Formation of Photosensitizing Crystalline C<sub>60</sub> Particles by Ink-Jet Method

doi:10.4236/wjnse.2012.22014

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World Journal of Nano Science and Engineering, 2012, 2, 110-115

http://dx.doi.org/10.4236/wjnse.2012.22014 Published Online June 2012 (http://www.SciRP.org/journal/wjnse)

Formation of Photosensitizing Crystalline C60

Particles by Ink-Jet Method

Masahito Ban, Fusako Sasaki

Systems Engineering Major, Graduate School, Nippon Institute of Technology, Saitama, Japan

Email: [email protected]

Received March 26, 2012; revised April 24, 2012; accepted May 8, 2012

ABSTRACT

The crystalline fullerene C60 particles were formed and immobilized on poly(dimethylsiloxane) (PDMS) substrates un-

der the various discharge conditions by an ink-jet method, and investigated for the reactive oxygen species (ROS) gen-

eration property under visible light irradiation. The particles were synthesized by discharging a toluene solution dis-

solved C60 and poly(methyl methacrylate) (PMMA) with the ink-jet spotting system. The ROS generation was evalu-

ated by comparisons of the fluorescence intensities measured for the formed particles under green laser irradiation and

in a dark room using fluorescent dyes, 2’,7’-dichlorofluorescein diacetate and dihydroethidium. The results of transmis-

sion electron microscope (TEM) observation showed that the formed particles consisted of crystalline C60. The optimal

ink-jet discharge conditions for synthesizing the particles to generate more ROS were found. In the case of the optimal

conditions, the structure in which the needle-like particles were three-dimensionally formed was confirmed. The surface

area of the crystalline C60 particles was calculated using the SEM observation results, and it was suggested that when

the needle-like finer particles were three-dimensionally formed under the optimal conditions, increasing the surface area

lead to an increase in the ROS generation amount.

Keywords: Fullerene; Particle; Ink-Jet; Reactive Oxygen Species

1. Introduction

Fullerenes, carbon allotropes, represent the unique phy-

sical and chemical properties, and have incited a consid-

erable hope of their potential for uses in biomedical ap-

plication. One of the biologically most relevant features

of fullerenes is mediating generation and quenching of

reactive oxygen species (ROS) under visible light irra-

diation [1]. As recent studies on ROS generation by

fullerenes, Sayes and co-workers reported that pure C60

brought into water by means of solvent extraction formed

water-stable crystalline aggregates (called nano-C60 or

nC60) in the size of about 100 nm to generate high amount

of ROS, and kill both normal and tumor cells at extre-

mely low concentrations [2]. Also, by Markovic and co-

workers, the cytotoxicity/ROS production of different

nC60 suspensions prepared using tetrahydrofuran, ethanol

and water were examined for investigating the mecha-

nisms of the cytotoxicity [3]. In this way, many studies

on ROS generation by C60 have been performed using

solutions with colloidal dispersions of C60. While, we

have up to date studied an method to immobilize C60 at

the specific locations on the surface of a polymer sub-

strate, for the purpose of applying the photosensitizing

properties of C60 as the function of microfluidic and mi-

croarray chips, called μ-TAS (micro total analysis sys-

tems). The μ-TAS is a technology having the ability to

perform the chemical operations such as mixing, reaction

and separation by inserting a small amount of solution

into various microstructures formed on the chip, and has

recently become a candidate for the biological applica-

tions [4].

On the other hand, an ink-jet technology has been

widely used for not only the direct-writing in the printing

field, but also the manufacturing of micro-optical parts

and polymer electronics [5]. One of important features of

the versatile ink-jet method is modifying a surface locally

by means of discharging extremely small amount of solu-

tion on the local area. As for the applications of the

ink-jet method to μ-TAS, the microarrays with covalent

attachment of DNA were fabricated by discharging 5’-

terminal-thiolated oligonucleotides to a glass surface using a

Bubble Jet method [6], and very recently Abe and co-

workers demonstrated “all-inkjet-printed” microfluidic

multianalyte chemical sensing paper for the simultaneous

determination of pH, total protein and glucose in clini-

cally relevant concentration ranges for urine analysis [7].

