversibility of the developed fluorescence probes. The fluores-
cence of AuNPs functionalized with PZPER was measured in MOPS buffer in three cycles
between 25 and 80 °C (Figure 9).
Figure 8. (a) Fluorescence spectra of AuNPs (1
×
10
12
NP/mL) functionalized with PZPER
(
λex
= 532 nm) in MOPS buffer (1 mM, pH 5.35) at variable temperatures, (b) the correlation between
the temperature and fluorescence of AuNPs functionalized with PZPER.
The intensity of the fluorescence at 590 nm can be used to determine the temperature
based on the linear calibration curve (Figure 8b). We were able to determine the amount
of PZPER attached to AuNPs. The solution of AuNPs was treated at pH 1 with a HCl
solution for 30 min. Then, the precipitated gold was separated, and the solution was taken
for fluorescence measurement. Based on the calibration curve at pH 1, the concentration
of PZPER released from AuNPs was 5 mM, which corresponds to 16% of all carboxylic
groups functionalized with PZPER (see Supporting Information, page S2).
We also verified the reversibility of the developed fluorescence probes. The fluores-
cence of AuNPs functionalized with PZPER was measured in MOPS buffer in three cycles
between 25 and 80 ◦C (Figure 9).

Materials 2024,17, 1097 8 of 13
Materials 2024, 17, x FOR PEER REVIEW 8 of 12
Figure 9. Fluorescence spectra of AuNPs (1 × 1012 NP/mL) functionalized with PZPER (λex = 532 nm)
in MOPS buffer (1 mM, pH 5.35) at 25 (1 cycle (black), 2 cycle (green), 3 cycle (blue)) and 50 °C (1
cycle (orange), 2 cycle (purple), 3 cycle (red)) (three cycles).
The behavior of AuNPs functionalized with PZPER in cyclic changes in temperature
is unusual. When the temperature is increased to 50 °C and then cooled to 25 °C, the flu-
orescence does not return to the starting intensity (black). However, in the next cycle (Fig-
ure 9, cycle 2 and 3), the signals in the experimental error are the same. The intensity of
fluorescence at 50 °C is almost identical for all three cycles. Therefore, PZPER is not re-
leased when the temperature changes. When the functionalized AuNPs were purified, the
fluorescence of the removed unbound PZPER was much higher than that of the remaining
AuNPs with PZPER. The unusual starting fluorescence at 25 °C may occur because the
PZPER folds into and disappears inside the PEG layer. When the temperature was increased
and then decreased, the PZPER chromophore did not return to the starting point at which
its fluorescence was partially quenched. In the next cycles, the behavior of PZPER occurred
as expected. This phenomenon is reproducible with fresh AuNPs. When AuNPs were heated
and cooled to room temperature, all the cycles were the same as for cycles 2 and 3.
3.3. Functionalization of AuNRs with PZPER
The I-Gene project (hps://i-geneproject.eu/, accessed on 8 January 2024) is focused
on gene editing triggered by laser light irradiation of nanotransducers. The plasmonic
gold nanoparticles can absorb the light and rapidly convert it into heat via a series of pho-
tophysical processes. The gold nanorods (AuNRs) with appropriate size can be quite use-
ful in potential in vivo applications, where tissue absorption in the near-infrared window
(650–900 nm) is minimal and provides optimal light penetration. We intended to develop
AuNRs functionalized with PZPER to provide a thermal probe for the quick assessment
of the conversion of laser light into heat. These measurements could provide promising
conditions for AuNRs based transducers developed for thermal promoted DNA cleavage.
AuNRs were functionalized in similar way as AuNPs (experimental procedures are in-
cluded in the Supporting Information, page S5). The UV–Vis spectrum and TEM image
are presented in Figure 10.
Figure 9. Fluorescence spectra of AuNPs (1
×
10
12
NP/mL) functionalized with PZPER
(
λex
= 532 nm) in MOPS buffer (1 mM, pH 5.35) at 25 (1 cycle (black), 2 cycle (green), 3 cycle
(blue)) and 50 ◦C (1 cycle (orange), 2 cycle (purple), 3 cycle (red)) (three cycles).
