Barbiturate squaraine dyes as fluorescent probes for serum albumins detection

Article abstract of DOI:10.1016/j.jphotochem.2020.112710

Three indolenine-based barbiturate squaraine dyes were synthesized, characterized and subjected to photophysical studies, including their affinity with human serum albumin and bovine serum albumin as protein models in phosphate buffer solution. All dyes successfully interact with both proteins with high affinity binding constants. It was found that dyes with hydrophobic substituents had superior binding constants with both proteins. The fluorescence intensity of all dyes increased in the presence of both proteins which allowed the determination of detection and quantifications limits in the tens of nanomolar, using a protein concentration of 0–3.5 μM. Concerning to the study on the binding sites of the synthesized dyes using the warfarin and ibuprofen markers, the results of this study suggest that, one dye binds to both BSA binding sites while the two others dyes binds only to site I, and that all three dyes bind to both HSA binding sites.

Full text of DOI:10.1016/j.jphotochem.2020.112710

Journal Pre-proof
Barbiturate squaraine dyes as fluorescent probes for serum albumins
detection
Vanessa S.D. Gomes, Helena M.R. Gonc¸alves, Renato E.F. Boto,
Paulo Almeida, Lucinda V. Reis
PII:
S1010-6030(20)30509-8
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112710
JPC 112710
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
30 March 2020
12 June 2020
13 June 2020
Please cite this article as: Gomes VSD, Gonc¸alves HMR, Boto REF, Almeida P, Reis LV,
Barbiturate squaraine dyes as fluorescent probes for serum albumins detection, Journal of
Photochemistry and amp; Photobiology, A: Chemistry (2020),
doi: https://doi.org/10.1016/j.jphotochem.2020.112710
This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.
© 2020 Published by Elsevier.

Barbiturate squaraine dyes as fluorescent probes for serum albumins
detection
Vanessa S. D. Gomes1, Helena M.R. Gonçalves1,2, Renato E. F. Boto3,4, Paulo
Almeida3,4, Lucinda V. Reis1,5*
1Chemistry Centre of Vila Real (CQ-VR), University of Trás-os-Montes and Alto Douro, Quinta de
Prados, 5001-801, Vila Real, Portugal;
2REQUIMTE, Instituto Superior de Engenharia do Porto, 4200-072 Porto, Portugal;
3Health Sciences Research Centre (CICS-UBI), University of Beira Interior, Av. Infante D. Henrique,
6201-506 Covilhã, Portugal
4Chemistry Department, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001, Covilhã,
Portugal
5Chemistry Department, University of Trás-os-Montes and Alto Douro, Quinta de Prados, 5001-801, Vila
Real, Portugal
* Chemistry Department and CQ-VR; School of Life and Environmental Sciences, University of Trás-
os-Montes e Alto Douro (UTAD); Quinta de Prados; 5001-801 Vila Real; Portugal.
Tel.: +351 259350225; Fax: +351 29350480; E-mail address: lucinda.reis@utad.pt
Highlights
 Three squaraine cyanine dyes possessing in their structures moieties derived from
barbituric acid was synthesized and fully characterized.
 The potential as probes for HSA and BSA detection of the three squaraine cyanine
dyes was studied.
 The binding constants showed that the dyes have a strong interaction and high
affinity for both proteins.
 The Zeta potential values revealed that the dyes are quite stable in aqueous media.

Abstract
Three indolenine-based barbiturate squaraine dyes were synthesized, characterized and
subjected to photophysical studies, including their affinity with human serum albumin
and bovine serum albumin as protein models in phosphate buffer solution. All dyes
successfully interact with both proteins with high affinity binding constants. It was found
that dyes with hydrophobic substituents had superior binding constants with both
proteins. The fluorescence intensity of all dyes increased in the presence of both proteins
which allowed the determination of detection and quantifications limits in the tens of
nanomolar, using a protein concentration of 0 – 3.5 µM. Concerning to the study on the
binding sites of the synthesized dyes using the warfarin and ibuprofen markers, the results
of this study suggest that, one dye binds to both BSA binding sites while the two others
dyes binds only to site I, and that all three dyes bind to both HSA binding sites.
Keywords: Barbiturate, Squaraine dyes, Near-IR-emitting, Fluorescent probes, Serum
albumins
1. Introduction
In the biomedical diagnosis area, the determination of serum albumin (SA) concentration
is a fundamental parameter to assess the patient's health. It provides a secure prognosis
for development of some diseases in both healthy and apparently healthy individuals [1].
SA like human serum albumin (HSA) and bovine serum albumin (BSA) are a family of
water-soluble globular proteins, within which SA is the major protein in blood plasma.
Over the years has been the most studied class of proteins due to their properties, such as
stability and ability to bind and transport several ligands to specific sites [2, 3]. Albumins
have an essential role in the maintenance of various metabolic processes, such as,
determining plasma oncotic pressure, antagonizing the activity of toxins and controlling
the anti-oxidant properties of plasma, among others [4-7]. Therefore, changes in albumin
concentration can be used as early stage biomarker for several disorders, such as
cardiovascular disease, liver injury, glomerular failure, diabetes, among others [8, 9].
Given its relevance in biological systems, its quantitative determination in fluids is of
great importance in clinical diagnosis. Nowadays there are several detection methods in
used, namely colorimetric techniques (Lowry and Bradford method), immunoassays,

Surface-Enhanced Raman Scattering (SERS),
cyclic
voltammetry and
chemiluminescence. However, these techniques have several drawbacks, the main one
being the need of specific equipment and specialized personnel, which increases the cost
efficiency of the processes, along with difficult sample preparation protocols [10-12].
The search for new methods that may overcome these disadvantages and allow rapid,
low-cost and non-invasive assessment is a constantly developing area. One of the
appealing alternatives is the fluorescence spectroscopy. This technique is a powerful tool
that offers unique advantages, e.g, simplicity, excellent sensitivity and compatibility with
both live cells and physiological assays and cost efficiency [13, 14]. Several fluorescence-
based methods are being developed especially in the clinical and biological research areas
[15, 16].
Polymethine dyes are a class of compounds that have unique properties, namely, sharp
and intense absorption in the red-near infrared (NIR) region, high extinction coefficient,
intense fluorescence and moderate photoconductivity [17, 18]. These characteristics
make them a target of interest for both technological and biological applications,
particularly as organic solar cell sensitizers [19, 20], photoconductive materials [21, 22],
photodynamic therapy photosensitizers [23, 24], as well as fluorescent probes and labels
[25-28]. Some squaraine dyes features a two-photon excited fluorescence effect (TPEF),
what makes them very appealing for use in bioimaging because it allows to obtain deeper
and clearer images [29].
Here it is presented for the first time the synthesis of two innovative squaraine dyes with
a barbiturate group that were successfully used for SA fluorescence detection. We also
used the dye 8a previously reported by some of us [23], which had not yet been used in
this type of tests. The binding mechanism that BSA and HSA have in the presence of
increasing amounts of the three dyes were fully explored. Moreover, since the
fluorescence enhancement provided by the formation of the dye-protein complex was
linear and, as such, it was possible to determine the system detection limit (DL),
quantification limit (QL) and sensitivity.
2. Materials and Methods
Chemicals and reagents (e.g. 2,3,3-trimethylindolenine (1), 3,4-dihydroxycyclobut-3-en-
1,2-dione, barbituric acid (6a), thiobarbituric acid (6b), BSA, HSA, warfarin and
ibuprofen, were purchased from commercial suppliers and used without further