In this study, by discharging a fullerene solution using

the ink-jet method, crystalline C60 particles were synthe-

sized and immobilized at the specific locations on poly

M. BAN, F. SASAKI 111

(dimethylsiloxane) (PDMS) substrates, and evaluated for

the ROS generation under visible light irradiation [8]. In

addition, the effects of the discharge conditions and the

structure of formed particles on the ROS generation were

investigated [9].

2. Experimental

2.1. Ink-Jet Discharging

A picojet 2000-CW ink-jet spotting system (Microjet Co.

Ltd., Japan) having the ability to discharge extremely

small amount of a solution was used in this study. This

system is equipped with a three-axis and microposition-

ing system of 1 μm accuracy, and a piezo-driven nozzle

with 30 μm in diameter. A stroboscopic camera system

allows visual monitoring to adjust piezo voltages and

pulse durations for reliable droplet ejection. The vertical

separation between the nozzle and the substrate was

typically 0.5 mm. As the solution, a fullerene solution

was prepared by dissolving C60 (Frontier carbon corpora-

tion: nanom purple ST) of 35 mg and poly(methyl

methacrylate) (PMMA) of about 10 mg in toluene of 40

ml. PDMS flat substrates were fabricated with SILPOT

184 W/C (Dow Corning Toray Co. Ltd.). As the discharge

conditions, the number of droplets per one discharge,

“droplets per discharge”, was varied from 10 to 20, and

the droplets were discharged 10 to 40 times onto the

same spots on the PDMS substrates. The product of

droplets per discharge and the number of discharges is

expressed as “total droplets”. The 11 × 11 spots were

arranged with the distance of 250 μm between the centers

to form the shape of a square.

2.2. Structural Evaluation

The surfaces of the PDMS substrates discharged the

fullerene solution by the ink-jet spotting system were

observed by a scanning electron microscope (SEM). A

transmission electron microscope (TEM) was used for

observations of the fullerene solution discharged on a

TEM grid using the ink-jet spotting system.

2.3. ROS Generation Evaluation

The production of ROS was determined by measuring

the fluorescence intensities emitted by fluorescent dyes;

2’,7’-dichlorofluorescein diacetate (DCF-DA) with 488

nm excitation and 530 nm emission, and dihydroethi-

dium (DHE) with 488 nm excitation and 600 nm emis-

sion. The evaluation was performed by the following

procedure. First, DCF-DA and DHE were soluted in pure

water to obtain a DCF-DA solution (6.0 μM) and a DHE

solution (2.3 μM), respectively. Second, each solution

was degassed by flowing argon gas in it at a flow rate of

200 sccm for 1h. The PDMS substrates (5 × 5 × 1t mm)

with the areas spotted by the ink-jet system were im-

mersed in the DCF-DA and DHE solutions in Quartz

cells. And then green lasers (532 nm, 1 mW) were irradi-

ated to the spotted areas on the substrates up to 4 h (“irra-

diation”), and fluorescence intensities were measured by

using the fluorescence spectrometer (Hitachi: F-7000) at

given times. As the comparison, the spotted PDMS sub-

strates immersed in the DCF-DA and DHE solutions in a

dark room were evaluated up to 4 h in the same manner

(“dark”), and pure water was measured as a control

(“pure water”).

3. Results and Discussion

3.1. Structural Evaluation

Figure 1(a) shows a typical image obtained from the

SEM observation of the area spotted on a PDMS sub-

strate by the ink-jet spotting system. In this case, ten

droplets were discharged 40 times onto the same area. As

the comparison, the fullerene solution of 10 μl was pi-

petted off onto a PDMS substrate, shown in a typical

SEM image of Figure 1(b). It is obvious from Figure

1(a) that fine needle-like particles with a few μm size

were numerously precipitated on the substrate. As can be

seen in Figure 1(b), the particles formed by the pipetting

had much larger size, and were likely the aggregate con-

sisted of a number of particles. The results indicated that

(a)

(b)

Figure 1. Typical SEM images of the area (a) Spotted on a

PDMS substrate with the discharge conditions, the droplets

per discharge of 10 and the total droplets of 400, by the

ink-jet spotting system; and (b) Pipetted off a fullerene so-

lution of 10 μl onto a PDMS substrate.

