Abstract Terbium ions (Tb3+) with unique photophysical properties have been utilized to

develop biosensors with low background and high sensitivity. In this study, the Ag+-
sensitized luminescence of Tb3+-DNA complexeswas uncovered. The luminescence of
Tb3+-DNA complexes could be enhanced by more than 30 times in the presence of Ag+,
whenTb3+was boundwith poly(G) and poly(T)whereas not with other homopolymers. This
research confirmed that the sensitization resulted from the interaction of Ag+ with certain
bases involved in DNA, not just with the reported certain G-quadruplex sequence. The
coordination of Ag+ to guanine and thymine bases was expected to increase their rigidities,
form Tb3+-DNA-Ag+ ternary structures, and thus enhance energy transfer from guanine and
thymine to Tb3+. These findings benefit the development of sensitive luminescence probes
for various nucleic acids-related targets.

Introduction It is known that the rare-earth lanthanide Tb3+ (terbium ion) has fascinating
luminescence properties, such as long-lifetimes, narrow emission bandwidths, large Stokes
shifts, and high resistance to photo-bleaching, which prequalify it for practical bioanalysis
[1,2]. The luminescence of aqueous Tb3+ isweak, due to lowabsorption cross sections and
nonradiative deactivation. Upon binding to appropriate organic ligands, Tb3+ exhibits a
striking luminescence enhancement, when excited in the absorption bands of ligands. In the
process, energy is funnelled into Tb3+ excited states by nonradiative energy transfer, which is
referred to as “antenna effect” [3,4]. Proteins and nucleic acids as ligands have received
special interest, as their triplet energy states overlap the resonance energy levels. The
fortuitously match makes Tb3+ an extremely valuable probe in awide variety of biomolecular
systems [5,6]. Accompanying with the development of nucleic acid technology, Tb3+ has
been employed to study the metal binding sites in nucleic acids, the structure of nucleic acids,
and to develop nucleic acidsbased sensors [7–13]. It is desirable to find highly luminescent
Tb3+ complexes in order to develop simple and sensitive analyticalmethods. However,
interactions between Tb3+ and nucleosides, nucleotides and nucleic acids are more complex
than expected. Cytosines enhance the Tb3+ luminescence apparently, whereas other bases (A,
T, and G) do not. Conversely, the mononucleotide GMP leads to great enhancement of the
Tb3+ emission, which is not observed for other nucleotides [14, 15]. Itwas shown that the
coordination to O2 and N3 of cytosinewas responsible for such enhancement of Tb3+
luminescence. In the case of nucleotides and nucleic acids, Tb3+ could bindwith two sites:
the phosphate moieties and electron donor groups on the nucleoside bases [14, 16]. It is
reported that certain transitionmetal ions can promote efficient energy transfer from ligands
to lanthanide ions while the exact mechanism is still ambiguous [17–19]. Due to the
advantages of the electron donor-rich deoxyribonucleic acid (DNA, a type of nucleic acids),
such as versatility, high stability and low cost, the interactions between DNA and various
metal ions received much attention [20]. It is reported that Ag+ has certain complexing ability
to DNA bases [19,21,22]. Therefore, we speculated that Ag+ possibly effected the energy
transfer from DNA to Tb3+, andwould subsequently change Tb3+ luminescence. Very
recently, Tan et al. have reported Ag+-mediated conformational change of terbium ion-
promoted G-quadruplex [23]. However, the research was restricted to a G-quadruplex
sequence. It still remains unknown whether it is the case for other DNA sequences and what
is the effect of different DNA bases involved in DNA sequences. Herein, we investigated the
effect of Ag+ on luminescence of Tb3+ coordinatedwith different DNA homopolymers.More
than 30-times luminescence enhancement was observed for Tb3+-poly(G) and Tb3+- poly(T)
complexes in the presence of Ag+, whereas not for Tb3+- poly(A) and Tb3+-poly(C)
complexes. The luminescence enhancement of Tb3+-DNA complexeswas attributed to
efficient energy transfer from guanine and thymine bases to Tb3+ due to the coordination of
Ag+ with guanine and thymine bases involved in DNA sequences. 2. Materials and Methods
2.1. Reagents and Instruments DNAwas obtained fromGENEWIZ Inc. (Suzhou, China). The
DNA sequences are as follows: 5′-GGGGGGGGGGGG-3′ (Poly(G)), 5′-TTTTTTTTTTTT-3′
(Poly(T)), 5′-AAAAAAAAAAAA-3′ (Poly(A)) and 5′-CCCCCCCCCCCC-3′ (Poly(C)).
DNA stock solutions were first preparedin ultrapure water, then concentrations were
accurately quantified using 260 nm UV absorbance, and finally stored at −20 °C before use.
Terbium(III) chloride hexahydrate (99.99%) was purchased from Energy Chemical
(Shanghai, China). Tb3+ stock solutions were prepared in ultrapurewater and stored at 4 °C.
AgNO3 of primary reagent, L-cysteine of biological reagent, 2-amino-2-(hydroxymethyl)-
1,3-propanediol (Tris) of biological reagent, acetic acid (HAc) of analytical grade, and other
chemical reagents of analytical grade were purchased from Sinopharm Chemical Reagent Co.
Ltd. (Shanghai, China). Heavy metal salts were dissolved in diluted acetic acid to avoid
precipitation, and the prepared stock solutions were stored away from light. The amino acid
solutions were prepared freshly on the day of use. Tris-HAc buffer (5 mM, pH 7.4) was
prepared from Tris base solution by adjusting the pHwith diluted acetic acid and diluting the
solution to a certain volume. Milli-Q water (18.2 MΩ) was used to prepare solutions.
DNA concentrations were detected using an Eppendorf biophotometer (Eppendorf,
Hamburg, Germany). Luminescence spectra were performed on a F-4600 fluorescence
spectrometer (Hitachi, Tokyo, Japan) with a quartz cuvette atambient temperature. The
luminescence excitationwavelengthwas fixed at 290nmand emission spectrawere collected
from450 to 650nmat 1nmintervals using a 350nmfilter. Excitation spectra of Tb3+ were
obtained by collecting emission with a maximum at the peak wavelength of ca. 547 nm. The
slits for excitation and emissionwere set at 5 and 5nm, respectively. The fitting of
fluorescence data was carried out by using the Origin 8.0 software. Circular Dichroism (CD)
measurements were carried out on a Chirascan-plus circular dichroism spectrometer (Applied
Photophysics Ltd., Surrey, UK) with a quartz cuvette of 1.0 cm path length. The bandwidth
was 0.5 nm, and the time per point was 0.5 s. Three scans from 220 nm to 320 nm at 1 nm
intervals were accumulated and averaged.