The behavior of AuNPs functionalized with PZPER in cyclic changes in temperature
is unusual. When the temperature is increased to 50
◦
C and then cooled to 25
◦
C, the
fluorescence does not return to the starting intensity (black). However, in the next cycle
(Figure 9, cycle 2 and 3), the signals in the experimental error are the same. The intensity
of fluorescence at 50
◦
C is almost identical for all three cycles. Therefore, PZPER is not
released when the temperature changes. When the functionalized AuNPs were purified, the
fluorescence of the removed unbound PZPER was much higher than that of the remaining
AuNPs with PZPER. The unusual starting fluorescence at 25
◦
C may occur because the
PZPER folds into and disappears inside the PEG layer. When the temperature was increased
and then decreased, the PZPER chromophore did not return to the starting point at which
its fluorescence was partially quenched. In the next cycles, the behavior of PZPER occurred
as expected. This phenomenon is reproducible with fresh AuNPs. When AuNPs were
heated and cooled to room temperature, all the cycles were the same as for cycles 2 and 3.
3.3. Functionalization of AuNRs with PZPER
The I-Gene project (https://i-geneproject.eu/, accessed on 8 January 2024) is focused
on gene editing triggered by laser light irradiation of nanotransducers. The plasmonic gold
nanoparticles can absorb the light and rapidly convert it into heat via a series of photo-
physical processes. The gold nanorods (AuNRs) with appropriate size can be quite useful
in potential
in vivo
applications, where tissue absorption in the near-infrared window
(650–900 nm) is minimal and provides optimal light penetration. We intended to develop
AuNRs functionalized with PZPER to provide a thermal probe for the quick assessment
of the conversion of laser light into heat. These measurements could provide promising
conditions for AuNRs based transducers developed for thermal promoted DNA cleavage.
AuNRs were functionalized in similar way as AuNPs (experimental procedures are in-
cluded in the Supporting Information, page S5). The UV–Vis spectrum and TEM image are
presented in Figure 10.

Materials 2024,17, 1097 9 of 13
Materials 2024, 17, x FOR PEER REVIEW 9 of 12
(a) (b)
Figure 10. (a) UV–Vis spectrum of AuNRs functionalized with PZPER, (b) TEM image of AuNRs
functionalized with PZPER.
We p erfor med fluorescence measurement of AuNRs functionalized with PZPER. Un-
fortunately, the observed fluorescence was very low. The measurement at variable tem-
perature provided unreliable results, and the florescence did not correlate with tempera-
ture changes (supporting information, page S8, Figure S13). The insufficient aachment
of PZPER to AuNRs can provide low fluorescence. We determined the amount of the
PZPER aached to AuNRs. The solution of AuNRs functionalized with PZPER was treated
at pH 1 with HCl solution for 30 min. The precipitated gold was separated, and the solu-
tion was taken for fluorescence measurement. Based on the calibration curve at pH 1, the
concentration of PZPER released from AuNPs was 0.85 µM, which corresponds to 8% of
all carboxylic groups being functionalized with PZPER (see the supporting information,
page S6). The presence of the aached PZPER was confirmed; so, we expected the fluores-
cence to be mu ch high er. We s uspect ed tha t PZPER fluorescence was quenched by AuNRs.
We a lso performe d fluorescence measurement of AuNRs functionalized with thiol
(HSPEG3000COOH), PZPER 0.85 µM, and a mixture of AuNRs and PZPER (Figure 11).
Figure 11. Fluorescence spectra of AuNRs (16.9 × 1014 NR/mL) functionalized with HSPEG3,000COOH
(black), PZPER (0.85 µM) (red), and a mixture of AuNRs (16.9 × 1014 NR/mL) and PZPER (0.85 µM)
(blue) (λex = 532 nm) in MOPS buffer (1 mM, pH 5.35) at 25 °C.
The AuNRs without PZPER obviously did not show any fluorescence (Figure 11
black), and the solution with PZPER (0.85 µM) demonstrated relatively high fluorescence
at the concentration determined for functionalized AuNRs (Figure 11 red). However the
fluorescence of the AuNRs (16.9 × 1014 NR/mL) and PZPER (0.85 µM) mixture was very
low. It looks like the presence of gold nanorods is able to efficiently decrease the fluores-
cence of PZPER whether it is aached or not aached to the surface of AuNRs.
The fluorescence of PZPER is much lower (ca. 100 times) when it is aached to AuNPs
or AuNRs. The concentration of aached PZPER was 5 µM for AuNPs and 0.85 µM for
AuNRs. The UV–Vis spectrum of functionalized AuNRs (Figure 10a) showed plasmon
peaks at 524 and 807 nm. Fluorescence spectra of AuNRs (16.9 × 1014 NR/mL)
Figure 10. (a) UV–Vis spectrum of AuNRs functionalized with PZPER, (b) TEM image of AuNRs
functionalized with PZPER.
