# Optical Biomedical Diagnostics: Sensors with  Optical Response Based on Two-Photon Excited  Luminescent Dyes for Biomolecules Detection

Authors: V. M. Yashchuk; S. M. Yarmoluk; V. Yu. Kudrya; M. Yu. Losytskyy; V. P. Tokar; V. M. Kravchenko; V. B. Kovalska; A. O. Balanda; D. V. Kryvorotenko
Journal: Advances in Optical Technologies
 (2008)

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## Abstract

The spectral properties of novel styryl dyes developed for the biomacromolecules (such as DNA) detection and imaging were investigated. The energy structures of dye molecules were examined. The spectral data prove that dyes aggregate and interact with DNA. The essential increase of the fluorescence intensity of dyes in the presence of DNA was observed. The photostability and phototoxic influence on the DNA of several styryl dyes were studied by analyzing absorption, fluorescence, and phosphorescence spectra of these dyes and dye-DNA systems. Changes of the optical density value of dye-DNA solutions caused by the irradiation were fixed in the DNA and dye absorption wavelength regions. Fluorescence emission of dye-DNA complexes upon two-photon excitation at wavelength 1064 nm with the 20-nanosecond pulsed YAG:Nd3+ laser and at 840 nm with the 90 famtosecond pulsed Ti:sapphire laser was registered. The values of two-photon absorption cross-sections of dye-DNA complexes were evaluated.


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## Body

## 1. Introduction

Fluorescent probes are widely used in biological
and medical studies for the detection of biological molecules and fluorescent
imaging of biological objects [1]. The two important properties of these probes
permitting their application for fluorescent detection are (1) strong affinity
of the probe binding to the biological molecule of interest, and (2) sharp
enhancement of the fluorescence intensity of the probe upon such binding.
Commonly, the fluorescence of the probes is excited with either ultraviolet
(UV) or visible light, so that the probe molecule is excited by one absorbed
photon (single photon excitation). The single photon excitation (SPE) approach
has several disadvantages. First, the light in UV and visual regions (250–700 nm) cannot penetrate deep into the biological tissue, being both absorbed and
scattered, and hence is not useful for the tissue fluorescent scanning. The
probes with absorption in the region 700–1000 nm, where the majority of
biological objects are transparent, are not easy to design, and if designed
such probes would fluoresce in infrared (IR) region, which is not convenient
for observation. Second, using SPE
(especially with UV light) causes significant damage of the studied biological
objects, not only in the point of detection, but also on the whole way of the
beam. Third, when used for three-dimensional (3D) scanning microscopy, SPE
results in rather restricted resolution in the two directions perpendicular to
the beam and in very poor resolution along the direction parallel to the beam
propagation.The problems mentioned above could be overcome
with the help of two-photon excitation (TPE) of the fluorescent probe. By TPE,
which is a nonlinear process, one probe molecule is excited by two photons
absorbed simultaneously (or, to be more precise, in a very short period of
time). Hence, the TPE efficiency is proportional to the square of the
excitation beam intensity, which permits the localization of the excitation
only at the focusing point, and not on the whole way of the beam. Moreover, the
wavelength of the exciting beam for TPE is about 2 times higher than this for
SPE beam for the same probe, which permits excitation of visual fluorescence in
the transparency region of biological objects (700–1000 nm), besides, the
higher excitation wavelength means the lower scattering of excitation beam. Thus,
using two-photon excitation of fluorescent probes in procedures of biological
objects detection permits deeper penetration of exciting beam into the tissue, excitation
of visual fluorescence in near infrared spectral region, where the majority of
biological objects are transparent, decreased photodamage of the studied object,
and obtaining of well-resolved three-dimensional image of biological object [2, 3].The efficiency of TPE of a molecule at the
constant excitation intensity is determined by the value of two-photon
absorption cross-section. The higher the value of two-photon absorption
cross-section is, the lower excitation intensity is required to obtain the same
fluorescence intensity. And lower excitation intensity means (1) cheaper and
simpler equipment needed and (2) lower photodamage of the sample. Thus, the
successful application of the TPE for the detection of biological molecules
requires the fluorescent probes with (1) high affinity to the biological
molecules of interest, and (2) high value of the two-photon absorption cross
section. Unfortunately, such probes are not developed at all for the moment.It is important that the luminescent biomedical
probe-sensor could be applied to the study of the living cells (e.g., with the
fluorescent microscopy). It was shown in a number of papers that the widely
used fluorescent probes photochemically destroy the biological objects (DNA,
RNA, etc.). So probes have to be nonphototoxic. Besides, the probe should be
photostable so that the biomolecule could be studied for the long enough period
of time without the probe damage. The phototoxic influence of the dye molecule
on the DNA or RNA can take place either directly via the excitation energy transfer from the dye to the
nucleotide bases or indirectly via the third molecule (e.g., by the triplet
excitation energy transfer to the oxygen molecule resulting in the generation
of the toxic singlet oxygen [4, 5]). It was shown [6–15] that the absorption
bands (connected with the first electronic transition) of the DNA, RNA, and
nucleotide bases are located in the near UV spectral region with the maximum near 260 nm.
At the same time, the corresponding absorption bands of the majority of dyes
used as luminescent probes are located in the visual spectral region (>400 nm) [1]. The fact that the first excited singlet and triplet energy
levels of dyes are situated essentially lower than correspondent levels of any
nucleotides does not allow the excitation energy transfer from the dye to the
DNA thus making impossible the direct phototoxic influence of the dye on the
DNA. Nevertheless, the molecules such as porphyrines or other pigments that
destroy the DNA indirectly by the generation of singlet oxygen are well known
and used in the photodynamic therapy.In the presented work we study the styryl dyes as
the probes for the TPE fluorescent detection of DNA. Styryl dyes are known as
having high two-photon absorption cross-section values [2, 16]; one of the
benzothiazolium styryls was shown to give strong fluorescence upon TPE by
1064 nm irradiation [17]. On the other hand, significant fluorescent
response on the dsDNA presence is observed for these dyes [18–21]. Recently
series of monomer and homodimer benzothiazolium styrylcyanines was synthesized
and studied for their efficiency as TPE nucleic acids sensitive dyes. It was
shown that dyes modified with spermine-like linkage/tail group demonstrate
increased sensitivity to DNA [19]. They have low intrinsic emission intensity
and enhance their fluorescence intensity up to three orders of magnitude in
presence of DNA. We believe that the influence of spermine-like linkage/tail
group on the dye sensitivity to DNA is due to the additional interaction of the
positively charged nitrogen atom with the DNA. Complexes of studied dyes with
DNA also demonstrate intensive emission upon the TPE [19].In the presented work, two-photon excited
fluorescence of monomer and homodimer styrylcyanine dyes based on
benzothiazole, naphtathiazole, benzoimidazole, pyridinium, quinoline, and
4-oxo-thieno[2,3-d]pyrimidinium heterocyclic residues and containing charged
spermine-like linkage/tail groups was studied in the presence of DNA. Besides,
for several dyes the fluorescent and phosphorescent properties at low
temperature, as well as photochemical stability and phototoxicity to the DNA,
were investigated.