2.2. Luminescence Measurements In a typical procedure, DNA stock solutions (25 μM)
were first incubated at 50 °C for 10 min, and then cooled down at ambient temperature. After
that, DNA stock solutions (3 μL) were diluted with 130.5 μL Tris-HAc buffer (5 mM, pH
7.4), and Tb3+ (12 μL, 25 μM) and Ag+ (4.5 μL, 200 μM) were added to the resulted
solutions and mixed, respectively. The mixtureswere incubated for 10min at ambient
temperature before luminescence measurements. In the experiment about reversibility,
different concentrations of cysteine were added 10 min after the addition of Ag+, and the
mixtures were incubated for another 10 min at ambient temperature before luminescence
measurements.

2.3. Circular Dichroism (CD) Measurements For CDmeasurements, first, DNA stock
solutions (24 μL, 50 μM) were diluted with 256.2 μL Tris-HAc buffer (5mM, pH 7.4), then
the resulted solutions were incubated at 50 °C for 10 min, and subsequently cooled down at
ambient temperature. Finally, Tb3+ (4.8 μL, 1 mM) and Ag+ (6 or 15 μL, 1mM)were added
to the solutions and mixed, respectively. For control samples, Milli-Q water was added to
make volume up to 300 μL. The mixtureswere incubated for 10min at ambient temperature
before circular dichroism measurements. Spectra of Tris-HAc buffer were also measured
under the same conditions and subtracted for baseline corrections.

