Time-depended Quenching Behaviors of Multi-dye Systems by
Single-Walled Carbon Nanotubes
Ying Tan and Kazuo Umemura
Department of Physics, Graduate School of Science, Tokyo University of Science, Japan
Keywords: Time-Depended, Quench, Multi-Fluorophore, T-Test, Single-Walled Carbon Nanotubes (SWNT).
Abstract: A fluorescence quenching phenomenon of fluorophores or dyes could be observed with single-walled
carbon nanotubes (SWNTs) suspension. In this study, uranine (Ur) and Rhodamin B (RB), were employed
as fluorophores to research time-depended quenching behaviors of multi-fluorophore system by SWNT
suspension with single-stranded DNA (SWNT-T30). The effect of “simultaneous quenched” scheme (Ur
and RB were quenched at the same time) and “un-simultaneous quenched” scheme (Ur was quenched to
equilibrium state, then RB was added; or RB first quenched, then Ur added) to the quenching properties of
multi-fluorophore system by SWNT-T30 were compared and researched. The quench equilibrium could be
realized in 10 min, for both single and multi-quenching system. Quenching balance and quenching
efficiency of each dye basically was not impacted obviously, whether there was another dye existed in the
same solution or not. The results or the methodology was important for the future multi-sensor design and
application, as it is essential to check the dynamic balance of quenching behavior and confirm that if the
quenching performance of target or hunter of sensor (or multi-sensor) was influenced by other constituents.
1 INTRODUCTION
Carbon nanotubes, especial single-walled carbon
nanotubes (SWNT) suspensions, have attracted
remarkable research interest from first presented in
2003 by Zhang et al., (2003:2). The unique physical,
chemical, optical, electrical properties induced by
the extremely small size make them suitable for a
wide range of biological (Lu et al., 2009: 21),
medical (Prato et al., 2008: 41), environmental
(Pelencia et al., 2015: 407) and electronic (Chitta et
al., 2007:111) applications. The fluorescence
quenching phenomenon of dyes/fluorophores (Li et
al., 2011: 21; Cao et al., 2009: 21) by the nearby
SWNT was found to be universal for a variety of
fluorophores or dyes, and is thought to be utilized
for biosensor applications (Sgobba et al., 2009: 38).
In our previous work, multi-fluorophore system’s
quenching properties and competitive adsorption
model on SWNT surface were studied. As the
succeeding work, here we discussed the time-
depended quenching performances of multi-
fluorophore system, via constantly fluorescent
monitoring. As considering more than one
fluorophore existed in the quenching system,
“simultaneously quenched” process (two
fluorophores were quenched at the same time) and
“un-simultaneously quenched” process (one
fluorophore added first and reached to equilibrium
state, then another fluorophore added) were designed,
then batch assays were carried out. Quenching
efficiencies gained from batch assays were also
investigated via T-test analysis, which is a statistical
method to be used to determine if two sets of data
are significantly different from each other.
Usually, in the system to be detected, more than
one constituent existed which maybe interfere or
influent the photosensitizer or aptamer of the SWNT
sensor platform. Furthermore, despite enormous
number of works focused on fluorescence quenching
by carbon nanotubes, there have been few
experimental efforts reported in the literature
attempting to evaluate the time-depended multi-
fluorescence quenching performance. Thus, the
methodology and results in this work could be useful
to confirm the dynamic equilibrium of quenching
behavior of fluorophores and if the quenching
performance of target or photosensitizer of sensor
(or multi-sensor) was influenced by other
constituents, which was necessary and essential for
nanosensor design and applications.
Tan, Y. and Umemura, K.
Time-depended Quenching Behaviors of Multi-dye Systems by Single-Walled Carbon Nanotubes.
DOI: 10.5220/0006650802310235
In Proceedings of the 11th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2018) - Volume 1: BIODEVICES, pages 231-235
ISBN: 978-989-758-277-6
Copyright © 2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
231
2 EXPERIMENTAL
Single-walled carbon nanotubes(SWNT) produced
by the high-pressure carbon monoxide (HiPco)
method were purchased from Raymor Nanotech
(Canada) as purified powders. Single stranded DNA
with thymine 30 mers (T30) were purchased from
Life Technologies Japan, Ltd., Tokyo, Japan.