We performed fluorescence measurement of AuNRs functionalized with PZPER. Un-
fortunately, the observed fluorescence was very low. The measurement at variable tempera-
ture provided unreliable results, and the florescence did not correlate with temperature
changes (Supporting Information, page S8, Figure S13). The insufficient attachment of
PZPER to AuNRs can provide low fluorescence. We determined the amount of the PZPER
attached to AuNRs. The solution of AuNRs functionalized with PZPER was treated at
pH 1 with HCl solution for 30 min. The precipitated gold was separated, and the solution
was taken for fluorescence measurement. Based on the calibration curve at pH 1, the
concentration of PZPER released from AuNPs was 0.85
µ
M, which corresponds to 8% of
all carboxylic groups being functionalized with PZPER (see the Supporting Information,
page S6). The presence of the attached PZPER was confirmed; so, we expected the flu-
orescence to be much higher. We suspected that PZPER fluorescence was quenched by
AuNRs. We also performed fluorescence measurement of AuNRs functionalized with thiol
(HSPEG3000COOH), PZPER 0.85 µM, and a mixture of AuNRs and PZPER (Figure 11).
Materials 2024, 17, x FOR PEER REVIEW 9 of 12
(a) (b)
Figure 10. (a) UV–Vis spectrum of AuNRs functionalized with PZPER, (b) TEM image of AuNRs
functionalized with PZPER.
We p erfor med fluorescence measurement of AuNRs functionalized with PZPER. Un-
fortunately, the observed fluorescence was very low. The measurement at variable tem-
perature provided unreliable results, and the florescence did not correlate with tempera-
ture changes (supporting information, page S8, Figure S13). The insufficient aachment
of PZPER to AuNRs can provide low fluorescence. We determined the amount of the
PZPER aached to AuNRs. The solution of AuNRs functionalized with PZPER was treated
at pH 1 with HCl solution for 30 min. The precipitated gold was separated, and the solu-
tion was taken for fluorescence measurement. Based on the calibration curve at pH 1, the
concentration of PZPER released from AuNPs was 0.85 µM, which corresponds to 8% of
all carboxylic groups being functionalized with PZPER (see the supporting information,
page S6). The presence of the aached PZPER was confirmed; so, we expected the fluores-
cence to be mu ch high er. We s uspect ed tha t PZPER fluorescence was quenched by AuNRs.
We a lso performe d fluorescence measurement of AuNRs functionalized with thiol
(HSPEG3000COOH), PZPER 0.85 µM, and a mixture of AuNRs and PZPER (Figure 11).
Figure 11. Fluorescence spectra of AuNRs (16.9 × 1014 NR/mL) functionalized with HSPEG3,000COOH
(black), PZPER (0.85 µM) (red), and a mixture of AuNRs (16.9 × 1014 NR/mL) and PZPER (0.85 µM)
(blue) (λex = 532 nm) in MOPS buffer (1 mM, pH 5.35) at 25 °C.
The AuNRs without PZPER obviously did not show any fluorescence (Figure 11
black), and the solution with PZPER (0.85 µM) demonstrated relatively high fluorescence
at the concentration determined for functionalized AuNRs (Figure 11 red). However the
fluorescence of the AuNRs (16.9 × 1014 NR/mL) and PZPER (0.85 µM) mixture was very
low. It looks like the presence of gold nanorods is able to efficiently decrease the fluores-
cence of PZPER whether it is aached or not aached to the surface of AuNRs.
The fluorescence of PZPER is much lower (ca. 100 times) when it is aached to AuNPs
or AuNRs. The concentration of aached PZPER was 5 µM for AuNPs and 0.85 µM for
AuNRs. The UV–Vis spectrum of functionalized AuNRs (Figure 10a) showed plasmon
peaks at 524 and 807 nm. Fluorescence spectra of AuNRs (16.9 × 1014 NR/mL)
Figure 11. Fluorescence spectra of AuNRs (16.9
×
10
14
NR/mL) functionalized with
HSPEG3,000COOH (black), PZPER (0.85
µ
M) (red), and a mixture of AuNRs (16.9
×
10
14
NR/mL)
and PZPER (0.85 µM) (blue) (λex = 532 nm) in MOPS buffer (1 mM, pH 5.35) at 25 ◦C.