purification unless otherwise noted. 1-Hexyl-2,3,3-trimethylindolin-1-ium iodide (2)
[30], 3,4-dibutoxycyclobut-3-en-1,2-dione (4) [31], 3-butoxy-4-[(1-hexyl-3,3-
dimethylindol-3H-ylidene)methyl]-cyclobut-3-en-1,2-dione
(5)
[23],
1,3‑ diphenylthiourea (6c3) [32], 1,3-diphenyl-2-thiobarbituric acid (6c5) [32],
triethylammonium 2-[(1-hexyl-3,3-dimethylindolin-2-ylidene)methyl]-4-oxo-3-(2,4,6-
trioxohexahydro-5-pyrimidinyliden)cyclobut-1-en-1-olate (7a) [23] and 4-[(1-hexyl-3,3-
dimethyl-3H-indol-1-ium-2-yl)methylidene]-2-[(1-hexyl-3,3-dimethylindolin-2-
ylidene)methyl]-3-(2,4,6-trioxohexahydro-5-pyrimidinyliden)cyclobut-1-en-1-olate (8a)
[23] were prepared according to the literature procedure.
All reactions were monitored by thin-layer chromatography (TLC) on aluminium plates
with 0.25 mm of silica gel (Merck 60 F254). Melting points (m.p.) were measured in a
hot plate binocular microscope apparatus (URA Technic, Portugal) and were uncorrected.
Visible (Vis) spectra were obtained on a Perkin Elmer Lambda 25 instrument; max in
nanometers (nm). The infrared (IR) spectra were recorded on a UNICAM Research Series
FTIR spectrophotometer using KBr pellets; ʋmax in cm-1. The bands were described as s
(strong), m (medium) or w (weak). Nuclear magnetic resonance of proton (1H NMR) and
nuclear magnetic resonance of carbon-13 (13C NMR) spectra were obtained at 298.15 K
1
on a NMR Brucker Avance III 400 spectrometer operating at 9.4 T, observing H at
400.13 MHz and 13C at 100.62 MHz. The solutions were prepared in CDCl3 and the
chemical shifts are expressed as δ (ppm) relative to tetramethylsilane (internal standard)
or to residual solvent signals and the coupling constant (J) values are given in hertz (Hz).
Splittings were described as s (singlet), d (doublet), t (triplet), qt (quintet) or combinations
of the above or m (multiplet). Chemical shifts of 13C NMR spectra are presented to the
hundredths because after rounding to the tenths the signals of some different carbons
would appear to the same chemical shift. The assignments of the carbon were made based
on Distortionless Enhancement by Polarization Transfer (DEPT 135) spectra. High
resolution electrospray ionization time-of-flight mass spectra (HRESI-TOFMS) were

recorded using a microTOF (focus) Brucker Daltonics spectrometer (C.A.C.T.I. at the
University of Vigo).
2.1. Synthesis and characterization of precursors and squaraine dyes
2.1.1. Synthesis
of
triethylammonium
2-[(1-hexyl-3,3-dimethylindolin-2-
ylidene)methyl]-4-oxo-3-(4,6-dioxo-2-thioxohexahydro-5-pyrimidinyliden)cyclobut-1-
en-1-olate (7b)
The title compound was prepared by a similar method described by Tatarets et al. [33],
from a reaction of the semisquaraine 5 (1.084 g, 2.74 mmol) and thiobarbituric acid (6b)
(0.593 g, 4.11 mmol) in ethanol (20 mL) in the presence of triethylamine (0.380 mL, 2.74
mmol). The mixture was stirred under reflux for 6 h and then was cooled on an ice bath
to allow the product to precipitate by the addition of diethyl ether. The resulting residue
was washed with heated diethyl ether, filtered under reduced pressure and used in the
next step without further purification.
2.1.2. Synthesis of triethylammonium 2-[(1-hexyl-3,3-dimethylindolin-2-ylidene)methyl]-
4-oxo-3-(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5-pyrimidinyliden)cyclobut-1-en-1-
olate (7c)
The title compound was synthetized analogously to 7b, from a reaction of the
semisquaraine 5 (0.585 g, 1.48 mmol) and compound 6c5 (0.657 g, 2.22 mmol) in ethanol
(15 mL) in the presence of triethylamine (0.205 mL, 1.48 mmol). The mixture was stirred
under reflux for 7 h and then was cooled on an ice bath to allow the product to precipitate
by the addition of diethyl ether. The resulting residue was washed with heated diethyl
ether, filtered under reduced pressure and used in the next step without further
purification.
2.1.3. Synthesis of 3-(4,6-dioxo-2-thioxotetrahydro-5-pyrimidinyliden)-4-[(1-hexyl-3,3-
dimethyl-3H-indol-1-ium-2-yl)methylidene]-2-[(1-hexyl-3,3-dimethylindolin-2-
ylidene)methyl]cyclobut-1-en-1-olate (8b)
The title dye was prepared by reaction of the semisquaraine dye 7b (0.92 g, 1.62 mmol)
with the ammonium quaternary salt 2 (0.60 g, 1.62 mmol) in a mixture (1:1 v/v) of n-
butanol/toluene (20 mL), stirred for 8 h at reflux in a Dean-Stark apparatus. The reaction
mixture was quenched with cold distilled water, and the organic layer, after separation by
decantation, dried with anhydrous Na2SO4 and the solvent removed under reduced

pressure. The obtained residue was purified by successive recrystallizations from CH2Cl2/
petroleum ether/ diethyl ether. Dark green crystals were recoiled and dried under reduced
pressure. Yield: 5%; m.p.: 259-260 ºC; Vis max (DMF): 652 nm, log ε = 5.17; Vis max
(PBS): 645 nm; IR ʋmax (KBr): 3420 (w, NH), 3067 (w, ArCH), 2955 (m, CH), 2926 (m,
CH), 2857 (m, CH), 1740 (m, C=O), 1597 (m, ArC=C), 1493 (s), 1454 (s), 1358 (m),
1288 (s), 1236 (w), 1219 (w), 1175 (m), 1153 (m), 1109 (s), 1057 (m), 1018 (w), 980 (w),
922 (m), 820 (w), 789 (w), 748 (w), 687 (w); 1H NMR (400.13 MHz, CDCl3) δ: 8.77 (2H,
s, NH, exchange with D2O), 7.38-7.34 (4H, m, ArH), 7.22 (2H, t, J = 7.4, ArH), 7.07 (2H,
d, J = 7.6, ArH), 6.29 (2H, s, CH=C), 4.00 (4H, t, J = 7.4, NCH2(CH2)4CH3), 1.82 (4H,
qt, NCH2CH2(CH2)3CH3), 1.75 (12H, s, C(CH3)2), 1.43 (4H, qt, N(CH2)2CH2(CH2)2CH3),
13
1.38-1.31 (8H, m, N(CH2)3(CH2)2CH3), 0.91 (6H, t, J = 6.8, N(CH2)5CH3); C NMR
(100.6 MHz, CDCl3) δ: 180.37, 174.91, 173.95, 173.01, 172.79, 160.62, 142.54, 141.86,
128.04 (ArCH), 124.85 (ArCH), 122.26 (ArCH), 110.37 (ArCH), 93.73 (CH=C), 88.41,
49.51 (C(CH3)2), 44.77 (NCH2), 31.30 (CH2), 26.86 (CH2), 26.63 (CH2), 26.42
(C(CH3)2), 22.49 (CH2), 14.01 (CH3); HRESI-TOFMS m/z: 690.3598 [M]+
(C42H50N4O3S calc. 690.3606).
2.1.4. Synthesis of 3-(4,6-dioxo-1,3-diphenyl-2-thioxotetrahydro-5-pyrimidinyliden)-4-
[(1-hexyl-3,3-dimethyl-3H-indol-1-ium-2-yl)methylidene]-2-[(1-hexyl-3,3-
dimethylindolin-2-ylidene)methyl]cyclobut-1-en-1-olate (8c)
The title dye was prepared by the reaction of the semisquaraine dye 5c (0.240 g, 0.334
mmol) with the ammonium quaternary salt 2 (0.126 g, 0.334 mmol) in a mixture (1:1 v/v)
of n-butanol/toluene (10 mL), stirred for 2 h at reflux in a Dean-Stark apparatus. After
work-up in the same way as barbiturate squaraine dye 8b, the obtained residue was
purified by alumina column chromatography (CH2Cl2/MeOH, 2%). We obtained dark
green crystals. Yield: 12%; M.p.: 250-251 ºC (dec.); Vis max (DMF): 647 nm, log ε =
5.16; Vis max (PBS): 650 nm; IR ʋmax (KBr): 3057 (w, ArCH), 2957 (w, CH), 2927 (m,
CH), 2860 (w, CH), 1741 (m, C=O), 1624 (m, ArC=C), 1491 (s), 1481 (s), 1454 (s), 1359
(s), 1290 (s), 1223 (w), 1179 (w), 1154 (w), 1129 (w), 1103 (m), 1054 (w), 1021 (w), 978
(w), 923 (w), 817 (w), 784 (w), 747 (w), 722 (w), 690 (w); 1H NMR (400.13 MHz, CDCl3)
δ: 7.46 (4H, t, J = 7.4, ArH), 7.39-7.31 (10H, m, ArH), 7.19 (2H, t, J = 7.4, ArH), 7.0 (2H,
d, J = 8.0, ArH), 6.32 (2H, s, CH=C), 3.86 (4H, t, J = 7.6, NCH2(CH2)4CH3), 1.73 (16H,
br s,+ NCH2CH2(CH2)3CH3), 1.34-1.27 (12H, m, N(CH2)2(CH2)3CH3), 0.91 (6H, t, J =