Acridine orange (AO) solution (1 mg/mL H
2
O),
Uranine (Ur, or sodium fluoresecein) and
Rhodamine B (RB) were purchased from Wako Pure
Chemical Industries, Ltd., Osaka, Japan.
Hybrids of T30 and SWNT (SWNT-T30): 1 mL of
T30 solution of deionized water (1 mg/mL) and 0.5
mg of SWNT powder were mixed and sonicated
using a probe-type sonicator in an ice-water bath for
90 min (VCX 130, Sonics & Materials, Inc., CT,
USA) with a 2-mm diameter tip at 3 W (Tahahashi
et al., 2004: 43; Nii et al., 2013: 106). The hybrid
suspension was centrifuged at 12,500 g for 3 h at
8ºC (high speed refrigerated micro centrifuge, MX-
150, TOMY, Japan). After centrifugation, the upper
75% of the volume was stored as the SWNT-ssDNA
suspension.
Ur-H
2
O and RB-H
2
O Solution: 12.4 µg of Ur and
15.8 µg of RB were dissolved in 1 mL of deionized
water, respectively to prepared 0.0124 mg/mL
(0.033mM) Ur-H
2
O solution and 0.0158 mg/mL
(0.033mM) RB-H
2
O solution.
Buffer Solution: 10 mM Tris (hydroxymethyl)
aminomethane-HCl (Tris) (pH = 8.0)
Fluorescence Measurement: The fluorescence
spectra of dyes, dye/SWNT-T30 were recorded for
different concentration by JASCO FP-6500
spectrophotometer with an excitation wavelength at
490 nm (for Ur) and 553 nm (for RB). Fixed amount
of dye-H
2
O origin solution and SWNT-T30 solution
were mixed and diluted with the buffer solution
(Tris, 10 mM) up to 2 mL to prepare the
fluorescence samples. During the time-depended
fluorescent monitoring, a rotator was used for
keeping the uniformity of sample system.
Vis-absorption (UV) Measurement: The excitation
wavelength for Ur systems and RB systems were
confirmed respectively via UV spectra recorded by
JASCO V-630 spectrophotometer.
Raman Measurement: Raman spectroscopy was
performed to examine the structural conditions of
SWNT. To prepare the samples, SWNT-T30
suspension was dropped to a glass slide and then
dried at room temperature. NRS-3200 Micro Raman
system (JASCO Corporation, Tokyo, Japan) was
used for the spectroscopy, with 632.8 nm He-Ne
laser excitation sources.
3 RESULTS AND DISCUSSION
3.1 Quenching Equilibrium and
Quenching Efficiency of Single- and
Multi- Fluorophore System
Figure 1: Time-depended fluorescent spectra of Ur and
RB in single-fluorophore(2µL)/SWNT-T30(10µL) system
and multi-fluorophore(2µL/2µL)/SWNT-T30(10µL)
system, respectively. All the samples were diluted to 2 mL
by Tris (10 mM). The excitation and emission wavelength
was set as 490 nm and 512 nm for Ur, 553 nm and 572 nm
for RB, respectively.
The quenching performance of Ur and RB were
confirmed via time-depended fluorescent spectra as
soon as the multi-fluorophore sample of
Ur(2µL)/RB(2µL)/SWNT-T30(10µL) was prepared.
As shown in Figure 1, the fluorescent intensity of Ur
(black) and RB (red) remained constantly after 10
min respectively, or the equilibrium state of Ur and
RB could be realized in 10 min. As comparison, we
also compared the performance of Ur and RB in
single-fluorophore quenching system (for instance,
Ur(2µL)/SWNT-T30(10µL) and RB(2µL)/SWNT-
T30(10µL)). The same proportion of fluorophore to
SWNT, the same concentrations of fluorophores and
SWNT-T30 were made sure for both single- and
multi-quenching systems. Less than 10 min was
spent to reach the quenching equilibrium for Ur
(blue) and RB (pink), similar with multi-fluorophore
system.