## 2. Methodology

The
total DNA from chicken erythrocytes was purchased from Sigma-Aldrich Inc (St. Louis, Mo, USA). The dye Dst-MdO (Figure1) was obtained by
the boiling of quaternary salt and p-dimethylaminobenzaldehyde
in acetic anhydride. Dyes Bos-1, DBos-21, DBos-24, DBos-25, DBos-28, and DBos-30
(Figure 1) were obtained as described in [19]. Dyes S-3, S-20, S-33, S-45, and
S-46 (Figure 1) were obtained as described in [21]. Dyes F, Sbt, Sil, Dst-6,
Dst-10, Dbt-10, and Dil-10 (Figure 1) were synthesized according to the
procedures described in [18]. The synthesis of dyes S-48, Tio-1, and S-40 (Figure 1) was described in [20]. The structures of the dyes were confirmed with H1NMR and element analysis.Figure 1
Chemical
structures of the studied dyes.The samples were
prepared in distilled water and 0.05 M TRIS-HCl buffer, pH 8.0. The concentrations of dye and
DNA were, respectively, 10−5 M and 6 × 10−5 M b.p. (base pairs) for the absorption
measurements and photodamage experiment, and 10−4 M
and 6 × 10−4 M b.p., respectively, for the low-temperature
luminescence measurements. For the low-temperature measurements, the prepared
solutions were poured out into the special cell so that the upper surface is
open, and then frozen at the temperature of the boiling nitrogen (77 K). The
excitation beam was directed to the open surface, and from the same surface the
luminescence was registered.The steady state
fluorescence and phosphorescence measurements were performed using
laboratory-designed equipment; absorption spectra were recorded with the help
of a Specord UV-VIS spectrophotometer. The photodamage of dyes and the DNA+dye
systems was performed by exposition of the corresponding solution to the
visible spectrum irradiation of the 1 kW Hg-lamp.The measurements were carried out at
77 K and ambient temperatures.The
TPE fluorescence measurements of the dyes in DNA presence were performed by
using the 1064 nm irradiation of the 20 nanoseconds pulsed YAG: Nd3+ laser as the excitation source. Experimental setup was used and two-photon
absorption cross-section  values calculation performed as described in [19].During
the TPE fluorescence measurements at wavelength 840 nm, a Ti:Sapphire laser (Mira Optima 900-F, Coherent Inc (Santa Clara, CA, USA).) pumped up with the cw Nd:YVO4 laser (Coherent) and generating
90 fs cosec2-shaped pulses with repetition rate 76 MHz was used.
Parameters of laser were monitored by autocorrelator (APE Autocorrelator mini) and power meter (Field Master GS,
LM-10, Coherent). The fluorescence light was detected at
right angle as it passed the telecentric system of two lenses and the entrance
slit (100 μ) of the spectrograph (500 mm Imaging Spectrograph SP-2558, Princeton Instruments - Acton (Acton, Mass, USA)). A CCD camera (CCD-Spec-10:256E/TEPLUS 1024 × 256 Open-electrode,
Marconi CCD 30-11) was used as a detector of TPE-fluorescence.


## 3. Results and Discussion

### 3.1. The study of SPE and TPE Fluorescence of
Dyes and Dye-DNA Complexes

Styryl dyes are known to be efficiently excited via the TPE [2, 16, 17]. Thus, the study
of the styryl dyes as possible fluorescent probes for the DNA was performed by
us as follows. First, wide series of styryl dyes based on different
heterocyclic residues were synthesized and studied as the SPE fluorescent
probes for DNA detection. Second, the dyes demonstrating both high value of
fluorescence intensity in DNA presence and sharp emission intensity increase
upon binding to DNA were studied in the TPE experiment.Recently, the series of novel monomer and
homodimer styrylcyanine dyes based on benzothiazole, naphtathiazole,
benzoimidazole, pyridinium, quinoline, and 4-oxo-thieno (2,3-d) pyrimidinium
heterocyclic residues (Figure1) was elaborated, synthesized, studied, and partly
reported [18, 20, 21]. The dyes demonstrated high DNA sensitivity, namely,
fluorescence intensity of dyes upon SPE enhanced up to thousand times upon
binding with DNA (Figure 2). The study of the dyes absorption and fluorescence
spectra at room temperature in water buffer solution as well as in the DNA
presence revealed that the spectra of majority of the studied dyes are
connected with the absorption and fluorescence of the separate styryl
chromophores. Meanwhile, for the styryl homodimer dyes containing the
spermine-like linker (DBos-21, DBos-24, DBos-25, DBos-28, DBos-30, S-45, and
S-46) the band corresponding to the aggregate of the styryl chromophores is
dominating in the absorption spectrum of the free dyes, while the addition of
the DNA to the solution leads to the decrease of the aggregate band and growth
of the monomer ones. At the same time, the mentioned aggregates were found to
be nonfluorescent at room temperature, thus the fluorescence spectra of the
mentioned dimers, as well as of all the studied dyes (both free and bound to
DNA), correspond to the emission of the separate styryl chromophores [19].
Hence, the strongly enhanced fluorescence of the studies styryls in DNA
presence is the emission of the styryl chromophores, bound to the DNA molecule.
The fixation of the separate chromophore on DNA (via either intercalation or
groove-binding) leads to the decrease in the rate constant of the
nonfluorescent deactivation of the singlet electronic excitation of the dye,
and thus to the increase of the dye fluorescence quantum yield [22].Figure 2
SPE fluorescence spectra of dyes in free state
and in DNA
presence.In order to enhance the dye-to-DNA affinity, some
of the studied dyes contained the spermine-like tail or linking group. It was
shown by us earlier [23] that the spermine-like tail group enhances the
constant of dye to DNA binding equilibrium (Kb)
in about three times (1.8 × 104 M−1 for Sbt and about 6 × 104 M−1 for S-20). We believe this enhancement to be connected with the
additional interaction of the charged spermine tail group with the DNA. It
should be mentioned that the cyanine dyes sensitive to DNA possess the Kb values of the same order
of magnitude as the benzothiazole styryl dyes Sbt and S-20 [24], though for
some of the cyanines (e.g., TO) the value of Kb was reported to be about 106 M−1[25].


### 3.2. The Effect of Dyes Aggregations on
Their Phosphorescent Properties

Phosphorescence
excitation and emission spectra of benzothiazole homodimer dyes solutions were
measured (Figure3). The energy of the lowest triplet level of dyes is about 1.85 eV (14900 cm−1). This value is less than the value of the lowest
triplet level among DNA bases thus the triplet electron excitation energy
transfer from DNA to the dye can take place and is still under study. The
phosphorescence lifetime was evaluated and amounted about 20 milliseconds.Figure 3
Fluorescence and phosphorescence emission
and excitation spectra of DBos-30 water solution at liquid
nitrogen temperature (C = 10−4 M).Fluorescence excitation
and emission spectra of benzothiazole homodimer dyes solutions at liquid
nitrogen temperature were obtained. Also fluorescence of dyes’ aggregates at
liquid nitrogen temperature was registered while at room temperature these
aggregates are virtually nonfluorescent. As far as fluorescence excitation
spectra of aggregates coincide with phosphorescence excitation spectra of dyes,
it was concluded that, namely, aggregates are mainly responsible for the
phosphorescence (Figure3). In order to exclude the interaction between the
dye-DNA complex and the molecular oxygen in the water solution the studying of
the dyes emission in vacuum is under research now.