13
6.6, N(CH2)5CH3); C NMR (100.6 MHz, CDCl3) δ: 180.65, 180.41, 175.60, 173.66,
172.69, 160.84, 142.86, 142.14, 141.86, 129.41 (ArCH), 129.22 (ArCH), 128.27 (ArCH),
128.00 (ArCH), 124.98 (ArCH), 122.52 (ArCH), 110.45 (ArCH), 94.45 (CH=C), 90.27,
49.63 (C(CH3)2), 45.03 (NCH2), 31.70 (CH2), 27.16 (CH2), 26.93 (CH2), 26.69
(C(CH3)2), 22.73 (CH2), 14.38 (CH3); HRESI-TOFMS m/z: 843.4302 [M+H]+
(C54H59N4O3S calc. 843.4285).
2.2. Preparation of dyes and protein solutions
The solutions were prepared by dissolution of dyes in dimethylformamide (DMF) to
obtain a stock solution with a concentration of 6.7×10-4M. The stock solutions of BSA
and HSA with a concentration of 14 µM were prepared in PBS (0.05M, pH 7.2). In order
to investigate protein/dye interactions, the DMF solutions of the dyes were diluted in PBS
to have a concentration of 2 µM of each dye and then was added different volumes of
BSA and HSA solutions to achieve the desired protein concentrations. All working
solutions were prepared immediately before the assays.
2.3. Spectroscopic measurements
The absorption spectra of dyes were recorded on a Lambda 25 UV/Vis
spectrophotometers (Perkin Elmer, USA) at room temperature (r.t.), using a spectral
range between 500-900 nm. The emission spectra were obtained on a Varian Cary Eclipse
fluorescence spectrophotometer (Agilent Technologies, USA). Spectroscopic
measurements were performed in a standard quartz cell (1 cm path length).
2.4. Determination of fluorescence quantum yield
The fluorescence quantum yield was determined according to the equation 1 [34]:
2
η
Grad
dye
ɸdye= ɸref
(
dye) (η
)
(1)
2
Grad
ref
ref
where the subscripts ref and dye denote reference and studied dyes respectively, Grad the
gradient from the plot integrated fluorescence intensity vs absorbance and η the refractive
index of the solvent (DMF). The reference used was zinc phthalocyanine (ɸF = 17% in
DMF) [35]. The emission spectra were obtained with excitation wavelength exc= 600
nm, excitation slit of 5 nm and emission slit of 10 nm.

2.5 Estimation of the dipole moments
The estimation of the ratio between the ground state and the excited state dipole moments
(µg and µe, respectively) was calculated using the solvatochromic method according to
the equations 2 and 3 [36].
(
)
풗풂 − 풗풇 = 풎ퟏ풇 휺, 풏 + 풄풐풏풔풕풂풏풕
(2)
(
)
( )
풗풂 + 풗풇 = −풎ퟐ풇 휺, 풏 + ퟐ품 풏 + 풄풐풏풔풕풂풏풕
(3)
where 풗풂 and 풗풇 represent the absorption and fluorescence band shifts in different
solvents that have a specific permittivity (휺) and refractive index (풏). The parameter
품(풏) is solvent dependent and can be calculated using the refractive index by the
following equation.
ퟒ
ퟑ
ퟐ
풏 −ퟏ
[
]
ퟐ
품(풏) =
(4)
ퟐ
(풏 +ퟐ)
The use of these parameters allows the calculation of the ground state and the exited state
dipole moments according to the following equation:
흁
풎 +풎
ퟏ
풆
ퟐ
ퟏ
=
(5)
흁
품
풎 −풎
ퟐ
2.6 Determination of Zeta Potential
The Zeta Potential measurements were performed in a Zeta PALS potential analyzer
(Brookhaven, USA) with parallel-plate platinum black electrodes spaced 5 mm apart and
a 10 mm path length rectangular organic glass cell. All the samples were measured using
a sinusoidal voltage of 80 V with a frequency of 3 Hz. This measure was determined in
solutions containing protein concentrations (BSA and HSA) in a range of 0-3.5 µM and
a 2 µM concentration of each dye. For each sample, a total of three values were
automatically given by the instrument, which were then averaged to give the final value
and the standard deviation.

2.7 Protein assays
In order to investigate protein-dye interactions the dye solutions were prepared in DMF
(due to the high insolubility in aqueous medium) and then diluted in PBS buffer. To this
solution, the protein stock solution was added and incubated for 1 hour under stirring, at
r.t.. In these experiments the dyes concentrations were maintained (2 µM) and the proteins
concentrations ranges from 0-3.5 µM. The fluorescence intensity of the final solutions
was measured using a exc= 580 nm, excitation slit of 10 nm and emission slit of 20 nm.
2.8. Binding parameters determination
The dissociation (푲푫 ) and binding (푲푩) constant values can give an idea about the
strength of binding and thereby throws some light on the mode of binding. The apparent
dissociation constant (푲푫) and the Hill’s coefficient (풏) of protein/dye system can be
estimated by Hill’s Plot using the data from fluorescence protein assays, by the following
equation [37]:
푸
[ ]
풍풐품 (ퟏ−푸) = 풍풐품 푲푫 + 풏풍풐품 푷
(6)
푭
where 푸 =
and [P] is the protein (BSA or HSA) concentration.
푭
풎풂풙
푸
The values of 푲푫 were obtained from the intercept of the plot of 풍풐품 (ퟏ−푸) vs 풍풐품[푷].
The binding constants can be obtained using the assays of protein/dye interaction data
through the modified Benesi-Hildebrand equation 7 [38]:
ퟏ
ퟏ
ퟏ
ퟏ
=
+
(7)
(
) [ ]
푷
푭 −푭
푭 −푭
푲
푭 −푭
 ퟎ
풙
ퟎ

ퟎ
푩
where 푭ퟎ, 푭풙 and 푭 are the fluorescence intensity in the absence of protein, at an
intermediate concentration of protein and at a concentration for complete interaction,
[ ]
respectively; 푲푩 is the binding constant and 푷 is the protein (BSA or HSA)
ퟏ
concentration. We obtained the value of binding constant plotting a graph of
vs
ퟎ
푭 −푭
풙
[ ]−ퟏ
푷
.