M. BAN, F. SASAKI

112

the ink-jet process was responsible for the formation of

finer particles. For the ink-jet spotting the volume of the

droplet discharged was extremely small, several pl, while,

in the case of conventional pipetting, approximately 0.1

μl at minimum, and therefore it is suggested that the

rapid evaporation of a solvent occurred to inhibit the

growth of particles. A typical SEM image of the ink-jet

spotted surface is shown in Figure 2(a), being magnified

than the image of Figure 1(a). It was demonstrated that

numerous needle-like particles with about 1 μm in di-

ameter seemed to be partially embedded in a film-like

residue, which might be remaining PMMA. Figure 2(b)

shows a perspective SEM image of same spotted surface

as Figure 2(a), indicating that the film-like PMMA resi-

due including the particles adhered properly to the

PDMS substrate. It was implied that the PMMA played

an important roll in immobilizing the particles on the

PDMS surface.

Figure 3 indicates the TEM observation results for (a)

particles on the TEM grid and (b) the magnification of

the edge (circle in (a)) of a particle. The particle shown

in Figure 3(a) had a needle or brush-like shape, which

was in good agreement with a typical shape of particles

formed on the PDMS substrates by various discharge

conditions in this study. Figure 3(b) suggests that the

lattice spacings between adjacent lattice plains are about

0.50 and 0.81 nm, corresponding to the (110) and (111)

plane spacings of face-center-cubic (fcc) C60 crystal, respec-

tively [10]. Therefore, the observation results implied

that the particles formed on the PDMS substrate (see

Figure 1(a) and Figure 2) consisted of crystalline C60.

(a)

(b)

Figure 2. Typical SEM images of the ink-jet spotted PDMS

substrate, (a) The surface and (b) The perspective view.

(a)

(b)

Figure 3. TEM observation images of (a) Particles on a

TEM grid and (b) The magnification of the edge of a parti-

cle seen in (a).

3.2. ROS Generation Evaluation

Figure 4 demonstrates the measurement results by a

fluorescence spectrometer, (a) and (b) in the cases of

using DCF-DA and DHE fluorescent dyes, respectively,

for the PDMS substrates formed crystalline C60 particles

under the conditions; the droplets per discharge of 10 and

the total droplets of 400. In Figure 4(a), the variations in

an intensity (I) of DCF-DA emitted green fluorescence

(488 nm) normalized to the initial intensity value (I0)

obtained at 0 h (before the measurement) are plotted as a

function of the measurement time. Concerning the crys-

talline C60 particle formed PDMS substrate irradiated a

green laser (irradiation) and the same PDMS substrate in

the dark room (dark), the values of I/I0 monotonously

increased with an increase in the measurement time, and

I/I0 for pure water maintained a virtually constant value.

In the case of “irradiation”, the increasing rate of I/I0

against the measurement time was larger than that of

“dark”, so that it was suggested that the crystalline C60

particles had the ability to generate ROS, hydrogen per-

oxide, by green laser irradiation. As is the case in Figure

4(a), Figure 4(b) indicates the variations of I/I0 obtained

from red fluorescence emission (600 nm) by DHE as a

function of the measurement time. I/I0 of “irradiation”

was higher than that of “dark”, implying that the crystal-

line C60 particles mediated the generation of ROS, su-

peroxide anion, under green laser irradiation. The reason

why I/I0 of not only “irradiation” but also “dark” were

M. BAN, F. SASAKI 113

(a)

(b)

Figure 4. Variations in intensities (I) of (a) DCF-DA and (b)

creased is thought to be an influence of oxidation of

sults of fluorescence measurements for the

DHE emitted fluorescences normalized to the initial inten-

sity values (I0) obtained at 0 h as a function of the meas-

urement time for the PDMS substrate spotted under the

conditions of 10 droplets per discharge and 400 total droplets.

DCF-DA and DHE by dissolved oxygen in a solution,

since the amount of the dissolved oxygen after the meas-

urement of 4 h indicated the value near that before the

degassing.