### 3.3. TPE Fluorescence of Dyes and Dye-DNA Complexes

The TPE study of the fluorescent properties of
the mentioned styryl dyes in the presence of DNA was performed.The dye-DNA complexes revealed intensive emission
upon two-photon excitation (TPE) at wavelength 1064 nm with the 20 nanoseconds
pulsed YAG:Nd3+ laser. Here the numerical results of dyes’ studying
upon the TPE in complexes with DNA are presented. The values of dyes’
two-photon absorption (TPA) cross section δwere
evaluated (see Table 1). The calculations of δwere
performed using the respective value for Rhodamine 6G [26]. As it is seen from
Table 1, the δvalues
of dye-DNA complexes are about 10−50 cm4 s.Table 1
The values of TPA cross sectionδof dyes
in DNA presence upon excitation at wavelength 1064 nm.
Dyeδ, 10−50 cm4⋅ sDyeδ, 10−50 cm4⋅ sS-202.3S331.1Bos-12.2D-1841.1DBos-211.7S400.2DBos-241.4S480.2DBos-251.7Tio10.2DBos-282.1Sil1.7DBos-301.8Dil-101.0S451.4S30.9S460.3S80.4Sbt1.7Din-100.4Dbt100.3S270.8F0.04Rhodamine 6G5.5Dst-60.6Dst-100.6Ds-100.4Dyes from the series with each type of
chromophore in complexes with DNA demonstrated emission upon TPE at 840 nm with
the 90 femtosecond pulsed
Ti:sapphire laser (see Figure4). TPA cross sections δat
wavelength 840 nm are under evaluation now.Figure 4
Fluorescence emission
spectra of dye-DNA water buffer solution and Rhodamine B ethanol
solution upon SPE and TPE (concentration of dyesC = 1.5 × 10−5 M).The possibility of the dye DBos-21 application
for the TPE fluorescent cell staining was studied. HeLa cells were incubated
for 1 hour with DBos-21 (5μM) and imaged using 880 nm excitation of the
femtosecond pulsed Ti:Sapphire laser (Tsunami, Spectra Physics). TPE
fluorescent image of the cells stained with DBos-21 is
shown in  Figure 5.Figure 5
TPE
fluorescent image of the HeLa cells stained with the dye DBos-21.
Fluorescent emission excited with the femtosecond pulsed
Ti:Sapphire laser, excitation wavelength 880 nm.The TPE fluorescent imaging study shows that the
styrylcyanine dimer dye DBos-21 containing the spermine-like linking group
successfully permeates through the living cell membrane. Besides, this dye
demonstrated mostly mitochondria staining, possibly because of high positive
charge of the dye.Thus, the styrylcyanine dimmer dyes containing
the spermine-like linking group could be successfully applied for the TPE
fluorescent imaging of the living cells.


### 3.4. The Study of Dyes Phototoxicity and Photostability

#### 3.4.1. The First Excited Singlet and Triplet Levels of Dyes

For the use of the dyes
as biomedical sensors for the DNA (or RNA) detection and imaging in the study
of the living objects the phototoxic influence of these dyes realized via the excitation energy transfer
from the dye to the DNA has to be absent. To avoid these processes the first
excited singlet and triplet levels of dyes must be situated lower than
correspondent levels of any nucleotide. To check this the optical absorption,
fluorescence, and phosphorescence spectra of the investigated dyes Bos-1, DBos-21,
DBos-24, DBos-25, DBos-28, and DBos-30 as well as correspondent dye+DNA systems
were studied. Some of these spectra (namely, for Bos-1, DBos-24 and DBos-30
dyes) are given in Figure6.The absorption (1,2), fluorescence (3,4), and
phosphorescence (5,6) spectra (3–6 at T = 77 K) of free styryl dyes
(1,3,5) and correspondent systems DNA+dye (2,4,6) of: (a) styryl
monomer Bos-1, (b) dimer DBos-24, and (c) dimer DBos-30.
(a)(b)(c)The absorption spectra
of the dye Bos-1 both in the presence and the absence of the DNA in visible spectral region
consist of the single band with the maxima near 17540 and 18470 cm−1, respectively
(Figure 6(a); curves 1,2). Since this band is the most long-wave band in the visible
spectral region and the corresponding fluorescence spectrum (Figure 6(a); curves 3,4)
obeys the mirror symmetry rule respectively to this absorption band, the latter  should be attributed to the
absorption transition to the first excited singlet level of the dye molecule. The shift between the maxima of the free dye and
the DNA+dye system spectra points to the dye interaction with the DNA. In the
absorption spectra of the dimer dyes DBos-24 and DBos-30 in presence of the DNA
the same band with the maximum near 18000 cm−1 (Figure 6(b); curve 2)
and 17750 cm−1 (Figure 6(c); curve 2), respectively, could be observed.
At the same time, the spectrum of DBos-24 in the DNA presence contains one more
band with the maximum at 22500 cm−1 shifted to the
short-wavelength region relatively to the main band. This short-wavelength band
with the maxima at 22200 and 20800 cm−1, respectively,
dominates in absorption spectra of both DBos-24 and DBos-30 in absence of the
DNA (Figures 6(b); curve 1, and 6(c); curve 1). Taking into account all the above-mentioned
results, the short-wavelength band could be attributed to the aggregates of the
styryl chromophore. Since for the dimer dyes the tendency to form aggregates is
much higher than for corresponding monomers [27], the short-wavelength band is
present in the spectra of DBos-24 and DBos-30 in free state, but is absent in the spectrum of
Bos-1 containing the same chromophore. At the same time, interaction of dimer
dyes with the DNA at low dye to the DNA concentrations ratio generally leads to
the fixation of separate chromophores on the DNA molecule, thus resulting in
decrease in the aggregates concentration in solution [27]. This explains the
decrease in the short-wavelength band contribution to the absorption spectrum
of DBos-24 in presence of the DNA, as well as the disappearance of this band in
the same spectrum of DBos-30.The fluorescence of the
dyes Bos-1, DBos-24, and DBos-30, both in free form and in DNA presence, at the
room temperature was measured in [19]. The maximum of these spectra was
situated near 17000 cm−1 similarly to the spectra
presented at Figures 6(a), 6(b), 6(c) (curves 3,4). It was shown in [19] with the help of fluorescence
excitation spectra that the fluorescence band with the maximum near 17000 cm−1 belongs to the emission of the single styryl chromophores. Thus, the absorption
and fluorescence spectra of the dyes Bos-1, DBos-24, and DBos-30 in DNA presence could be
used for the calculation of the position of the singlet electronic excited
energy level of the nonaggregated chromophore of these dyes, bound to DNA.The
positions of the first excited singlet level of the investigated dyes in
nonaggregated form in the presence of the DNA were obtained by intersection of
the absorption and fluorescence spectra curves and given in Table2.Table 2
Positions of singlet (S) and triplet (T) levels of the
dyes in presence of the DNA, cm−1.
CompoundBos-1DBos-21DBos-24DBos-25DBos-28DBos-30S (293 K)182501818018120181201828017940S (77 K)191001880019530191601868019110T (77 K)147901482014240147301475014890The
values of singlet and triplet energy levels of the nucleotides obtained by us
in [28, 29] are presented in Table 3. The comparison of these data with data
given in Table 2 removes all doubts that even the triplet level of any
nucleotide is situated much upper than the singlet level of any investigated
dye. That is why the irradiation of the dye in the visual spectral region
exciting the first singlet electronic level of the dye cannot be resulted in
the excitation energy transfer from the dye to the DNA.Table 3
Positions of singlet (S1) and triplet (T1) levels of the
nucleotides, cm−1.
CompounddCMPdGMPdTMPdAMPS133090330303353034490T126630263202616025950