2.9. Determination of binding site
The determination of the binding sites of the synthesized dyes to the tested proteins was
performed using a method already described by Luo et al. [4]. Assays were carried out
with the different synthesized dyes at a concentration of 2µM, with BSA and HSA at a
concentration of 3 µM and with different concentrations of warfarin and ibuprofen (0-
100µM).
2.10 Determination of detection limit (DL) and quantification limit (QL)
The detection limit (DL) and the quantification limit (QL) are important parameters in
the validation of the method. These parameters were calculated using a calibration curve
obtained from the plot of maximum fluorescence intensity as a function of the protein
concentration and then we applied the follow equation formulas:
ퟑ.ퟑ풙푺.푫.
푫푳 =
푸푳 =
(8)
(9)
풌
ퟏퟎ풙푺.푫.
풌
where 푺. 푫. is the standard deviation of blank and 풌 is the slope of the curve equation
[39].
2.11 Determination of complex dye-protein lifetimes
Lifetime data were obtained using the maximum excitation and emission of each dye at
room temperature using frequency domain interrogation. For each case an appropriate
wavelength LED was used to excite the sample by direct irradiation. The luminescence
emission was guided by a 600-m Y fiber coupler. Luminescence was collected by the
same optical fiber and lead to detection by a variable gain amplified silicon photodetector
(PDA36A from Thorlabs, Inc.) set at 30 dB amplification and 785 kHz bandwidth. The
signal obtained by the detector was fed into a lock-in amplifier (SR844 from SRS) where
it was compared with a reference signal from the modulation source. The lifetime study
was performed for individual dyes and for the complex dye-proteins. For the last it was
used the protein concentration ranged from 0-3.5 µM. When a two-exponential decay was
obtained the medium lifetime was calculated using the following expression: 흉풎
=

풇ퟏ흉ퟏ + 풇ퟐ흉ퟐ where 풇ퟏ and 풇ퟐ are the fractional intensities and 흉ퟏ and 흉ퟐ represent
the obtained lifetimes [40].
3. Results and discussion
3.1. Synthesis of squaraine dyes
The synthesis of the squaraine dyes is presented in Scheme 1. As far as the author’s
knowledge, synthesis and spectroscopic characterization of squaraine dyes 8b and 8c are
being presented here for the first time. Indolenine-based barbiturate squaraine dyes with
different barbituric acid derivatives, such as thiobarbituric acid (8b) and 1,3-diphenyl-2-
thiobarbituric acid (8c) positionated on the four-membered central ring were synthesized
by a multistep reaction (Scheme 1). Dye 8a, although already reported in a recent work
published by some of us [23], has never been evaluated for its potential use as a
fluorescent probe for proteins detection.
Scheme 1: Schematic representation of the barbiturate squaraine dyes 8a-c synthesis. Conditions: i)
acetonitrile, reflux for 5 days; ii) n-butanol, reflux for 4h; iii) EtOH/Et3N, r.t., overnight; iv) CH2Cl2, r.t.

for 3h; v) acetyl chloride, 60°C, 1h; vi) EtOH/Et3N, reflux, vii) n-butanol/ toluene (1:1, v/v), Dean-Stark
apparatus.
The barbiturate dyes were synthetized using a similar procedure previously described
[33], e.g., the squaric acid (3) was refluxed with n-butanol, resulting the dibutylsquarate
(4), that reacted with the quaternary ammonium salt (2), which was obtained through the
alkylation of 2,3,3-trimethylindolenine (1) with an excess of iodohexane, to afford the
monosubstituted intermediate 5. Afterwards, the semisquaraines 7a-c were obtained upon
reaction of the monosubstituted intermediate 5 with barbituric acid (6a) or a derivative
thereof (6b or c), and were used in the next reaction without further purification. Finally,
the desired barbiturate squaraine dyes 8a-c were obtained through a base-catalyzed
condensation reaction between the respective semisquaraine intermediate 7a-c and the
quaternary ammonium salt 2 in a n-butanol/toluene (1:1 v/v) mixture using a Dean-Stark
apparatus.
Although barbituric (6a) and thiobarbituric acid (6b) were used as commercially
available, 1,3-diphenyl-2-thiobarbituric acid (6c) was obtained by a two-step
methodology already described in the literature [32] (Scheme 1, highlighted box). This
barbituric acid derivative was obtained by the reaction between the 1,3-diphenylthiourea
(6c3), which was achieved by the reaction of phenylisothiocyanate (6c1) with aniline (6c2)
in dichloromethane, and malonic acid (6c4), in the presence of acetyl chloride.
The structures of synthetized dyes were confirmed by standard spectroscopy methods,
namely 1H NMR, 13C NMR, IR and HRMS spectrometry. The carbon atoms attributions
were based in DEPT 135 spectra (Fig S1-11 Supplementary Data),
3.2. Photophysical properties of the synthetized dyes
The photophysical properties were studied in organic solvents with different polarities
(Table 1). The absorption and emission spectra for the three synthetized dyes in the
different solvents are shown in Fig. 1 a-f. As it is possible to see from the figure analysis
both absorbance and fluorescence emission are solvent sensitive. Moreover, the observed
shift in each solvent is different according to the dye.

Table 1. Photophysical results from UV-VIS/NIR absorption and fluorescence emission of synthesized
dyes (8a-c) in different solvents.
Dyes Solvent λabs (nm) λem (nm)*
Ɛ (M -1. cm -1) Stokes shift (nm) ɸF (%)
PBS
650
632
658
644
645
630
652
641
650
630
647
644
708
653
681
663
700
654
675
661
708
653
673
663
4.31 × 104
9.74 × 104
1.35 × 105
1.04 × 105
5.76 × 104
1.30 × 105
1.47 × 105
1.54 × 106
6.61 × 104
1.55 × 105
1.46 × 105
1.84 × 105
58
21
23
19
55
24
23
20
58
23
26
19
-
-
4.8
MeOH
DMF
CHCl3
PBS
MeOH
DMF
CHCl3
PBS
8a
8b
8c
-
-
-
5.6
-
-
-
8.1
-
MeOH
DMF
CHCl3
* λexc 580 nm, excitation and emission slits 10 nm.
Fig. 1. Absortion spectra (a-c, corresponding to compound 8a-c, respectively) and normalized emission
spectra (d-f, corresponding to compound 8a-c, respectively) of synthetized dyes in four different solvents.
The fluorescent intensities in PBS are practically not measurable (FI = 13 and 16 for dye 8a; FI = 14 for
dye 8b and FI = 12 for dye 8c)
It is possible to perform a deconvolution of the absorption peak and obtain two distinct
spectral signals one at greater relative wavelength to the monomer band, and other that
can be attributed to H-aggregates at a shorter wavelength [41, 42] (absorption band shifted
hypsochromic) closely followed by the monomer band (Fig. S12, Supplementary Data).
The monomer band is sharp and strong for all dyes, which is consistent to the typical

optical transitions associated with the high delocalization of the π-system [15]. The
maximum emission wavelength shift observed in the different solvents is an indication
that the dyes are solvatochromic. Each dye had a distinct behavior in the solvents that
needs to be further analyzed according to their molecular structure. Both 8a and 8b have
electron withdrawing ring substituents, the carbonyl and sulfur groups, nonetheless they
exhibit some relevant optoelectronic differences since oxygen electronegativity is
superior. Indeed, when considering the maximum wavelength shift between dyes
absorption and emission in the different solvents it is possible to see that 8a has a more
pronounced wavelength shift in the absorption peak from methanol to DMF (26 nm) than
the emission (18 nm). On the other hand and, considering the same solvents, in 8b, even
though it seems to have the same trend, the shifts are too close to be distinguished (22 nm
and 21 nm, respectively), probably due to the stability that the ring substituents provide
to the dye in the ground state. Indeed, when the absorption peak is more affected than the
emission it is possible to say that the ground state energy distribution of the dye is affected
by the solvents, and this is most probably related to the polar nature of the dye in this state
[43]. On the other hand, 8c has an electron withdrawing sulfur group and a phenyl group,
which in this case, should act as an electron donating substituent. The electron donating
ability of the phenyl group is superior than the one obtained by hydrogen substituents (8a
and b), in this sense the electrons π mobility in the excited state should be higher and the
dye is less polar in the ground state. In this case the spectral shift in the absorption (17
nm) is less to the emission (20 nm), which provides further evidence that the ground state
energy is not particularly affected by the solvents. In order to confirm the influence that
the absorbed photon has on the charge’s redistribution and its consequence on the
conformational change of the excited state, the dipole moment of the ground and excited
state were calculated according to the solvatochromic method described by Koutek [44].
This method is based on the dyes absorption and emission spectral shift in different
polarity solvents. The reason between the dipole moments of the excited (μe) and ground-
state (μg) (Table S1, Supplementary Data) indicates that in all the synthetized dyes the
dipole moment of the excited state is higher than in the ground state. Moreover, this
reason has a descending trend from 8a to 8c (0.5; 0.19; 0.089, respectively). In such cases,
the relaxed excited state S1 is energetically stabilized relative to the ground state S0 and a
significant red shift of the fluorescence will be observed [36].