As the re

rticles formed at various discharge conditions, the ratios

of “irradiation” to “dark” for the increasing rates of in-

tensities in 3 h are shown in Figure 5 as functions of

both the droplets per discharge and the total droplets; (a)

and (b) in the cases of using DCF-DA and DHE fluores-

cent dyes, respectively. Note that an increase in the ratio

means an increase in the amount of generated ROS; hy-

drogen peroxide and superoxide anion in the cases of (a)

and (b), respectively. The figure indicated that the larger

ratios were obtained in the case of the discharges under

the conditions in the droplets per discharge of 10 to 20

and the total droplets of 400 compared to the other dis-

charge conditions. The results revealed that the optimal

discharge conditions to form the particles for generating

more ROS existed. Figure 6 shows typical images ob-

tained from the SEM observations of the crystalline C60

particles formed on the PDMS substrates represented as

A, B and C in Figure 5(a). Namely, the droplets per dis-

charge and the total droplets are A 10 and 100, B 10 and

(a)

(b)

Figure 5. Ratios of “irradiation” to “dark” for the increasing

rates of intensities in 3 h as functions of both the droplets per

discharge and the total droplets; (a) and (b) in the cases of

using DCF-DA and DHE fluorescent dyes, respectively.

(a) (b)

(c)

Figure 6. Typical SEM obseion images of the crystalline

00, and C 40 and 800, respectively. Figure 6(a) shows

rvat

C60 particles formed on the PDMS substrates represented

as (a), (b) and (c) in Figure 5(a). The droplets per discharge

and the total droplets are (a) 10 and 100; (b) 10 and 400;

and (c) 40 and 800, respectively.

that needle-shaped particles with 0.5 to 1.0 μm in diame-

ter and several μm length were formed on the substrate.

The particles seen in Figure 6(b) had finer needle-like

shape and were much numerously formed than those in

M. BAN, F. SASAKI

114

Figure 6(a). For Figure 6(c), the particles having a shape of

relatively grain were synthesized, and formed sticking to

each other. Therefore, it is noted that at the optimal dis-

charge conditions for generating more ROS, that is to say,

the droplets per discharge of 10 to 20 and the total drop-

lets of 400, the finer needle-like particles were three-di-

mensionally formed as seen in Figure 6(b). Additionally,

the SEM observation results of all ink-jet spotted PDMS

substrates suggested that the particles formation state was

transformed from planar into three-dimensional structure,

and from needle-shaped into grain-shaped particles with

an increase in both the droplets per discharge and the

total droplets.

To investigate the relation between the formation state

les formed in the various

particles and the ROS production, the total surface

area of the crystalline C60 particles formed on the PDMS

substrate was estimated by the following calculation pro-

cedure. The total amount of the fullerene solution dis-

charged was calculated by the product of an amount of

one droplet (4.2 pl, supposing that the diameter was 20

μm) and the number of total droplets. The weight of all

crystalline C60 particles formed on the PDMS substrate

was calculated with the total discharged amount and the

concentration of the C60 dissolved in the solution, 35

mg/ml. Here, the weight of one crystalline C60 particle

was obtained using the density of C60, 1.73 g/cm3 [11],

and the sizes of the crystalline C60 particles measured

from the SEM images. Assuming that all dissolved fulle-

rene in the fullerene solution was precipitated as the crys-

talline C60 particles, the number of the crystalline C60

particles formed on the PDMS substrate was obtained

from the weight of one crystalline C60 particle and the

calculated weight of all crystalline C60 particles formed

on the PDMS substrate. Multiplying the surface area of

one crystalline C60 particle, which was calculated from

the size measured using the SEM observation results, by

the number of the crystalline C60 particles, the total sur-

face area of the crystalline C60 particles formed on the

PDMS substrate was obtained.

For the crystalline C60 partic

scharge conditions, the variation of the ratio of in-

creasing rate in the case of using DCF-DA (see Figure 5

(a)) as a function of the calculated total surface area of

the particles is shown in Figure 7. The figure revealed

that there appeared to be two tendencies (plots of group I

and II) of the ratio of increasing rate against the total

surface area of the particles. That is to say, for group I

and II, the ratios of increasing rate were increased and

almost unchanged with an increase in the surface area of

the particles, respectively. The crystalline C60 particles

belonging to group I had a three-dimensionally formed

finer structure as observed in the image of Figure 6(b),

and it was suggested that larger exposed particle sur-

face led to more production of ROS. Meanwhile, for the

Figure 7. Variation in the ratio of increasing rate as a fnc-

rystalline C60 particles in group II the increment of the

4. Conclusion

particles were formed on the PDMS

5. Acknowledgements

xpress many thanks to Mr.

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The dotted lines are drawn to guide the eye.

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The crystalline C60

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