#### 3.4.2. Effect of Irradiation of Dyes and the DNA+Dye Systems on Their Optical Properties

It is known that the phototoxic influence of a
luminescent dye probe on the DNA results in the spectral response of the
DNA+dye system. This response is connected with the DNA macromolecule damage
and is observed as the change of the optical density of the DNA absorption band
(i.e., situated at 260 nm and corresponds to
the S0→S1 electronic transition) in the
DNA+dye system under excitation in the dye absorption band (i.e., situated at
400–500 nm and corresponds to its S0→S1 transition). Besides, the
investigation of the optical density changes of the dye band under the free dye
excitation at the wavelength of the same band is the way to study the dye
photostability. The fact that the DNA absorption band connected with S0→S1 transition is located far from
the same band of majority of dyes and the value of optical density of the
absorption band connected with the dye S0→S2 transition (located near
260 nm) is much less than corresponding value of the DNA band gives the
ground to study the dynamics of the absorption bands optical density changes
almost independently for a dye and for the DNA. In our paper, the
investigations results of the absorption spectra of the elaborated styryls and
the respective DNA+dye complexes (that were recorded with the aim to study both
the dye photostability and its phototoxicity influence on the DNA) during the
irradiation of the investigated compounds solutions by visible light of 1 kW
Hg-lamp were described.As it is shown in Figure7 the gradual decrease of D of the absorption spectra bands is
observed for free dyes under the irradiation time increase (curve 1). This
phenomenon is connected with the damage of dyes π-electron systems under irradiation and is the
evidence that Bos-1 and DBos-30 are not photostable in free form. The gradual
decrease of D of the dyes absorption
spectra bands in the DNA presence takes place too but essentially slower than
for free dyes. Moreover, the decrease rate of D of the DNA+DBos-30 system (Figure 7(b); curve 2) is lower than of
the system DNA+Bos-1 (Figure 7(a); curve 2). It means that dimer DBos-30 bound
to the DNA is more photostable than monomer Bos-1 bound to the DNA. Comparing
the dependencies of optical density on irradiation time of investigated styryl
dyes with the same dependence of thiazole orange (TO) dye (Figure 7, curve 4), it is
shown that both Bos-1 and DBos-30 dyes (bound to the DNA) are more photostable
even than TO dye (bound to the DNA). The rise of the dyes photostability under
binding to the DNA occurs, in our opinion, because of the formation of the
tough spatial fixation of a dye molecule after the intercalation of this dye in
the DNA macromolecule. As the result, the DNA macromolecule screens the dye
molecule against external influences and decreases the contacts between the dye
molecules and dissolved molecules of oxygen. The last is transferred into the
singlet excited electronic state under the direct excitation of the dye and
becomes an active oxidant [30]. The results obtained in this paper agree with
the data obtained in [31] on investigations of the cyanine dyes. Since the
constant of binding to the DNA for the dimer is often higher than for the
monomer with the same π-electron system [32, 33], the number of dye molecules bound
with the DNA is higher for the dimer DBos-30 than for the monomer Bos-1. Thus
the DNA macromolecule screens the dimer molecules rather than the monomer
molecules that cause the difference in Bos-1 and DBos-30 photostability in the
DNA presence.The dependence of optical density
(obtained from optical absorption spectra) on irradiation time of
dyes: (a) Bos-1 and (b) DBos-30 (1—free dye, 2—DNA+dye,
3—the DNA maximum 260 nm, 4—TO with the DNA).
(a)(b)The dependencies of the DNA band maximum optical densityD on time of dye irradiation (Figure 7; curve 3)
for the DNA+Bos-1 and DNA+DBos-30 systems were investigated with the aim to
study phototoxic influence of the investigated dyes on the DNA. According to
our experimental data the value of the optical density D of the investigated dyes absorption band
connected with the S0→S2 transition is changed negligible
during all the time of irradiation. As it is shown in Figure 7 the value of D is changed within 3% at 260 nm
out of its intact value that may be connected with these negligible changes of
the dyes S0→S2 band and experimental errors. It
is known that the DNA destruction is either the double strand untwisting or the
strand cutting that is reverse to the hypochromic effect, the complete
untwisting resulting in the 37% increase in D value at 260 nm [34].
That is why the 3%-changes obtained by us cannot be connected with the
significant DNA damage and can be included in experimental errors.So, Bos-1 and DBos-30 dyes are nonphototoxic for the DNA
as the first approximation and can be used as the optical biomedical sensors
for the DNA detection and imaging. On the other hand, the DNA protects these
dyes against the photodamage.As is shown in Figure6(b), the dimer DBos-24 absorption band consists of two bands. Comparing
Figures 6(a) and 6(b) it can be seen that the long-wavelength band (18500 cm−1)
is located close to the monomer Bos-1 band and corresponds to nonaggregated
state. The short-wave band (23100 cm−1), in our opinion, is
connected with aggregates (the similar case was observed for cyanine dyes [35, 36]). Using optical absorption spectra of DBos-24 dye and the DNA+DBos-24
system, the dependencies of the optical density D on irradiation time for these molecular systems are obtained
(Figures 8(a), 8(b)). The aggregates band of the free dye decreased gradually during all
the irradiation time while the nonaggregated dyes band increased slightly at
first (Figure 9). In our opinion, it is connected with the fact that aggregates
are damaged and turned into nonaggregated state. Since the D value of dye DBos-24 decreases by 40–50% after 600 minutes of
irradiation, this dye is more photostable than Bos-1 and DBos-30.The dependence of optical
density (obtained from optical absorption spectra) on irradiation
time of dimer dye DBos-24: (a) free dye, (b) bond to the DNA
(1—at 23100 cm−1, 2—at 18500 cm−1,
3—the DNA maximum at 260 nm).
(a)(b)Figure 9
The
absorption spectra of dimer DBos-24 during irradiation (free dye:
1—intact, 2–210 minutes, 3–540 minutes,
4–600 minutes; dye with DNA: 5—intact,
6–240 minutes, 7–540 minutes).For the DNA+DBos-24
system, the optical density of aggregate band (Figure9) decreases gradually with the irradiation
time increasing but D of
nonaggregated dyes band (in presence of the DNA) remains constant. In our
opinion, it is connected with the fact that nonaggregated dyes intercalate into
the DNA macromolecule while aggregates bind outside the DNA chain. As it was
mentioned above, during the irradiation the molecules of environmental oxygen
are transferred in the singlet excited electronic state and become an active
oxidant [30]. Generated singlet oxygen damages the aggregated dyes that are
outside the DNA chain. The DNA screens nonaggregated dyes that are intercalated
in the DNA macromolecule (between the DNA strands) and protect them from the influence of singlet
oxygen and, therefore, the dyes molecules are not damaged.As it is shown (Figure8(b);
curve 3), the increase (up to by 30%) of D of the DNA absorption spectra bands is observed for the DNA+DBos-24
system under the irradiation time increase. In our opinion, this phenomenon is
caused by the untwisting or cutting of the strands of the DNA macromolecule
which contacts with the molecules of DBos-24 (this phenomenon is reverse to the
hypochromic effect). These results agree with 37% increase for the complete DNA
untwisting [34]. Thus the presence of DBos-24 dye causes the DNA damage, that
is, DBos-24 showed phototoxic influence on the DNA. But dyes phototoxicity
appears not only in the untwisting or cutting of the DNA strands. For the
DNA+Dst-MdO system, the value of optical density of the DNA absorption band
decreases by 10% (Figure 10). We suppose it is connected with the direct DNA π-electron systems damage that is caused by Dst-MdO
molecules.Figure 10
The dependence of optical density of the DNA maximum in
the DNA+Dst-MdO system.So, DBos-24 and Dst-MdO dyes could be possibly used in the photodynamic therapy for the
DNA damage.


## 3.1. The study of SPE and TPE Fluorescence of
Dyes and Dye-DNA Complexes

Styryl dyes are known to be efficiently excited via the TPE [2, 16, 17]. Thus, the study
of the styryl dyes as possible fluorescent probes for the DNA was performed by
us as follows. First, wide series of styryl dyes based on different
heterocyclic residues were synthesized and studied as the SPE fluorescent
probes for DNA detection. Second, the dyes demonstrating both high value of
fluorescence intensity in DNA presence and sharp emission intensity increase
upon binding to DNA were studied in the TPE experiment.Recently, the series of novel monomer and
homodimer styrylcyanine dyes based on benzothiazole, naphtathiazole,
benzoimidazole, pyridinium, quinoline, and 4-oxo-thieno (2,3-d) pyrimidinium
heterocyclic residues (Figure1) was elaborated, synthesized, studied, and partly
reported [18, 20, 21]. The dyes demonstrated high DNA sensitivity, namely,
fluorescence intensity of dyes upon SPE enhanced up to thousand times upon
binding with DNA (Figure 2). The study of the dyes absorption and fluorescence
spectra at room temperature in water buffer solution as well as in the DNA
presence revealed that the spectra of majority of the studied dyes are
connected with the absorption and fluorescence of the separate styryl
chromophores. Meanwhile, for the styryl homodimer dyes containing the
spermine-like linker (DBos-21, DBos-24, DBos-25, DBos-28, DBos-30, S-45, and
S-46) the band corresponding to the aggregate of the styryl chromophores is
dominating in the absorption spectrum of the free dyes, while the addition of
the DNA to the solution leads to the decrease of the aggregate band and growth
of the monomer ones. At the same time, the mentioned aggregates were found to
be nonfluorescent at room temperature, thus the fluorescence spectra of the
mentioned dimers, as well as of all the studied dyes (both free and bound to
DNA), correspond to the emission of the separate styryl chromophores [19].
Hence, the strongly enhanced fluorescence of the studies styryls in DNA
presence is the emission of the styryl chromophores, bound to the DNA molecule.
The fixation of the separate chromophore on DNA (via either intercalation or
groove-binding) leads to the decrease in the rate constant of the
nonfluorescent deactivation of the singlet electronic excitation of the dye,
and thus to the increase of the dye fluorescence quantum yield [22].Figure 2
SPE fluorescence spectra of dyes in free state
and in DNA
presence.In order to enhance the dye-to-DNA affinity, some
of the studied dyes contained the spermine-like tail or linking group. It was
shown by us earlier [23] that the spermine-like tail group enhances the
constant of dye to DNA binding equilibrium (Kb)
in about three times (1.8 × 104 M−1 for Sbt and about 6 × 104 M−1 for S-20). We believe this enhancement to be connected with the
additional interaction of the charged spermine tail group with the DNA. It
should be mentioned that the cyanine dyes sensitive to DNA possess the Kb values of the same order
of magnitude as the benzothiazole styryl dyes Sbt and S-20 [24], though for
some of the cyanines (e.g., TO) the value of Kb was reported to be about 106 M−1[25].