3.3 Zeta potential of the dyes in the presence of BSA and HSA
It is fairly known that common squaraine dyes forms aggregates in water that quenches
their fluorescence and act as a major shortcoming when it is intended the development of
sensitive probes for biological molecules [15]. The dyes here described where chemically
engineered with the intent to try to overcome this drawback. Before we start with
interaction tests between dye and proteins and, in order to better access the stability of the
compounds in PBS, a zeta potential assay was developed where each dye was diluted in
PBS and then placed in contact with increasing amounts of the studied proteins: BSA and
HSA.
The results are present in Figure 2a, b and shows that even before the protein addition all
Fig. 2. Zeta potential profile of the three squaraine dyes (8a (black), 8b (red) and 8c (blue)) in the presence
of BSA (a) and HSA (b) in a concentration range of 0-3.5 µM. (The blue dotted line was placed to highlight
the aggregation point).
dyes where stable in PBS presenting negative zeta potential values (-33 mV, -48 mV, -44
mV, for 8a-c, respectively). It was expected that they all exhibit different zeta potential
values in PBS, since they have distinct substituents that exert a unique effect on the
molecule overall charge distribution. What is interesting to notice is that depict the PBS
dissolution they are all far from the aggregation zone (close to 0 mV), thereby
demonstrating a higher stability in this common biological buffer than the existing
squaraine dyes reported in literature [43].
3.4. Dye-protein association

The non-fluorescent aggregates typical of the squaraine dyes in PBS solutions are often
described in the literature [2, 6, 43, 45]. However, reports of their fluorescence in this
medium are quite scarce [43]. The synthetized dyes here described also exhibit non-
fluorescent aggregates in PBS prior to protein addition (Fig. 3a-c). Upon the addition of
BSA (Fig. 3a-c) or HSA (Fig. 4a-c) the complex dye-proteins emit at about 650nm and
the emission intensity is directly proportional to the concentration of protein used. It is
also interesting to notice that the new adduct dye/protein has a fluorescence intensity 35%
higher with BSA than HSA.
Fig. 3. Fluorescence emission spectra of compounds 8a (a), 8b (b) and 8c (c) in PBS with varied BSA
concentrations (0-3.5 µM) for a fixed dye concentration of 2 µM. (d) Maximum fluorescence emission
intensity of dyes (8a (black), 8b (red), 8c (blue)) as a function of BSA concentration in PBS.

Fig. 4. Fluorescence emission spectra of compounds 8a (a), 8b (b) and 8c (c) in PBS with varied HSA
concentrations (0-3.5 µM) for a fixed dye concentration of 2 µM. (d) Maximum fluorescence emission
intensity of dyes (8a (black), 8b (red), 8c (blue)) as a function of HSA concentration in PBS.
In order to better understand the binding mechanism between the synthetized squaraine
dyes and the proteins the Hill’s plot was analyzed (Fig. S13 a, b, Supplementary Data).
Its possible to see that for BSA all dyes exhibit a linear trend, that allowed the
determination of the Hills coefficient (푛) and the apparent dissociation constant (퐾퐷)
(Table 2). According to the obtained results it is possible to say that the interaction
between the dyes and BSA is governed by a cooperative mechanism, which is the most
common process underlying a large range of biochemical and physiological processes
[36, 46]. Moreover, the apparent dissociation constant values provide information on the
dye concentration that saturates 50% of the protein binding sites. Considering the data on
Table 2, the dye that saturates the protein binding sites at the lowest concentration is 8a.
On the other hand it is possible to add a 55% higher concentration of 8c (compared to 8a)
to obtain the same saturation of the protein binding sites. It is also interesting to notice
that both 8a and 8c exhibit a positive cooperativity (n>1) and that 8b system has a
negative cooperativity (n<1) [47], e.g, when dyes 8a and 8c bind to BSA in one of its

binding sites, the other binding site are affected by this change. This is also quite visible
in the Zeta potential plot (Fig. 2 a, b), where there is a change in the protein conformation
that affects the overall surface charge at a similar dye concentration (0.4 µM) for 8a and
8c. This phenomenon is less pronounced for 8b, which is consistent with negative
cooperativity where there is a decrease in the binding affinity once the dye bounds to one
of the sites [47].
Table 2- Parameters obtained from the Hill’s Plot.
Dyes
Hill’s Coefficient (n)
KD (µM)
R2
1.29±0.07
0.892±0.06
1.05±0.08
0.628±0.04
0.827±0.04
1.14±0.05
0.97374
0.95761
0.95284
8a
8b
8c
Even though the two studied proteins, BSA and HSA, are quite similar, indeed the main
difference in their composition is that HSA has only one tryptophan residue, rather than
BSA that has two [45, 47], their interaction with the synthetized dyes is quite different
(Fig. S13 a,b, Supplementary Data). The Hill’s plots for the assays with HSA exhibits a
polynomial fit with n=3. This deviation from the typical linear trend of this plot is
consistent with the fact that for HSA the binding mechanism cannot be described as
cooperative. This deviation from linearity has been described by Adair [47, 48] that
hypothesized that cooperativity is dye saturation dependent and, as such, it is possible to
have polynomial fits when the cooperativity is not 1:1 proportion.
The binding constants values further supported the obtained data considering the binding
mechanism between the dyes and the proteins. According to the obtained data (Fig. 5a,
b) the binding constants are higher for HSA (KB=9.97x105 M; 7.32x105 M; 1.27x106 M
for 8a-c, respectively) than BSA (KB=3.60x105 M; 6.21x105 M; 4.44x105 M, for 8a-c,
respectively) which is an indication that the dyes have better chemical/structural affinity
towards HSA than BSA. Nonetheless, with the exception of compound 8c with HSA, the
binding constants are all on the same order of magnitude (≈105 M). This order of
magnitude is consistent with an intercalative binding, that seems feasible considering the
dyes π system [38]. Moreover, the parabolic fitting observed for both BSA and HSA with
all dyes (Fig. 5a, b) which is an indication of a 1:2 complexation between the dye and the
protein [6].

Fig. 5. Benesi-Hildebrand plots for the binding of squaraine dyes (8a (black),8b (red),8c (blue)) to BSA (a)
and HSA (b).
The comparison of the obtained binding constants in between dyes, makes it is possible
to observe that 8c has more affinity towards HSA than the other two dyes. This maybe
related to two different and concomitant factors. The phenyl substituent provides a
molecular stucture more rigid than in the other dyes, but is also confers an hydrophobic
nature that can better interact with the hidrophobic moety of both proteins [36].
In order to acquire insight knowledge of the binding site affinity of the dyes for the two
proteins, displacement experiments were performed using warfarin and ibuprofen. It is
known that warfarin has a high affinity towards binding site I of both proteins and
ibuprofen to binding site II [49, 50]. Taking this into account an experiment was designed
to understand to which binding site the dyes have preference for. The results demonstrate
that the synthesized compounds behave differently depending on the protein used. In the
case of BSA (Fig.6a-c), in warfarin assays, there is a gradual increase in the percentage
of inhibition for compounds 8a and 8c, with a rate of 24% and 38% respectively for the
100 µM concentration of warfarin. For compound 8b only when the highest concentration
of warfarin is used, there is a slight increase in the rate of inhibition (8%). When ibuprofen
is used, compound 8a has an increased inhibition rate, with the opposite effect for
compounds 8b and 8c. These results suggest that compound 8a binds to both BSA sites
while compound 8b and 8c binds only to site I.