Moreover, we also could see that the fluorescent
intensity of Ur at the equilibrium state in both multi-
fluorophore/SWNT-T30 system (black or blue) and
single-fluorophore/SWNT-T30 system (red or pink)
0 5 10 15 20 25 30
1400
1500
2000
2500
3000
3500
4000
4500
5000
Fluorescent Intensity
Time (min)
Ur (Ur/RB/SWNT-T30)
RB(Ur/RB/SWNT-T30)
Ur (Ur/SWNT-T30)
RB(RB/SWNT-T30)
BIODEVICES 2018 - 11th International Conference on Biomedical Electronics and Devices
232
did not fluctuated obviously, at approximate 4500-
4600 (Ur) or 1550-1570 (RB). It was illustrated that
under the existence of another fluorophore (for
example, Ur), the absorption coverage of one
fluorophore (for instance, RB) onto SWNT surface
seemed not be dramatically influenced compared
with the case of single-fluorophore/SWNT-T30
quenching system.
Quenching efficiency was further calculated and
researched via T-test analysis. The fluorescent
intensity of Ur and RB with and without SWNT-T30
in both single-fluorophore/SWNT-T30 system and
multi-fluorophore/SWNT-T30 system were obtained,
then the corresponding quenching efficiency was
compared. All the intensity was measured after the
prepared samples set for 30 min to reach equilibrium
state. As T-test is a statistical hypothesis test which
could be used with small sample sizes (usually less
than 30 samples) to determine if two sets of data
are significantly different from each other, each
experiment here was carried out 10 times for
determination.
As listed in table 1, 13.0±1.0 % and 12.0±0.5 %
emission of Ur was quenched by same content of
SWNT-T30 with and without RB exist, respectively;
as far as RB, about 42.3±1.2 % and 44.9±1.5 % of
fluorescence was restrained in single- and multi-
quenching systems. The degree of freedom (P value)
of T-test was calculated via TTEST function of
EXCEL software based on 10 quenching efficiency
values to confirm whether the quenching behavior of
Ur or RB in single- and multi-quenching system is
different.
In function TTEST (array1, array2, tails, type),
the data of array 1 and array 2 came from the
quenching efficiencies of single- and multi-
fluorophore system, respectively; tails was set as 2
(two-tailed test); type was set as 2 (normal
distribution). The P value obtained from Ur and RB
was 0.0206 and 0.0367, less than 0.05 (usually the
statistical significance is set as 0.05), illustrated that
the quenching performances of both Ur and RB were
not identical in different quenching systems.
We presumed that Ur showed a mild higher
quenching efficiency in single-fluorophore system,
while RB in multi-quenching system exhibited a
little stronger quenching efficiency; although the
variances of corresponding efficiency values were
seemingly slight.
Table 1: Quenching efficiency and T-test result of Ur and RB quenching system by SWNT-T30.
Ur
RB
Quenching efficiency
(%)
P value
Quenching efficiency
(%)
P value
Ur(2µL)/SWNT-T30(10µL)
13.0 ± 1.1
0.0206
-
0.0367
RB(2µL)/SWNT-T30(10µL)
-
42.3 ± 3.4
Ur(2µL)/RB(2µL)/SWNT-T30(10µL)
12.0 ± 0.5
45.0 ± 1.5
Quenching efficiency = [1- (intensity with SWNT-T30)/(intensity without SWNT-T30)] × 100%
: P value is the degree of freedom of T-test, in function TTEST (array1, array2, tails, type), the data of array 1 and array 2 come from the
quenching efficiencies of samples of single-fluorophore system and multi-fluorophore system; tails was set as 2 (two-tailed test); type was
set as 2 (normal distribution), if P value is more than the statistical significance (usually 0.05), the array 1 and array 2 are assumed to be
equal.
Table 2: Fluorescent intensity and T-test results of converse-sequent quenching systems.
RB
intensity
P value
intensity
P value
Ur + RB + SWNT-T30
30 min
1454 ± 57
/
0.6388
4656 ± 165
/ 0.8818
Ur + SWNT
30 min
+ RB
30 min
1441 ± 41
/ 0.8279
4105 ± 137
/ 1.71E-06
RB + SWNT
30 min
+ Ur
30 min
1445 ± 20
/ 0.7014
4019 ± 105
/ 9.39E-08
The exciting wavelength and emitting wavelength of Ur, AO, RB was set as (490 nm and 512 nm) and (553 nm and 572 nm), respectively.