## 3.2. The Effect of Dyes Aggregations on
Their Phosphorescent Properties

Phosphorescence
excitation and emission spectra of benzothiazole homodimer dyes solutions were
measured (Figure3). The energy of the lowest triplet level of dyes is about 1.85 eV (14900 cm−1). This value is less than the value of the lowest
triplet level among DNA bases thus the triplet electron excitation energy
transfer from DNA to the dye can take place and is still under study. The
phosphorescence lifetime was evaluated and amounted about 20 milliseconds.Figure 3
Fluorescence and phosphorescence emission
and excitation spectra of DBos-30 water solution at liquid
nitrogen temperature (C = 10−4 M).Fluorescence excitation
and emission spectra of benzothiazole homodimer dyes solutions at liquid
nitrogen temperature were obtained. Also fluorescence of dyes’ aggregates at
liquid nitrogen temperature was registered while at room temperature these
aggregates are virtually nonfluorescent. As far as fluorescence excitation
spectra of aggregates coincide with phosphorescence excitation spectra of dyes,
it was concluded that, namely, aggregates are mainly responsible for the
phosphorescence (Figure3). In order to exclude the interaction between the
dye-DNA complex and the molecular oxygen in the water solution the studying of
the dyes emission in vacuum is under research now.


## 3.3. TPE Fluorescence of Dyes and Dye-DNA Complexes

The TPE study of the fluorescent properties of
the mentioned styryl dyes in the presence of DNA was performed.The dye-DNA complexes revealed intensive emission
upon two-photon excitation (TPE) at wavelength 1064 nm with the 20 nanoseconds
pulsed YAG:Nd3+ laser. Here the numerical results of dyes’ studying
upon the TPE in complexes with DNA are presented. The values of dyes’
two-photon absorption (TPA) cross section δwere
evaluated (see Table 1). The calculations of δwere
performed using the respective value for Rhodamine 6G [26]. As it is seen from
Table 1, the δvalues
of dye-DNA complexes are about 10−50 cm4 s.Table 1
The values of TPA cross sectionδof dyes
in DNA presence upon excitation at wavelength 1064 nm.
Dyeδ, 10−50 cm4⋅ sDyeδ, 10−50 cm4⋅ sS-202.3S331.1Bos-12.2D-1841.1DBos-211.7S400.2DBos-241.4S480.2DBos-251.7Tio10.2DBos-282.1Sil1.7DBos-301.8Dil-101.0S451.4S30.9S460.3S80.4Sbt1.7Din-100.4Dbt100.3S270.8F0.04Rhodamine 6G5.5Dst-60.6Dst-100.6Ds-100.4Dyes from the series with each type of
chromophore in complexes with DNA demonstrated emission upon TPE at 840 nm with
the 90 femtosecond pulsed
Ti:sapphire laser (see Figure4). TPA cross sections δat
wavelength 840 nm are under evaluation now.Figure 4
Fluorescence emission
spectra of dye-DNA water buffer solution and Rhodamine B ethanol
solution upon SPE and TPE (concentration of dyesC = 1.5 × 10−5 M).The possibility of the dye DBos-21 application
for the TPE fluorescent cell staining was studied. HeLa cells were incubated
for 1 hour with DBos-21 (5μM) and imaged using 880 nm excitation of the
femtosecond pulsed Ti:Sapphire laser (Tsunami, Spectra Physics). TPE
fluorescent image of the cells stained with DBos-21 is
shown in  Figure 5.Figure 5
TPE
fluorescent image of the HeLa cells stained with the dye DBos-21.
Fluorescent emission excited with the femtosecond pulsed
Ti:Sapphire laser, excitation wavelength 880 nm.The TPE fluorescent imaging study shows that the
styrylcyanine dimer dye DBos-21 containing the spermine-like linking group
successfully permeates through the living cell membrane. Besides, this dye
demonstrated mostly mitochondria staining, possibly because of high positive
charge of the dye.Thus, the styrylcyanine dimmer dyes containing
the spermine-like linking group could be successfully applied for the TPE
fluorescent imaging of the living cells.


## 3.4. The Study of Dyes Phototoxicity and Photostability

### 3.4.1. The First Excited Singlet and Triplet Levels of Dyes

For the use of the dyes
as biomedical sensors for the DNA (or RNA) detection and imaging in the study
of the living objects the phototoxic influence of these dyes realized via the excitation energy transfer
from the dye to the DNA has to be absent. To avoid these processes the first
excited singlet and triplet levels of dyes must be situated lower than
correspondent levels of any nucleotide. To check this the optical absorption,
fluorescence, and phosphorescence spectra of the investigated dyes Bos-1, DBos-21,
DBos-24, DBos-25, DBos-28, and DBos-30 as well as correspondent dye+DNA systems
were studied. Some of these spectra (namely, for Bos-1, DBos-24 and DBos-30
dyes) are given in Figure6.The absorption (1,2), fluorescence (3,4), and
phosphorescence (5,6) spectra (3–6 at T = 77 K) of free styryl dyes
(1,3,5) and correspondent systems DNA+dye (2,4,6) of: (a) styryl
monomer Bos-1, (b) dimer DBos-24, and (c) dimer DBos-30.
(a)(b)(c)The absorption spectra
of the dye Bos-1 both in the presence and the absence of the DNA in visible spectral region
consist of the single band with the maxima near 17540 and 18470 cm−1, respectively
(Figure 6(a); curves 1,2). Since this band is the most long-wave band in the visible
spectral region and the corresponding fluorescence spectrum (Figure 6(a); curves 3,4)
obeys the mirror symmetry rule respectively to this absorption band, the latter  should be attributed to the
absorption transition to the first excited singlet level of the dye molecule. The shift between the maxima of the free dye and
the DNA+dye system spectra points to the dye interaction with the DNA. In the
absorption spectra of the dimer dyes DBos-24 and DBos-30 in presence of the DNA
the same band with the maximum near 18000 cm−1 (Figure 6(b); curve 2)
and 17750 cm−1 (Figure 6(c); curve 2), respectively, could be observed.
At the same time, the spectrum of DBos-24 in the DNA presence contains one more
band with the maximum at 22500 cm−1 shifted to the
short-wavelength region relatively to the main band. This short-wavelength band
with the maxima at 22200 and 20800 cm−1, respectively,
dominates in absorption spectra of both DBos-24 and DBos-30 in absence of the
DNA (Figures 6(b); curve 1, and 6(c); curve 1). Taking into account all the above-mentioned
results, the short-wavelength band could be attributed to the aggregates of the
styryl chromophore. Since for the dimer dyes the tendency to form aggregates is
much higher than for corresponding monomers [27], the short-wavelength band is
present in the spectra of DBos-24 and DBos-30 in free state, but is absent in the spectrum of
Bos-1 containing the same chromophore. At the same time, interaction of dimer
dyes with the DNA at low dye to the DNA concentrations ratio generally leads to
the fixation of separate chromophores on the DNA molecule, thus resulting in
decrease in the aggregates concentration in solution [27]. This explains the
decrease in the short-wavelength band contribution to the absorption spectrum
of DBos-24 in presence of the DNA, as well as the disappearance of this band in
the same spectrum of DBos-30.The fluorescence of the
dyes Bos-1, DBos-24, and DBos-30, both in free form and in DNA presence, at the
room temperature was measured in [19]. The maximum of these spectra was
situated near 17000 cm−1 similarly to the spectra
presented at Figures 6(a), 6(b), 6(c) (curves 3,4). It was shown in [19] with the help of fluorescence
excitation spectra that the fluorescence band with the maximum near 17000 cm−1 belongs to the emission of the single styryl chromophores. Thus, the absorption
and fluorescence spectra of the dyes Bos-1, DBos-24, and DBos-30 in DNA presence could be
used for the calculation of the position of the singlet electronic excited
energy level of the nonaggregated chromophore of these dyes, bound to DNA.The
positions of the first excited singlet level of the investigated dyes in
nonaggregated form in the presence of the DNA were obtained by intersection of
the absorption and fluorescence spectra curves and given in Table2.Table 2
Positions of singlet (S) and triplet (T) levels of the
dyes in presence of the DNA, cm−1.
CompoundBos-1DBos-21DBos-24DBos-25DBos-28DBos-30S (293 K)182501818018120181201828017940S (77 K)191001880019530191601868019110T (77 K)147901482014240147301475014890The
values of singlet and triplet energy levels of the nucleotides obtained by us
in [28, 29] are presented in Table 3. The comparison of these data with data
given in Table 2 removes all doubts that even the triplet level of any
nucleotide is situated much upper than the singlet level of any investigated
dye. That is why the irradiation of the dye in the visual spectral region
exciting the first singlet electronic level of the dye cannot be resulted in
the excitation energy transfer from the dye to the DNA.Table 3
Positions of singlet (S1) and triplet (T1) levels of the
nucleotides, cm−1.
CompounddCMPdGMPdTMPdAMPS133090330303353034490T126630263202616025950