In the assays with HSA (Fig. 6d-f) an increase in the % inhibition is observed for all the
dyes, with the addition of warfarin and ibuprofen. Upon addition of 100 µM of warfarin
the fluorescence of the complex dye-protein was inhibited by 39%, 26% and 32% for 8a,
8b and 8c, respectively. For a concentration of 100 µM of ibuprofen the inhibition rate is
57%, 38% and 45% for 8a, 8b and 8c, respectively. These results lead to the conclusion
that all dyes bind to both HSA binding sites. Since the dyes demonstrated the ability to
bind to both sites, an experiment was carried out, where there was a simultaneous
blocking of the sites, using warfarin and ibuprofen at 50 µM. It is possible to observe that
blocking the two sites leads to more pronounced decrease in the fluorescence intensity
than when blocking only one binding site. For compound 8a, with HSA, there is an
inhibition rate of 66%, for 8b 54% and for compound 8c 42%. It is interesting to notice
that despite the use of both blocking agents in a concentration much higher (100 µM) than
the one used for the dyes (2 µM), the fluorescence emission is still verified. This is an
indication that not only the synthetized dyes can bind to both sites, but also that their
binding affinity is higher than the inhibitors usually used to block the sites.
Fig. 6. Inhibition rate of fluorescence intensity for the dye/BSA complex (a-c, corresponding respectively
to compound 8a-c) and dye/HSA complex (d-f, corresponding respectively to compound 8a-c) by the
addition of site-specific markers (warfarin and ibuprofen, 2-100 μM).

3.5. Dye-Protein Sensing System
Despite the different affinities towards the two used proteins all dyes revealed a consistent
linear trend between the protein concentration and the maximum fluorescence intensity
(Fig. 3d and 4d) that allowed the development of an efficient sensing system in PBS. In
Table 3 is possible to see that the DL and QL of both proteins changes with the dye. The
dye that has the lowest sensing parameters is 8a, followed by 8c and the 8b. The values
here determined are in agreement to the ones previously reported in the literature [43, 51].
Upon the determination of the DL and QL using the fluorescence intensity a similar
procedure was performed using the fluorescence lifetime (Table 3). It was found that all
the dyes had a mono-exponential decay, which is consistent with these organic dyes [25].
Moreover, 8c had the lowest lifetime (0.22 ns) when compared to 8a (0.42 ns) and 8b
(0.34 ns) (Table S2, Supplementary Data), which was to be expected considering the
previous calculus of the excited and ground-state dipole moment. On the other hand the
complex dye-protein had a two-exponential lifetime decay profile for all dyes in both
proteins. Interestingly when in contact with increasing amounts of BSA and HSA, the
medium lifetime of all dyes exhibit a similar trend than the one previously observed for
fluorescence intensity that could also be linearized in order to obtain the sensing
parameters (Fig. 7, Table 3). The 8a binding to BSA and HSA resulted in a fluorescence
lifetime increase of 60% and 54%, respectively. On the other hand, 8b had an increase of
65% and 59% and 8c had a boost of 85% and 70%, respectively. This lifetime behavior
is consistent to the one observed by Markova et al. (2013) [40], where squaraine dyes
with different substituents had a distinct lifetime increase when binding to the HSA
protein. The DL and QL obtained through the linearization of the lifetime with the protein
concentration is slightly lower and has a better sensitivity. This was to be expected, since
the fluorescence intensity measurements are more prone to fluctuations that can affect the
method precision. Nonetheless they are in the same order of magnitude, further
confirming that both techniques effectively reflect the relationship between the proteins
concentration and the dyes and can be used indiscriminately.
Table 3. Values of detection limit (DL), quantification limit (QL) and sensitivity of the sensitizing dyes
(8a- c) with BSA and HSA, calculated using the fluorescence intensitya and the medium lifetimeb.
BSA
Dyes
DLa
QLa
Sensitivitya
DLb
QLb
Sensitivityb

66
199
719
567
2.19x105
1.20x105
2.45x104
42
43
46
129
133
140
1.75x102
1.84x102
3.67x102
8a
8b
8c
237
187
HSA
Dyes
DLa
QLa
Sensitivitya
DLb
QLb
Sensitivityb
62
199
1.46x105
52
157
1.42x102
8a
8b
8c
156
152
474
461
7.77x104
1.11x104
34
42
104
126
1.44x102
1.50x102
Fig. 7. Fluorescence lifetime of compounds 8a (black), 8b (red) and 8c (blue) in PBS with varied BSA (a)
and HSA (b) concentrations (0-3.5 µM) for a fixed dye concentration of 2 µM.
4. Conclusions
Three squaraine dyes containing moieties derived from barbituric acid have been
synthesized and their potential as probes for HSA and BSA detection was studied. The
dyes have high absorption and emission efficiencies at wavelengths in the Vis/NIR region
with high extinction coefficients with an order of magnitude of 105 M-1 cm-1. The Zeta
potential values reveal that the compounds here presented, are quite stable in aqueous
media, unlike what has been previously described for other squaraine dyes. The sensing
ability of these new dyes was tested towards BSA and HSA proteins. According to the
obtained data the synthesized compounds form a complex with the proteins that result in
a fluorescence intensity enhancement with increasing protein amounts. Furthermore, the

dye-protein complex is responsible for an up rise of a new Gaussian band in a different
wavelength of the dye. Furthermore, the binding constants show that the dyes have a
strong interaction and high affinity for both proteins. Regarding the study on the binding
sites of dyes to BSA and HSA proteins we can conclude that dye 8a binds to both BSA
sites while dyes 8b and 8c binds only to site I, and that all dyes bind to both HSA binding
sites. The fluorescence and lifetime behavior of all dyes in contact with the proteins could
be linearized, which allowed the determination of DL and QL using both techniques. This
feature makes these barbiturate dyes particularly appealing for bioimaging applications
using the fluorescence lifetime and two-photon excited fluorescence techniques.
Author Statement
The authors and their involvement on the work are: Vanessa S. D. Gomes performed of the chemical
and photochemical experiments and data analysis, contributed with the data discussion and wrote part of
the original draft of the manuscript; Helena M.R. Gonçalves performed the photochemical assays, played
a role with the data discussion and wrote part the original draft of the manuscript; Renato E. Boto and
Paulo Almeida acquired the NMR data and revised the manuscript; Lucinda V. Reis contributed with
manuscript writing and revision, with the conception and study design of organic chemistry synthesis, with
the analysis and discussion of spectroscopic and photochemical data, and critically revised the manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
We acknowledge the European Investment Funds by FEDER/COMPETE/POCI under
project POCI-01-0145-FEDER-007491 (CICS-UBI) and Funds by FCT – Portuguese
Foundation for Science and Technology, under the projects UID/QUI/UI0616/2019 (CQ-
VR). This work was also supported by funds from the Health Sciences Research Center
(CICS-UBI) through National Funds by FCT – Foundation for Science and Technology
(UID/Multi/00709/2019). H.M.R. Gonçalves was funded by project PTDC/BTM-
MAT/30858/2017. FEDER funds through the POCI and by National Funds through FCT
- Foundation for Science and Technology under the NORTE-01-0145-FEDER-030858
and by the European Union (FEDER funds through Compete) and National Funds