: P value is the degree of freedom of T-test, in function TTEST (array1, array2, tails, type), tails was set as 2 (two-tailed test); type was set
as 2 (normal distribution), if P value is more than the statistical significance (usually 0.05), the array 1 and array 2 are assumed to be equal.
/
0.6388: in function TTEST (array1, array2, tails, type), the data of array 1 and array 2 come from the fluorescent intensity records
of assay and assay ; 0.6388 is the P value calculated from intensities of assay and assay .
Time-depended Quenching Behaviors of Multi-dye Systems by Single-Walled Carbon Nanotubes
233
3.2 Simultaneous Quench” and
Un-Simultaneous Quench”
Considering the competitive adsorption between two
kinds of fluorophores, it was admired that if the
addition order of two fluorophores alters the
quenching equilibrium, because sometimes the
multi-detection application cannot satisfy the
simultaneous quench condition. Here three
experiment schemes were designed, e.g., 1) 2 µL Ur
and 2 µL RB were mixed and diluted firstly, then 10
µL of SWNT-T30 was added (the final mixture
volume was 2 mL), set for 30 min and the
fluorescent intensity of Ur and RB was measured
respectively; 2) 2 µL Ur and 10 µL SWNT-T30 was
mixed and diluted, set for 30 min; then 2 µL RB was
added, set for 30 min (the final mixture volume was
2 mL) and the fluorescent intensity was measured; 3)
2 µL RB and 10 µL SWNT-T30 was mixed and
diluted, set for 30 min; then 2 µL Ur was added into
mixture, set for 30 min (the final mixture volume
was 2 mL) and the fluorescent intensity was
measured. Each experiment above was carried out in
10 times for determination. The final fluorescent
intensity and quenching performance of Ur and RB
in three systems were listed in Table 2.
The final Ur intensity after quenched by SWNT-
T30 from “simultaneous added” model was
calculated as 1541 ± 57, less than those from “un-
simultaneous added” model ( and ,1441 ± 41
and 1445 ± 20). In order to confirm if the 3
distributions could be regarded from the same one,
T-test analysis was carried out for paired samples of
assay and , and , and , respectively.
P values (the freedom degree) were extracted as
0.8279, 0.7014 and 0.6388, all above 0.05, the
threshold of statistical significance, demonstrated Ur
performed similar quenching property in the three
quenching situations. Different results were gained
for RB, an average intensity of 4656 ± 165 in
“simultaneous added” model was more than those of
4105 ± 137 in “Ur added first” model and 4019 ±
105 in “RB added first” model. The P value of
0.8818 gained via comparing the two “un-
simultaneous added” models /, larger than 0.05,
suggested that there was not difference for optical
properties of RB in multi-fluorophore quenching
systems in which whether RB was quenched first or
not. However, extremely low P values, 1.71E-06 and
9.39E-08, obtained by comparing “simultaneous
added” model with un-simultaneous added” model
( or ), indicated the independent photo-
performance of RB in two models; more RB
molecules attached to the SWNT surface and lower
photo-luminescence was observed in the “un-
simultaneous added” model.
Thus, Ur showed similar quenching performance
in both “simultaneous added” model and “un-
simultaneous added” model. For RB, it was
seemingly indicated that in “un-simultaneous added”
model, SWNT-T30 could not quench RB as much as
“simultaneous added” model; while in two “un-
simultaneous added” model, the RB added sequence
had no effects on the quenching performance of RB.
We made a hypothesis that the adsorption of
fluorophore onto SWNT surface was a dynamic
balance process, equal number of fluorophore
molecules attach onto and detach from SWNT
surface at the same time. In the “un-simultaneous
added” model, for example, before RB added, there
had been an equilibrium state of adsorption and
desorption of Ur onto SWNT surface; when RB
added into the quenching system, several sites of Ur
was replaced by RB during the Ur molecules
departed from SWNT surface. Perhaps RB exhibited
stronger scramble ability over Ur for SWNT surface
occupation, which could also be verified by the
quenching efficiency result of single- and multi-
fluorophore quenching system (see section 3.1 and
Table 1), RB showed slighter higher quenching
efficiency in multi-system than single-system while
Ur appeared a contrary tendency.