### 3.4.2. Effect of Irradiation of Dyes and the DNA+Dye Systems on Their Optical Properties

It is known that the phototoxic influence of a
luminescent dye probe on the DNA results in the spectral response of the
DNA+dye system. This response is connected with the DNA macromolecule damage
and is observed as the change of the optical density of the DNA absorption band
(i.e., situated at 260 nm and corresponds to
the S0→S1 electronic transition) in the
DNA+dye system under excitation in the dye absorption band (i.e., situated at
400–500 nm and corresponds to its S0→S1 transition). Besides, the
investigation of the optical density changes of the dye band under the free dye
excitation at the wavelength of the same band is the way to study the dye
photostability. The fact that the DNA absorption band connected with S0→S1 transition is located far from
the same band of majority of dyes and the value of optical density of the
absorption band connected with the dye S0→S2 transition (located near
260 nm) is much less than corresponding value of the DNA band gives the
ground to study the dynamics of the absorption bands optical density changes
almost independently for a dye and for the DNA. In our paper, the
investigations results of the absorption spectra of the elaborated styryls and
the respective DNA+dye complexes (that were recorded with the aim to study both
the dye photostability and its phototoxicity influence on the DNA) during the
irradiation of the investigated compounds solutions by visible light of 1 kW
Hg-lamp were described.As it is shown in Figure7 the gradual decrease of D of the absorption spectra bands is
observed for free dyes under the irradiation time increase (curve 1). This
phenomenon is connected with the damage of dyes π-electron systems under irradiation and is the
evidence that Bos-1 and DBos-30 are not photostable in free form. The gradual
decrease of D of the dyes absorption
spectra bands in the DNA presence takes place too but essentially slower than
for free dyes. Moreover, the decrease rate of D of the DNA+DBos-30 system (Figure 7(b); curve 2) is lower than of
the system DNA+Bos-1 (Figure 7(a); curve 2). It means that dimer DBos-30 bound
to the DNA is more photostable than monomer Bos-1 bound to the DNA. Comparing
the dependencies of optical density on irradiation time of investigated styryl
dyes with the same dependence of thiazole orange (TO) dye (Figure 7, curve 4), it is
shown that both Bos-1 and DBos-30 dyes (bound to the DNA) are more photostable
even than TO dye (bound to the DNA). The rise of the dyes photostability under
binding to the DNA occurs, in our opinion, because of the formation of the
tough spatial fixation of a dye molecule after the intercalation of this dye in
the DNA macromolecule. As the result, the DNA macromolecule screens the dye
molecule against external influences and decreases the contacts between the dye
molecules and dissolved molecules of oxygen. The last is transferred into the
singlet excited electronic state under the direct excitation of the dye and
becomes an active oxidant [30]. The results obtained in this paper agree with
the data obtained in [31] on investigations of the cyanine dyes. Since the
constant of binding to the DNA for the dimer is often higher than for the
monomer with the same π-electron system [32, 33], the number of dye molecules bound
with the DNA is higher for the dimer DBos-30 than for the monomer Bos-1. Thus
the DNA macromolecule screens the dimer molecules rather than the monomer
molecules that cause the difference in Bos-1 and DBos-30 photostability in the
DNA presence.The dependence of optical density
(obtained from optical absorption spectra) on irradiation time of
dyes: (a) Bos-1 and (b) DBos-30 (1—free dye, 2—DNA+dye,
3—the DNA maximum 260 nm, 4—TO with the DNA).
(a)(b)The dependencies of the DNA band maximum optical densityD on time of dye irradiation (Figure 7; curve 3)
for the DNA+Bos-1 and DNA+DBos-30 systems were investigated with the aim to
study phototoxic influence of the investigated dyes on the DNA. According to
our experimental data the value of the optical density D of the investigated dyes absorption band
connected with the S0→S2 transition is changed negligible
during all the time of irradiation. As it is shown in Figure 7 the value of D is changed within 3% at 260 nm
out of its intact value that may be connected with these negligible changes of
the dyes S0→S2 band and experimental errors. It
is known that the DNA destruction is either the double strand untwisting or the
strand cutting that is reverse to the hypochromic effect, the complete
untwisting resulting in the 37% increase in D value at 260 nm [34].
That is why the 3%-changes obtained by us cannot be connected with the
significant DNA damage and can be included in experimental errors.So, Bos-1 and DBos-30 dyes are nonphototoxic for the DNA
as the first approximation and can be used as the optical biomedical sensors
for the DNA detection and imaging. On the other hand, the DNA protects these
dyes against the photodamage.As is shown in Figure6(b), the dimer DBos-24 absorption band consists of two bands. Comparing
Figures 6(a) and 6(b) it can be seen that the long-wavelength band (18500 cm−1)
is located close to the monomer Bos-1 band and corresponds to nonaggregated
state. The short-wave band (23100 cm−1), in our opinion, is
connected with aggregates (the similar case was observed for cyanine dyes [35, 36]). Using optical absorption spectra of DBos-24 dye and the DNA+DBos-24
system, the dependencies of the optical density D on irradiation time for these molecular systems are obtained
(Figures 8(a), 8(b)). The aggregates band of the free dye decreased gradually during all
the irradiation time while the nonaggregated dyes band increased slightly at
first (Figure 9). In our opinion, it is connected with the fact that aggregates
are damaged and turned into nonaggregated state. Since the D value of dye DBos-24 decreases by 40–50% after 600 minutes of
irradiation, this dye is more photostable than Bos-1 and DBos-30.The dependence of optical
density (obtained from optical absorption spectra) on irradiation
time of dimer dye DBos-24: (a) free dye, (b) bond to the DNA
(1—at 23100 cm−1, 2—at 18500 cm−1,
3—the DNA maximum at 260 nm).
(a)(b)Figure 9
The
absorption spectra of dimer DBos-24 during irradiation (free dye:
1—intact, 2–210 minutes, 3–540 minutes,
4–600 minutes; dye with DNA: 5—intact,
6–240 minutes, 7–540 minutes).For the DNA+DBos-24
system, the optical density of aggregate band (Figure9) decreases gradually with the irradiation
time increasing but D of
nonaggregated dyes band (in presence of the DNA) remains constant. In our
opinion, it is connected with the fact that nonaggregated dyes intercalate into
the DNA macromolecule while aggregates bind outside the DNA chain. As it was
mentioned above, during the irradiation the molecules of environmental oxygen
are transferred in the singlet excited electronic state and become an active
oxidant [30]. Generated singlet oxygen damages the aggregated dyes that are
outside the DNA chain. The DNA screens nonaggregated dyes that are intercalated
in the DNA macromolecule (between the DNA strands) and protect them from the influence of singlet
oxygen and, therefore, the dyes molecules are not damaged.As it is shown (Figure8(b);
curve 3), the increase (up to by 30%) of D of the DNA absorption spectra bands is observed for the DNA+DBos-24
system under the irradiation time increase. In our opinion, this phenomenon is
caused by the untwisting or cutting of the strands of the DNA macromolecule
which contacts with the molecules of DBos-24 (this phenomenon is reverse to the
hypochromic effect). These results agree with 37% increase for the complete DNA
untwisting [34]. Thus the presence of DBos-24 dye causes the DNA damage, that
is, DBos-24 showed phototoxic influence on the DNA. But dyes phototoxicity
appears not only in the untwisting or cutting of the DNA strands. For the
DNA+Dst-MdO system, the value of optical density of the DNA absorption band
decreases by 10% (Figure 10). We suppose it is connected with the direct DNA π-electron systems damage that is caused by Dst-MdO
molecules.Figure 10
The dependence of optical density of the DNA maximum in
the DNA+Dst-MdO system.So, DBos-24 and Dst-MdO dyes could be possibly used in the photodynamic therapy for the
DNA damage.