3. Results and Discussions

3.1. Luminescence Responses of Tb3+-DNA Complexes to Ag+

The intrinsic emission of free Tb3+ is very weak in aqueous solution (data not shown),
because of low absorption cross sections and nonradiative deactivation. When Tb3+ is
coexisted with single- stranded DNA, the luminescence of Tb3+ displays different changes,
depending on the sequences of DNA. Fig. 1 indicates the luminescence emission spectra of
Tb3+-homopolymers containing different bases in the absence and presence of Ag+. Four
characteristic emission peaks of Tb3+ were observed in the wavelength range of 485–500
nm, 540– 555 nm, 580–595 nm and 615–625 nm, respectively (Fig. 1A and B) [3]. The most
intense emission is located in the range of 540–555 nm. In the absence of Ag+, the addition
of poly(G) to the Tb3+ solution led to an obvious increase (ca. 12-fold at ca. 547 nm) in the
Tb3+ luminescence, compared with that of free Tb3+ [24]. However, the presence of other
homopolymers produced almost no enhancement of Tb3+ emission in the test conditions.
After adding Ag+ to the Tb3+-homopolymer solutions, the emission was significantly
enhanced for poly(G) and poly(T), compared with that of the corresponding Tb3+-
homopolymers, respectively. Among the four emission peaks, the intensity at ca. 547 nm was
enhanced by more than 30 times for both poly(G) and poly(T). However, no enhancement
was observed after adding Ag+ into Tb3+-poly(C) and Tb3+-poly(A) solutions. Besides, the
maximum emission peak exhibits a gradual red shift from 545 nm to 548 nm for Tb3+-
poly(G) with the increase in the concentration of Ag+, which is invisible for Tb3+-poly(T).
For Tb3+-DNA complexes, the luminescence enhancement of Tb3+ relies on energy transfer
from bases to Tb3+. Herein, the further substantial increase in luminescence due to the
presence of Ag+ demonstrates the coordination of Ag+ with DNA and it is reasonable to
conclude that more efficient energy transfer from poly(G) and poly(T) to Tb3+ is therefore
achieved. Besides, it only takes several minutes for this system to reach the maximum
luminescence (Fig. 2), which is significant for designing fast-response sensors. Excitation
spectra of the ligand-sensitized Tb3+ luminescence will generally resemble the absorption
spectra of the ligand energy donor moieties [3]. As shown in Fig. 3, the excitation spectra of
Tb3+-homopolymer- Ag+ complexes basically mimic the excitation spectra of Tb3+-
homopolymer complexes and the absorption of the corresponding nucleotides in the 250–
300nmrange. Therefore, it can be concluded that in the presence of Ag+, the luminescence
enhancement also resulted from energy transfer from nucleic bases to Tb3+. However, the
efficient sensitization by Ag+ is limited to guanine and thymine bases involved in DNA.
Besides, it is confirmed that the sensitization could be achieved with DNA sequences
containing guanine or thymine bases, not just with the reported certain G-quadruplex
sequence. This is the first report that Ag+ enhanced energy transfer from poly(T) to Tb3+.