The AuNRs without PZPER obviously did not show any fluorescence (Figure 11
black), and the solution with PZPER (0.85
µ
M) demonstrated relatively high fluorescence
at the concentration determined for functionalized AuNRs (Figure 11 red). However
the fluorescence of the AuNRs (16.9
×
10
14
NR/mL) and PZPER (0.85
µ
M) mixture was
very low. It looks like the presence of gold nanorods is able to efficiently decrease the
fluorescence of PZPER whether it is attached or not attached to the surface of AuNRs.
The fluorescence of PZPER is much lower (ca. 100 times) when it is attached to AuNPs
or AuNRs. The concentration of attached PZPER was 5
µ
M for AuNPs and 0.85
µ
M for
AuNRs. The UV–Vis spectrum of functionalized AuNRs (Figure 10a) showed plasmon

Materials 2024,17, 1097 10 of 13
peaks at 524 and 807 nm. Fluorescence spectra of AuNRs (16.9
×
10
14
NR/mL) func-
tionalized with PZPER were recorded with
λex
= 532 nm. The overlapping of excitation
wavelength with AuNRs plasmon (524 nm) and the low concentration of PZPER (0.85
µ
M)
resulted in a lower fluorescence of AuNRs in comparison with the similar functional-
ized AuNPs.
4. Conclusions
In this work, we successfully synthesized AuNPs and AuNRs functionalized with
PZPER. We demonstrated the possibility of temperature determination based on the in-
tensity of fluorescence-developed gold nanoparticles. Based on fluorescence spectra, the
intensity of emission at 590 nm can be related to the temperature, pH, and ionic strength of
the environment. From this point of view, the developed functionalized AuNPs should be
calibrated, and the conditions (concentration of particles, pH and ionic strength) should be
strictly controlled to determine the temperature.
Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi.
com/article/10.3390/ma17051097/s1. Figure S1. UV–Vis spectra of PZPER (50
µ
M) in water at
25, 50, 60, 70, 75, 80 and 85
◦
C. Figure S2. UV–Vis spectra of AuNPs functionalized with PZPER
(5
µ
M) in MOPS buffer (1 mM, pH 5.35) in 3 cycles 25 and 50
◦
C. Figure S3. The linear calibration
curve of PZPER at pH 1. Figure S4. UV–Vis spectrum of AuNRs stabilized with CTAB in water.
Figure S5. TEM image of AuNRs stabilized with CTAB/BDAC. Figure S6. Synthesis of AuNRs
functionalized with PZPER. Figure S7. TEM image of AuNRs stabilized with HS-PEG3,000-COOH.
Figure S8. UV–Vis spectrum of AuNRs (13.4
×
10
14
NR/mL) functionalized with PZPER in water.
Figure S9. TEM image of AuNRs functionalized with PZPER. Figure S10. The linear calibration
curve of PZPER at pH 1. Figure S11. UV–Vis AuNRs_PEG3000_COOH (without PZPER) (blue),
AuNRs_PEG3000_PZPER (functionalized with PZPER 0.85
µ
M) (red) and PZPER (0.85
µ
M) (green).
Figure S12. Fluorescence spectra of AuNRs (13.4
×
10
14
NR/mL black and 16.9
×
10
14
NR/mL red)
functionalized with PZPER (
λex
= 450 nm) in MOPS buffer (1 mM, pH 5.35) at room temperature.
Figure S13. Fluorescence spectra of AuNRs (16.9
×
10
14
NR/mL) functionalized with PZPER
(λex = 450 nm) in MOPS buffer (1 mM, pH 5.35) at variable temperature.
Author Contributions: The manuscript was written by D.W., P.B. and A.L. The synthesis of PZPER,
functionalized AuNPs, and spectroscopic measurements were carried out by A.L., J.D., A.M. and
A.D. The experimental work and the manuscript were supervised by P.B., V.R. and D.W. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the European Union’s Horizon 2020 Research and Inno-
vation Programme under grant agreement No 862714 (https://i-geneproject.eu/, accessed on 8
January 2024).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: The authors wish to acknowledge the Laboratory of Electron Microscopy, the
Nencki Institute of Experimental Biology, Polish Academy of Science, Warsaw, Poland for support
during the recording of TEM images of functionalized AuNPs and AuNRs.
Conflicts of Interest: The authors, A.L., J.D., A.M., A.D. and P.B. are employed by the company
ProChimia Surfaces Sp. z o.o. The remaining author declares that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a potential conflict
of interest.
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