## 3.4.1. The First Excited Singlet and Triplet Levels of Dyes

For the use of the dyes
as biomedical sensors for the DNA (or RNA) detection and imaging in the study
of the living objects the phototoxic influence of these dyes realized via the excitation energy transfer
from the dye to the DNA has to be absent. To avoid these processes the first
excited singlet and triplet levels of dyes must be situated lower than
correspondent levels of any nucleotide. To check this the optical absorption,
fluorescence, and phosphorescence spectra of the investigated dyes Bos-1, DBos-21,
DBos-24, DBos-25, DBos-28, and DBos-30 as well as correspondent dye+DNA systems
were studied. Some of these spectra (namely, for Bos-1, DBos-24 and DBos-30
dyes) are given in Figure6.The absorption (1,2), fluorescence (3,4), and
phosphorescence (5,6) spectra (3–6 at T = 77 K) of free styryl dyes
(1,3,5) and correspondent systems DNA+dye (2,4,6) of: (a) styryl
monomer Bos-1, (b) dimer DBos-24, and (c) dimer DBos-30.
(a)(b)(c)The absorption spectra
of the dye Bos-1 both in the presence and the absence of the DNA in visible spectral region
consist of the single band with the maxima near 17540 and 18470 cm−1, respectively
(Figure 6(a); curves 1,2). Since this band is the most long-wave band in the visible
spectral region and the corresponding fluorescence spectrum (Figure 6(a); curves 3,4)
obeys the mirror symmetry rule respectively to this absorption band, the latter  should be attributed to the
absorption transition to the first excited singlet level of the dye molecule. The shift between the maxima of the free dye and
the DNA+dye system spectra points to the dye interaction with the DNA. In the
absorption spectra of the dimer dyes DBos-24 and DBos-30 in presence of the DNA
the same band with the maximum near 18000 cm−1 (Figure 6(b); curve 2)
and 17750 cm−1 (Figure 6(c); curve 2), respectively, could be observed.
At the same time, the spectrum of DBos-24 in the DNA presence contains one more
band with the maximum at 22500 cm−1 shifted to the
short-wavelength region relatively to the main band. This short-wavelength band
with the maxima at 22200 and 20800 cm−1, respectively,
dominates in absorption spectra of both DBos-24 and DBos-30 in absence of the
DNA (Figures 6(b); curve 1, and 6(c); curve 1). Taking into account all the above-mentioned
results, the short-wavelength band could be attributed to the aggregates of the
styryl chromophore. Since for the dimer dyes the tendency to form aggregates is
much higher than for corresponding monomers [27], the short-wavelength band is
present in the spectra of DBos-24 and DBos-30 in free state, but is absent in the spectrum of
Bos-1 containing the same chromophore. At the same time, interaction of dimer
dyes with the DNA at low dye to the DNA concentrations ratio generally leads to
the fixation of separate chromophores on the DNA molecule, thus resulting in
decrease in the aggregates concentration in solution [27]. This explains the
decrease in the short-wavelength band contribution to the absorption spectrum
of DBos-24 in presence of the DNA, as well as the disappearance of this band in
the same spectrum of DBos-30.The fluorescence of the
dyes Bos-1, DBos-24, and DBos-30, both in free form and in DNA presence, at the
room temperature was measured in [19]. The maximum of these spectra was
situated near 17000 cm−1 similarly to the spectra
presented at Figures 6(a), 6(b), 6(c) (curves 3,4). It was shown in [19] with the help of fluorescence
excitation spectra that the fluorescence band with the maximum near 17000 cm−1 belongs to the emission of the single styryl chromophores. Thus, the absorption
and fluorescence spectra of the dyes Bos-1, DBos-24, and DBos-30 in DNA presence could be
used for the calculation of the position of the singlet electronic excited
energy level of the nonaggregated chromophore of these dyes, bound to DNA.The
positions of the first excited singlet level of the investigated dyes in
nonaggregated form in the presence of the DNA were obtained by intersection of
the absorption and fluorescence spectra curves and given in Table2.Table 2
Positions of singlet (S) and triplet (T) levels of the
dyes in presence of the DNA, cm−1.
CompoundBos-1DBos-21DBos-24DBos-25DBos-28DBos-30S (293 K)182501818018120181201828017940S (77 K)191001880019530191601868019110T (77 K)147901482014240147301475014890The
values of singlet and triplet energy levels of the nucleotides obtained by us
in [28, 29] are presented in Table 3. The comparison of these data with data
given in Table 2 removes all doubts that even the triplet level of any
nucleotide is situated much upper than the singlet level of any investigated
dye. That is why the irradiation of the dye in the visual spectral region
exciting the first singlet electronic level of the dye cannot be resulted in
the excitation energy transfer from the dye to the DNA.Table 3
Positions of singlet (S1) and triplet (T1) levels of the
nucleotides, cm−1.
CompounddCMPdGMPdTMPdAMPS133090330303353034490T126630263202616025950