3.2. CD Properties of Tb3+-DNA-Ag+ Complexes

To further investigate the luminescence enhancement mechanism, circular dichroism (CD)

spectroscopy was performed to characterize the interaction of Ag+ with different
homopolymers. As shown in Fig. 4, in the absence of metal ions, the CD spectra of poly(T),
poly(C), and poly(A) show small peaks, suggesting the predominant random-coil state.
Poly(G) exhibits an obvious ellipticity at 265 nm, indicating the formation of a number of G-
quadruplex structures [25]. When Tb3+ was present, the CD spectra of homopolymers
showed no remarkable change, except for a blue shift of the positive peak of poly(C) [26].
With the addition of Ag+, an eminent change was observed for poly(G), poly(T), and
poly(C), indicating the interaction of Ag+ with DNA bases. In case of poly(G), the positive
peak at 265 nmwas decreased when Ag+ was present, demonstrating that some G-quadruplex
structures were converted into random-coil states due to the coordination of Ag+ with
guanine bases [27]. In the absence of Ag+, it is considered that the binding of Tb3+ to both
phosphate groups and guanine bases (C6\\O and N7 sites) is responsible for the luminescence
enhancement of Tb3+ (Fig. S1) [14,16]. As Ag+ was bound exclusively to the C6\\O and N7
of guanine bases in poly(G) [21], the coordination site of Tb3+ may be migrated from C6\\O
and N7 to other donor sites (e.g. N1) of guanine bases. As for poly(T), the obvious red shift
of the peaks indicates the interaction of Ag+ with thymine bases. Previous reports suggested
that Ag+ could coordinate to N3 and C4\\O of thymine bases forming base pairs [28,29].
However the interaction is so weak that DNA duplex is unlikely to form [30]. Herein, the CD
spectra indicate the absence of DNA duplex in the presence of Ag+. It is likely that Tb3+ then
binds with the rest donor atom C2\\O of the thymine base and thus forms a ternary structure.
Above all, it could be reasonably inferred that the coordination of Ag+will increase the
rigidity of guanine and thymine, and thus eliminates vibrations of C_O andN\\H, minimizing
the radiationless deactivation [4,23,31,32]. Therefore more efficient energy transfer to Tb3+
was allowed in this ternary structure than that in the previous binary structure [14,16].
Given that the photophysics of nucleic bases is considerably too complex, more evidence is
required for the definite sensitization mechanism. In particular, poly(C) exhibited two
negative peaks at 269 nm and 228nmin the presence of Ag+, corresponding to the formation
of stable duplexeswith C\\Ag+\\C base pairs [33]. Since the binding of Tb3+ exclusively to
the phosphate bone of duplex does not enhance the Tb3+ luminescence [9,14], this could be
the reason why Ag+ has no effect on the luminescence of the Tb3+-poly(C) complex. As for
poly(A), the presence of Ag+ induced no obvious change on its CD spectrum in the test
conditions, in accordance with the luminescence change.
3.3. Effect of Tb3+ and Ag+ Concentration

Focusing on poly(G), the effect of Tb3+ concentration on the luminescence of Tb3+