Moreover, RB itself seemed attached onto
SWNT surface easier in the un-simultaneous
added” model than “simultaneous added” model. It
was try to be explained that in “simultaneous added”
model, Ur and RB molecules collided more
intensely, so that the attaching probability on SWNT
surface decreased steadily. Also in section 3.1 and
Table 1, RB showed slight higher coverage onto
SWNT surface in competitive system (or multi-
system) than un-competitive system (or single-
system), we thought perhaps it was a little difficult
for RB molecules detach from SWNT surface
because they were blocked by the Ur molecules
surrounded.
As far as the unchanged Ur appearance in all the
three quenching situations, it was proposed that
perhaps there was tiny differences, but the violation
could be ignored, as Ur exhibits a strong quench-
restrain ability to SWNT surface, based on our
previous work.
3.3 Raman Feature
Raman spectra of SWNT-T30 (black), SWNT-
T30/Ur (blue) and SWNT-T30/RB (red) were
measured for evaluating the effects of fluorophores
BIODEVICES 2018 - 11th International Conference on Biomedical Electronics and Devices
234
to SWNT surface. The volume ratio of SWNT-T30
origin solution to fluorophore origin solution was set
as 10:1. The G-band and D-band (see the arrows)
were clearly observed, the shapes were similar in all
the samples. As shown in the features, SWNT’s G
band shift of both SWNT-T30/Ur (blue) and SWNT-
T30/RB (red) located at 1580 cm
-1
, the same as that
of SWNT-T30 (black), maybe indicated that the
fluorophore-wrapping did not affect the vibrations of
SP
2
hybrid orbital of carbon atoms. Usually the
frequency of D band is sensitive to the lattice defects
of carbon atoms, here D band assigned to 1332 cm
-1
for both SWNT-T30 (black) and SWNT-T30/Ur
(blue), however, 2 split peaks (1336 cm
-1
and 1361
cm
-1
) appeared in the case of SWNT-T30/RB (red).
Perhaps some lattice deformation occurred during
RB attached onto SWNT surface compared with Ur.
Figure 2: Raman features of SWNT-T30, SWNT-T30/RB
and SWNT-T30/Ur.
4 CONCLUSIONS
Our analytical assays present time-depended
quenching behaviors of alone-fluorophore and multi-
fluorophore by SWNT-T30. All the quenching
equilibrium could be realized in 10 min. Ur showed
a mild higher quenching efficiency in single-
fluorophore system, while RB in multi-quenching
system exhibited a little stronger quenching
efficiency.
The effects of “simultaneous quenched” and “un-
simultaneous quenched” on the quenching properties
of Ur/RB/SWNT-T30 multi-quenching system were
researched. Ur showed similar quenching
performance in both “simultaneous added” model
and “un-simultaneous added” model; RB itself
seemed attached onto SWNT surface easier in the
un-simultaneous added” model than “simultaneous
added” model. In un-simultaneous added” mode,
the RB quenching sequence had no effects on the its
quenching performance. As shown in Raman results,
fluorophore-wrapping did not influent the vibrations
of SP
2
hybrid orbital of carbon atoms in SWNT;
some lattice deformation occurred during RB
attached onto SWNT surface compared with Ur. The
fundamental methodology and results in this work
were necessary and essential for multi-nanosensor
applications, and could be useful for the further and
extended application of fluorophores/dyes detection
and quantification in aqueous solution.
ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for
Scientific Research (26400436) of the Japan Society
for the Promotion of Science (JSPS).
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1000 1250 1500 1750 2000 2250
Raman Intensity
Raman Shift (cm
-1
)
SWNT-T30
SWNT-T30/RB (10:1)
SWNT-T30/Ur (10:1)
G
D
Time-depended Quenching Behaviors of Multi-dye Systems by Single-Walled Carbon Nanotubes
235