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

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

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

O and RB-H

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

O solution and 0.0158 mg/mL

(0.033mM) RB-H

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

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.

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.

intensity

P value

※

intensity

P value

※

① Ur + RB + SWNT-T30

30 min

→

1454 ± 57

②/③

＃

0.6388

4656 ± 165

②/③ 0.8818

② Ur + SWNT

30 min

→

+ RB

30 min

→

1441 ± 41

①/② 0.8279

4105 ± 137

①/② 1.71E-06

③ RB + SWNT

30 min

→

+ Ur

30 min

→

1445 ± 20

①/③ 0.7014

4019 ± 105

①/③ 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.

②/③

＃

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 ② and assay ③; 0.6388 is the P value calculated from intensities of assay ② and assay ③.

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 ① “simultaneous added” model was

calculated as 1541 ± 57, less than those from “un-

simultaneous added” model (② and ③,1441 ± 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 ① and ②, ① and ③, ② and ③, 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 ①

“simultaneous added” model was more than those of

4105 ± 137 in ② “Ur added first” model and 4019 ±

105 in ③ “RB added first” model. The P value of

0.8818 gained via comparing the two “un-

simultaneous added” models ②/③, 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 ① “simultaneous

added” model with “un-simultaneous added” model

(② or ③), 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

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

-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

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)

Time-depended Quenching Behaviors of Multi-dye Systems by Single-Walled Carbon Nanotubes

235