(FCT/MCTES) through Project UID/QUI/50006/2019. FCT is also acknowledged for the
PhD grant UMINHO/BD/43/2016 to Vanessa S. D. Gomes.
References
1.
Levitt, D.G. and M.D. Levitt, Human serum albumin homeostasis: a new look at the roles of synthesis,
catabolism, renal and gastrointestinal excretion, and the clinical value of serum albumin measurements.
International
journal
of
general
medicine,
2016.
9:
p.
229-255
DOI:
https://doi.org/10.2147/IJGM.S102819.
2.
3.
4.
Barbero, N., C. Butnarasu, S. Visentin, and C. Barolo, Squaraine Dyes: Interaction with Bovine Serum
Albumin to Investigate Supramolecular Adducts with Aggregation-Induced Emission (AIE) Properties.
Chemistry – An Asian Journal, 2019. 14(6): p. 896-903 DOI: https://doi.org/10.1002/asia.201900055.
Zhang, Y.-Z., Q.-F. Yang, H.-Y. Du, Y.-L. Tang, G.-Z. Xu, and W.-P. Yan, Spectroscopic Investigation
on the Interaction of a Cyanine Dye with Serum Albumins. Chinese Journal of Chemistry, 2008. 26(2):
p. 397-401 DOI: https://doi.org/10.1002/cjoc.200890076.
Luo, Z., B. Liu, K. Zhu, Y. Huang, C. Pan, B. Wang, and L. Wang, An environment-sensitive fluorescent
probe for quantification of human serum albumin: Design, sensing mechanism, and its application in clinical
diagnosis of hypoalbuminemia. Dyes and Pigments, 2018. 152: p. 60-66 DOI:
https://doi.org/10.1016/j.dyepig.2018.01.033.
5.
6.
7.
8.
9.
Tong, J., T. Hu, A. Qin, J.Z. Sun, and B.Z. Tang, Deciphering the binding behaviours of BSA using ionic
AIE-active fluorescent probes. Faraday Discussions, 2017. 196(0): p. 285-303 DOI:
https://doi.org/10.1039/C6FD00165C.
Jurek, K., J. Kabatc, and K. Kostrzewska, Fluorescent amino-substituted squaraine probes for bovine
serum
https://doi.org/10.1111/cote.12264.
Huang, S., F. Li, C. Liao, B. Zheng, J. Du, and D. Xiao, A selective and sensitive fluorescent probe for the
determination of HSA and trypsin. Talanta, 2017. 170: p. 562-568 DOI:
https://doi.org/10.1016/j.talanta.2017.01.034.
albumin.
Coloration
Technology,
2017.
133(2):
p.
170-177
DOI:
Li, P., Y. Wang, S. Zhang, L. Xu, G. Wang, and J. Cui, An ultrasensitive rapid-response fluorescent probe
for highly selective detection of HSA. Tetrahedron Letters, 2018. 59(14): p. 1390-1393 DOI:
https://doi.org/10.1016/j.tetlet.2018.02.065.
Lee, S., D.-B. Sung, S. Kang, S. Parameswaran, J.-H. Choi, J.S. Lee, and M.S. Han, Development of
Human Serum Albumin Selective Fluorescent Probe Using Thieno[3,2-b]pyridine-5(4H)-one Fluorophore
Derivatives.
Sensors
(Basel,
Switzerland),
2019.
19(23):
p.
5298
DOI:
https://doi.org/10.3390/s19235298.
10.
11.
Jing, X., C. Xiaozheng, Y. Shuqi, M. Zhaohui, W. Wei, and Z. Wenbin, Fluorescent Probes for Detection
of Protein: From Bench to Bed. Protein & Peptide Letters, 2018. 25(6): p. 548-559 DOI:
http://dx.doi.org/10.2174/0929866525666180531080624.
Liu, C., W. Yang, Q. Gao, J. Du, H. Luo, Y. Liu, and C. Yang, Differential recognition and quantification
of HSA and BSA based on two red-NIR fluorescent probes. Journal of Luminescence, 2018. 197: p. 193-
199 DOI: <https://doi.org/10.1016/j.jlumin.2018.01.021.

12.
13.
Sun, Q., W. Wang, Z. Chen, Y. Yao, W. Zhang, L. Duan, and J. Qian, A fluorescence turn-on probe for
human (bovine) serum albumin based on the hydrolysis of a dioxaborine group promoted by proteins.
Chemical Communications, 2017. 53(48): p. 6432-6435 DOI: https://doi.org/10.1039/C7CC03587J.
Wang, N., C. Xin, Z. Li, G. Zhang, L. Bai, Q. Gong, C. Xu, X. Han, C. Yu, L. Li, and W. Huang, A
reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-
induced
emission.
Dyes
and
Pigments,
2019.
163:
p.
425-432
DOI:
https://doi.org/10.1016/j.dyepig.2018.12.027.
14.
Yu, C., X. Li, F. Zeng, F. Zheng, and S. Wu, Carbon-dot-based ratiometric fluorescent sensor for detecting
hydrogen sulfide in aqueous media and inside live cells. Chemical Communications, 2013. 49(4): p. 403-
405 DOI: https://doi.org/10.1039/C2CC37329G.
15.
16.
Lakowicz, J.R., Topics in Fluorescence Spectroscopy: Principles. 2006: Springer US.
Suzuki, Y. and K. Yokoyama, Development of Functional Fluorescent Molecular Probes for the Detection
of Biological Substances. Biosensors, 2015. 5(2): p. 337-363 DOI: https://doi.org/10.3390/bios5020337.
Friães, S., A.M. Silva, R.E. Boto, D. Ferreira, J.R. Fernandes, E.B. Souto, P. Almeida, L.F.V. Ferreira,
and L.V. Reis, Synthesis, spectroscopic characterization and biological evaluation of unsymmetrical
aminosquarylium cyanine dyes. Bioorganic & Medicinal Chemistry, 2017. 25(14): p. 3803-3814 DOI:
https://doi.org/10.1016/j.bmc.2017.05.022.
17.
18.
19.
Fabian, J., H. Nakazumi, and M. Matsuoka, Near-infrared absorbing dyes. Chemical Reviews, 1992.
92(6): p. 1197-1226 DOI: https://doi.org/10.1021/cr00014a003.
Liu, L., J. Chen, Z. Ku, X. Li, and H. Han, Unsymmetrical squaraine sensitizers containing auxiliary
arylamine donor for NIR-harvesting on dye-sensitized solar cell. Dyes and Pigments, 2014. 106: p. 128-
135 DOI: https://doi.org/10.1016/j.dyepig.2014.03.008.
20.
21.
22.
23.
Matsui, M., Y. Haishima, Y. Kubota, K. Funabiki, J. Jin, T.H. Kim, and K. Manseki, Application of
benz[c,d]indolenine-based unsymmetrical squaraine dyes to near-infrared dye-sensitized solar cells. Dyes
and Pigments, 2017. 141: p. 457-462 DOI: https://doi.org/10.1016/j.dyepig.2017.02.024.
Weiss, D.S. and M. Abkowitz, Organic Photoconductors, in Springer Handbook of Electronic and
Photonic Materials, S. Kasap and P. Capper, Editors. 2017, Springer International Publishing: Cham.
p. 1-1 DOI: https://doi.org/10.1007/978-3-319-48933-9_37.
Law, K.-Y. and F.C. Bailey, Squaraine chemistry. A new approach to symmetrical and unsymmetrical
photoconductive squaraines. Characterization and solid state properties of these materials. Canadian
Journal of Chemistry, 1993. 71(4): p. 494-505 DOI: https://doi.org/10.1139/v93-070.
Lima, E., O. Ferreira, V.S.D. Gomes, A.O. Santos, R.E. Boto, J.R. Fernandes, P. Almeida, S.M.
Silvestre, and L.V. Reis, Synthesis and in vitro evaluation of the antitumoral phototherapeutic potential of
squaraine cyanine dyes derived from indolenine. Dyes and Pigments, 2019. 167: p. 98-108 DOI:
https://doi.org/10.1016/j.dyepig.2019.04.007.
24.
25.
Serpe, L., S. Ellena, N. Barbero, F. Foglietta, F. Prandini, M.P. Gallo, R. Levi, C. Barolo, R. Canaparo,
and S. Visentin, Squaraines bearing halogenated moieties as anticancer photosensitizers: Synthesis,
characterization and biological evaluation. European Journal of Medicinal Chemistry, 2016. 113: p. 187-
197 DOI: https://doi.org/10.1016/j.ejmech.2016.02.035.
Martins, T.D., M.L. Pacheco, R.E. Boto, P. Almeida, J.P.S. Farinha, and L.V. Reis, Synthesis,
characterization and protein-association of dicyanomethylene squaraine dyes. Dyes and Pigments, 2017.
147: p. 120-129 DOI: https://doi.org/10.1016/j.dyepig.2017.07.070.