homopolymer-Ag+ system was investigated. By titrating poly(G) with Tb3+, luminescence of
the system was determined in the absence and presence of Ag+, respectively. As shown in
Fig. 5, with the increase in Tb3+ concentration, the luminescence of Tb3+ was gradually
enhanced in the absence of Ag+, and furthermore sharply enhanced in the presence of Ag+
(12 μM). Excessive Tb3+ led to no obvious change in luminescence in the absence of Ag+
whereas a slightly decrease in the presence of Ag+. The luminescence enhancement factor
reached maximum at Tb3+ concentration ca 4 μM. Deduced from the quantitative
relationship between Tb3+ and nucleotides, Tb3+ possibly binds with each three nucleotides
in poly(G) [34]. Next, the effect of Ag+ concentration on luminescence sensitization was
investigated by titrating Tb3+-poly(G) and Tb3+-poly(T) with Ag+, respectively. Fig. 6
shows how the luminescence increases as the increase in Ag+ concentration (Fig. S2). The
luminescence dependence on Ag+ is logistic for both Tb3+-poly(G)-Ag+ and Tb3+-poly(T)-
Ag+ systems. The small lag at low Ag+ concentration may attribute to a certain extent of
cooperativity in Ag+ binding or to a small amount of base residues in the homopolymers
[35]. The luminescence of Tb3+-poly(G)- Ag+ system reaches maximum near 6 μM Ag+ for
0.5 μM poly(G), which is consistent with the 1:1 ratio that a guanine combines with Ag+
[21]. As for Tb3+-poly(T)-Ag+ system, it requires more than 12 μM Ag+ for 1 μMpoly(T) to
reachmaximum. Due to the relatively weak interaction of Ag+ with thymine bases,more Ag+
is necessary to compete with Tb3+ for thymine binding. In comparison, the luminescence
exhibits no obvious change upon titrating Ag+ into Tb3+-poly(A) or Tb3+-poly(C) solutions
(Fig. S3).
3.4. Effect of Other Metal Ions
To investigate the effect of other common metal ions on the luminescence of Tb3+-
homopolymer complexes, we tested luminescence of the Tb3+-poly(G) complex in the
presence of various metal ions with and without Ag+. As shown in Fig. S4, when metal ions
existed alone, Li+, Na+, K+, Mg2+, Ca2+, Mn2+, Cu2+, Ba2+, Cd2+, Zn2+, and Pb2+
induced no remarkable luminescence change comparing with the significant enhancement
induced by Ag+. When other metal ions coexistedwith Ag+,metal ions except Pb2+ had little
effect on the luminescence enhancement. This is consistent with the fact that Li+, Na+, K+
and other ions (except Pb2+) have comparatively weak influence on the structure of G-rich
sequences [25,36–39]. As Pb2+ and Ag+ both specifically coordinate with C6\\O and N7
sites of guanosine bases, the coordination of Ag+ could be suppressed [27,40]. Besides,
Pb2+ is known to lead to a remarkable decrease in luminescence of Tb3+-DNA complexes
[41]. Therefore, the luminescence enhancement induced by Ag+ was reduced when Pb2+
coexistedwith Ag+. The selective response to Ag+ over most of the other common metal ions
makes this system suitable for Ag+ detection.
3.5. Luminescence Reversibility and Photostability
Luminescence reversibility of the Ag+-sensitized Tb3+-homopolymer system was
investigated using cysteine to competitively bind with Ag+ in the case of poly(G). Cysteine
has been proved to possess a stronger binding affinity thanDNA bases towards Ag+
[42].When cysteine was added into the Tb3+-poly(G)-Ag+ system, the luminescence
was decreased as a function of cysteine with a plateau near 6 μM cysteine (Fig. 7). The
kinetic luminescence decrease could achieve stability within several minutes. The Tb3+-
homopolymer-Ag+ system also exhibits excellent selectivity towards cysteine, regardless of
amino acids existing singly or cysteine coexisting with other amino acids (Fig. S5). The
property of highly sensitive and selective luminescence response to cysteine makes it an ideal
probe for cysteine. Next, we evaluated the photostability of the Tb3+-homopolymer- Ag+
complex. As shown in Fig. S6, no decay was observed for luminescence of the Tb3+ poly(G)-
Ag+ complex under continuous illumination with a 150Wxenon lamp at 290 nm. The result
indicates that the Tb3+- homopolymer-Ag+ complex possesses high resistance to
photobleaching,which is helpful for analytics applications, since organic fluorophores are
subject to photobleaching.
4. Conclusions
In conclusion, we introduced a new way to sensitize the luminescence of Tb3+-DNA
complex. By coordination with DNA bases, Ag+ could induce significant luminescence
enhancement of Tb3+-poly(G) and Tb3+-poly(T) complexes. The coordination of Ag+ with
guanine or thyminewas expected to forma rigid ternary structure, thus induced efficient
energy transfer from guanine and thymine to Tb3+. This work will benefit the mechanism
study on the interaction of Tb3+ with ligands and the development of highly luminescent
complexes for biosensors and biological imaging [3,4,7–13,43]. The findings may be also
extended to other lanthanide ions for designing new materials.
Acknowledgements
The authors acknowledge the financial support from the National Natural Science Foundation
of China (Nos. 21575154, 21507156, 21628502).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.saa.2017.03.001