## 3.4.2. Effect of Irradiation of Dyes and the DNA+Dye Systems on Their Optical Properties

It is known that the phototoxic influence of a
luminescent dye probe on the DNA results in the spectral response of the
DNA+dye system. This response is connected with the DNA macromolecule damage
and is observed as the change of the optical density of the DNA absorption band
(i.e., situated at 260 nm and corresponds to
the S0→S1 electronic transition) in the
DNA+dye system under excitation in the dye absorption band (i.e., situated at
400–500 nm and corresponds to its S0→S1 transition). Besides, the
investigation of the optical density changes of the dye band under the free dye
excitation at the wavelength of the same band is the way to study the dye
photostability. The fact that the DNA absorption band connected with S0→S1 transition is located far from
the same band of majority of dyes and the value of optical density of the
absorption band connected with the dye S0→S2 transition (located near
260 nm) is much less than corresponding value of the DNA band gives the
ground to study the dynamics of the absorption bands optical density changes
almost independently for a dye and for the DNA. In our paper, the
investigations results of the absorption spectra of the elaborated styryls and
the respective DNA+dye complexes (that were recorded with the aim to study both
the dye photostability and its phototoxicity influence on the DNA) during the
irradiation of the investigated compounds solutions by visible light of 1 kW
Hg-lamp were described.As it is shown in Figure7 the gradual decrease of D of the absorption spectra bands is
observed for free dyes under the irradiation time increase (curve 1). This
phenomenon is connected with the damage of dyes π-electron systems under irradiation and is the
evidence that Bos-1 and DBos-30 are not photostable in free form. The gradual
decrease of D of the dyes absorption
spectra bands in the DNA presence takes place too but essentially slower than
for free dyes. Moreover, the decrease rate of D of the DNA+DBos-30 system (Figure 7(b); curve 2) is lower than of
the system DNA+Bos-1 (Figure 7(a); curve 2). It means that dimer DBos-30 bound
to the DNA is more photostable than monomer Bos-1 bound to the DNA. Comparing
the dependencies of optical density on irradiation time of investigated styryl
dyes with the same dependence of thiazole orange (TO) dye (Figure 7, curve 4), it is
shown that both Bos-1 and DBos-30 dyes (bound to the DNA) are more photostable
even than TO dye (bound to the DNA). The rise of the dyes photostability under
binding to the DNA occurs, in our opinion, because of the formation of the
tough spatial fixation of a dye molecule after the intercalation of this dye in
the DNA macromolecule. As the result, the DNA macromolecule screens the dye
molecule against external influences and decreases the contacts between the dye
molecules and dissolved molecules of oxygen. The last is transferred into the
singlet excited electronic state under the direct excitation of the dye and
becomes an active oxidant [30]. The results obtained in this paper agree with
the data obtained in [31] on investigations of the cyanine dyes. Since the
constant of binding to the DNA for the dimer is often higher than for the
monomer with the same π-electron system [32, 33], the number of dye molecules bound
with the DNA is higher for the dimer DBos-30 than for the monomer Bos-1. Thus
the DNA macromolecule screens the dimer molecules rather than the monomer
molecules that cause the difference in Bos-1 and DBos-30 photostability in the
DNA presence.The dependence of optical density
(obtained from optical absorption spectra) on irradiation time of
dyes: (a) Bos-1 and (b) DBos-30 (1—free dye, 2—DNA+dye,
3—the DNA maximum 260 nm, 4—TO with the DNA).
(a)(b)The dependencies of the DNA band maximum optical densityD on time of dye irradiation (Figure 7; curve 3)
for the DNA+Bos-1 and DNA+DBos-30 systems were investigated with the aim to
study phototoxic influence of the investigated dyes on the DNA. According to
our experimental data the value of the optical density D of the investigated dyes absorption band
connected with the S0→S2 transition is changed negligible
during all the time of irradiation. As it is shown in Figure 7 the value of D is changed within 3% at 260 nm
out of its intact value that may be connected with these negligible changes of
the dyes S0→S2 band and experimental errors. It
is known that the DNA destruction is either the double strand untwisting or the
strand cutting that is reverse to the hypochromic effect, the complete
untwisting resulting in the 37% increase in D value at 260 nm [34].
That is why the 3%-changes obtained by us cannot be connected with the
significant DNA damage and can be included in experimental errors.So, Bos-1 and DBos-30 dyes are nonphototoxic for the DNA
as the first approximation and can be used as the optical biomedical sensors
for the DNA detection and imaging. On the other hand, the DNA protects these
dyes against the photodamage.As is shown in Figure6(b), the dimer DBos-24 absorption band consists of two bands. Comparing
Figures 6(a) and 6(b) it can be seen that the long-wavelength band (18500 cm−1)
is located close to the monomer Bos-1 band and corresponds to nonaggregated
state. The short-wave band (23100 cm−1), in our opinion, is
connected with aggregates (the similar case was observed for cyanine dyes [35, 36]). Using optical absorption spectra of DBos-24 dye and the DNA+DBos-24
system, the dependencies of the optical density D on irradiation time for these molecular systems are obtained
(Figures 8(a), 8(b)). The aggregates band of the free dye decreased gradually during all
the irradiation time while the nonaggregated dyes band increased slightly at
first (Figure 9). In our opinion, it is connected with the fact that aggregates
are damaged and turned into nonaggregated state. Since the D value of dye DBos-24 decreases by 40–50% after 600 minutes of
irradiation, this dye is more photostable than Bos-1 and DBos-30.The dependence of optical
density (obtained from optical absorption spectra) on irradiation
time of dimer dye DBos-24: (a) free dye, (b) bond to the DNA
(1—at 23100 cm−1, 2—at 18500 cm−1,
3—the DNA maximum at 260 nm).
(a)(b)Figure 9
The
absorption spectra of dimer DBos-24 during irradiation (free dye:
1—intact, 2–210 minutes, 3–540 minutes,
4–600 minutes; dye with DNA: 5—intact,
6–240 minutes, 7–540 minutes).For the DNA+DBos-24
system, the optical density of aggregate band (Figure9) decreases gradually with the irradiation
time increasing but D of
nonaggregated dyes band (in presence of the DNA) remains constant. In our
opinion, it is connected with the fact that nonaggregated dyes intercalate into
the DNA macromolecule while aggregates bind outside the DNA chain. As it was
mentioned above, during the irradiation the molecules of environmental oxygen
are transferred in the singlet excited electronic state and become an active
oxidant [30]. Generated singlet oxygen damages the aggregated dyes that are
outside the DNA chain. The DNA screens nonaggregated dyes that are intercalated
in the DNA macromolecule (between the DNA strands) and protect them from the influence of singlet
oxygen and, therefore, the dyes molecules are not damaged.As it is shown (Figure8(b);
curve 3), the increase (up to by 30%) of D of the DNA absorption spectra bands is observed for the DNA+DBos-24
system under the irradiation time increase. In our opinion, this phenomenon is
caused by the untwisting or cutting of the strands of the DNA macromolecule
which contacts with the molecules of DBos-24 (this phenomenon is reverse to the
hypochromic effect). These results agree with 37% increase for the complete DNA
untwisting [34]. Thus the presence of DBos-24 dye causes the DNA damage, that
is, DBos-24 showed phototoxic influence on the DNA. But dyes phototoxicity
appears not only in the untwisting or cutting of the DNA strands. For the
DNA+Dst-MdO system, the value of optical density of the DNA absorption band
decreases by 10% (Figure 10). We suppose it is connected with the direct DNA π-electron systems damage that is caused by Dst-MdO
molecules.Figure 10
The dependence of optical density of the DNA maximum in
the DNA+Dst-MdO system.So, DBos-24 and Dst-MdO dyes could be possibly used in the photodynamic therapy for the
DNA damage.


## 4. Conclusions

Novel monomer and
homodimer styrylcyanine dyes based on benzothiazole, naphtathiazole,
benzoimidazole, pyridinium, quinoline, and 4-oxo-thieno[2,3-d]pyrimidinium
heterocyclic residues were elaborated, synthesized, and studied. The dyes
demonstrated rather high sensitivity to DNA.Monomer and homodimer styrylcyanine dyes
demonstrated fluorescence emission of high intensity upon TPE at wavelength of
840 nm and 1064 nm. TPA cross sections of dyes at the wavelength
1064 nm are of the 10−50 cm4 s order. The dyes based
on benzothiazole and benzoimidazole heterocycle generally demonstrate higher
TPA cross sections values as compared to the other studied dyes.Dyes with spermine-like linkage/tail groups
demonstrated higher sensitivity to DNA and have higher TPA cross sections than
dyes with the same chromophore but without respective groups.We can conclude that the studies dyes can be used
for SPE and TPE fluorescent DNA detection.The developed dyes are
rather photostable and mainly photochemicaly safe. The DBos-24 is most
photostable but manifests some phototoxicity. The dye Dst-MdO is rather
phototoxic; both these dyes could be possibly used in the photodynamic therapy.



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*Source: 908246-2008-07-13.xml*