26.
Pisoni, D.S., M.P. de Abreu, C.L. Petzhold, F.S. Rodembusch, and L.F. Campo, Synthesis,
photophysical study and BSA association of water-insoluble squaraine dyes. Journal of Photochemistry
and
Photobiology
A:
Chemistry,
2013.
252:
p.
77-83
DOI:
https://doi.org/10.1016/j.jphotochem.2012.11.013.
27.
28.
29.
Welder, F., B. Paul, H. Nakazumi, S. Yagi, and C.L. Colyer, Symmetric and asymmetric squarylium
dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis. Journal of
Chromatography B, 2003. 793(1): p. 93-105 DOI: https://doi.org/10.1016/S1570-0232(03)00367-2.
Butnarasu, C., N. Barbero, C. Barolo, and S. Visentin, Squaraine dyes as fluorescent turn-on sensors for
the detection of porcine gastric mucin: A spectroscopic and kinetic study. Journal of Photochemistry and
Photobiology B: Biology, 2020. 205: p. 111838 DOI: https://doi.org/10.1016/j.jphotobiol.2020.111838.
Rongxing Yi, P.D., Fangrui Lin, Binglin Shen, Zhigang Yang, Yihua Zhao, Liang Hong, Ying He,
Rui Hu, Jun Song, Junle Qu, and Liwei Liu, Fluorescence enhancement of small squaraine dye and its
two-photon excited fluorescence in long-term near-infrared I&II bioimaging Optics Express, 2019. 27(9):
p. 12360-12372 DOI: https://doi.org/10.1364/OE.27.012360.
30.
Pardal, A.C., S.S. Ramos, P.F. Santos, L.V. Reis, and P. Almeida, Synthesis and Spectroscopic
Characterisation of N-Alkyl Quaternary Ammonium Salts Typical Precursors of Cyanines. Molecules : A
Journal of Synthetic Chemistry and Natural Product Chemistry, 2002. 7(3): p. 320-330 DOI:
https://doi.org/10.3390/70300320.
31.
32.
33.
34.
35.
36.
Graça, V.C., M.S. Silva, L.V. Reis, F. Sousa, P. Almeida, J.A. Queiroz, and P.F. Santos,
Ethylenediamine-Derived Chromatographic Ligand to Separate BSA, Lysozyme, and RNase A.
Chromatographia, 2014. 77(21): p. 1529-1537 DOI: https://doi.org/10.1007/s10337-014-2749-y.
Serrano, J.L., P.F. Soeiro, M.A. Reis, R.E.F. Boto, S. Silvestre, and P. Almeida, Synthesis and process
optimization of symmetric and unsymmetric barbiturates C5-coupled with 2,1-benzisoxazoles. Molecular
Diversity, 2019 DOI: https://doi.org/10.1007/s11030-019-09937-4.
Tatarets, A.L., I.A. Fedyunyaeva, E. Terpetschnig, and L.D. Patsenker, Synthesis of novel squaraine
dyes and their intermediates. Dyes and Pigments, 2005. 64(2): p. 125-134 DOI:
https://doi.org/10.1016/j.dyepig.2004.05.003.
Shi, Y., C. Li, S. Liu, Z. Liu, J. Zhu, J. Yang, and X. Hu, Facile synthesis of fluorescent carbon dots for
determination of curcumin based on fluorescence resonance energy transfer. RSC Advances, 2015. 5(79):
p. 64790-64796 DOI: https://doi.org/10.1039/C5RA13404H.
Ogunsipe, A., D. Maree, and T. Nyokong, Solvent effects on the photochemical and fluorescence
properties of zinc phthalocyanine derivatives. Journal of Molecular Structure, 2003. 650(1): p. 131-140
DOI: https://doi.org/10.1016/S0022-2860(03)00155-8.
Patil, N.R., R.M. Melavanki, S.B. Kapatkar, N.H. Ayachit, and J. Saravanan, Solvent Effect on
Absorption and Fluorescence Spectra of Three Biologically Active Carboxamides (C1, C2 and C3).
Estimation of Ground and Excited State Dipole Moment from Solvatochromic Method Using Solvent
Polarity Parameters. Journal of Fluorescence, 2011. 21(3): p. 1213-1222 DOI:
https://doi.org/10.1007/s10895-010-0800-4.
37.
Małgorzata Maciążek-Jurczyk, J.R.-Z., Anna Sułkowska, Comparative Analysis of KP-HSA Complex
by Spectroscopic Methods. Acta Physica Polonica A, 2013. 123(4): p. 673-680 DOI:
http://doi.org/10.12693/APhysPolA.123.673.

38.
Jana, B., S. Senapati, D. Ghosh, D. Bose, and N. Chattopadhyay, Spectroscopic Exploration of Mode of
Binding of ctDNA with 3-Hydroxyflavone: A Contrast to the Mode of Binding with Flavonoids Having
Additional Hydroxyl Groups. The Journal of Physical Chemistry B, 2012. 116(1): p. 639-645 DOI:
https://doi.org/10.1021/jp2094824.
39.
40.
41.
42.
43.
Baird, R.B., A. D.Eaton, and E. W.Rice, Standard Methods for the Examination of Water and Wastewater
(SMWW). 2017, American Public Health Association, American Water Works Association, Water
Environment Federation DOI: https://doi.org/10.2105/SMWW.2882.216.
Markova, L.I., E.A. Terpetschnig, and L.D. Patsenker, Comparison of a series of hydrophilic squaraine
and cyanine dyes for use as biological labels. Dyes and Pigments, 2013. 99(3): p. 561-570 DOI:
https://doi.org/10.1016/j.dyepig.2013.06.022.
Bricks, J.L., Y.L. Slominskii, I.D. Panas, and A.P. Demchenko, Fluorescent J-aggregates of cyanine dyes:
basic research and applications review. Methods and Applications in Fluorescence, 2017. 6(1): p. 012001
DOI: https://doi.org/10.1088/2050-6120/aa8d0d.
Xu, Y., Z. Li, A. Malkovskiy, S. Sun, and Y. Pang, Aggregation Control of Squaraines and Their Use as
Near-Infrared Fluorescent Sensors for Protein. The Journal of Physical Chemistry B, 2010. 114(25): p.
8574-8580 DOI: 10.1021/jp1029536.
Wang, G., W. Xu, Y. Guo, and N. Fu, Near-infrared squaraine dye as a selective protein sensor based on
self-assembly. Sensors and Actuators B: Chemical, 2017. 245: p. 932-937 DOI:
https://doi.org/10.1016/j.snb.2017.01.172.
44.
45.
Koutek, B., Dipole moments in excited state. Statistical investigation of methods employing
solvatochromism. 1978 DOI: https://doi.org/10.1135/cccc19782368
Jisha, V.S., K.T. Arun, M. Hariharan, and D. Ramaiah, Site-Selective Binding and Dual Mode
Recognition of Serum Albumin by a Squaraine Dye. Journal of the American Chemical Society, 2006.
128(18): p. 6024-6025 DOI: https://doi.org/10.1021/ja061301x.
46.
47.
48.
49.
Silverstein, T.P., Principles of Physical Biochemistry (van Holde, Kersal E.; Johnson, W. Curtis; Ho, P.
Shing). Journal of Chemical Education, 1999. 76(4): p. 474 DOI: https://doi.org/10.1021/ed076p474.1.
Stefan, M.I. and N. Le Novère, Cooperative Binding. PLOS Computational Biology, 2013. 9(6): p.
e1003106 DOI: https://doi.org/10.1371/journal.pcbi.1003106.
Adair, G.S., W.t.c.o.A.V. Bock, and J. H. Field, The hemoglobin system: VI. The oxygen dissociation curve
of hemoglobin. Journal of Biological Chemistry, 1925. 63(2): p. 529-545.
Nicoletti, F.P., B.D. Howes, M. Fittipaldi, G. Fanali, M. Fasano, P. Ascenzi, and G. Smulevich,
Ibuprofen Induces an Allosteric Conformational Transition in the Heme Complex of Human Serum Albumin
with Significant Effects on Heme Ligation. Journal of the American Chemical Society, 2008. 130(35): p.
11677-11688 DOI: https://doi.org/10.1021/ja800966t.
50.
51.
Fielding, L., S. Rutherford, and D. Fletcher, Determination of protein–ligand binding affinity by NMR:
observations from serum albumin model systems. Magnetic Resonance in Chemistry, 2005. 43(6): p. 463-
470 DOI: https://doi.org/10.1002/mrc.1574.
Choudhury, R., S. Rajeshbhai Patel, and A. Ghosh, Selective detection of human serum albumin by near
infrared emissive fluorophores: Insights into structure-property relationship. Journal of Photochemistry
and
Photobiology
A:
Chemistry,
2019.
376:
p.
100-107
DOI:
https://doi.org/10.1016/j.jphotochem.2019.02.038.