Formation of Photosensitizing Crystalline C 60 Particles by Ink-Jet Method

The crystalline fullerene C60 particles were formed and immobilized on poly(dimethylsiloxane) (PDMS) substrates under the various discharge conditions by an ink-jet method, and investigated for the reactive oxygen species (ROS) generation property under visible light irradiation. The particles were synthesized by discharging a toluene solution dissolved C60 and poly(methyl methacrylate) (PMMA) with the ink-jet spotting system. The ROS generation was evaluated 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 transmission 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.


Introduction
Fullerenes, carbon allotropes, represent the unique physical and chemical properties, and have incited a considerable hope of their potential for uses in biomedical application.One of the biologically most relevant features of fullerenes is mediating generation and quenching of reactive oxygen species (ROS) under visible light irradiation [1].As recent studies on ROS generation by fullerenes, Sayes and co-workers reported that pure C 60 brought into water by means of solvent extraction formed water-stable crystalline aggregates (called nano-C 60 or nC 60 ) in the size of about 100 nm to generate high amount of ROS, and kill both normal and tumor cells at extremely low concentrations [2].Also, by Markovic and coworkers, the cytotoxicity/ROS production of different nC 60 suspensions prepared using tetrahydrofuran, ethanol and water were examined for investigating the mechanisms of the cytotoxicity [3].In this way, many studies on ROS generation by C 60 have been performed using solutions with colloidal dispersions of C 60 .While, we have up to date studied an method to immobilize C 60 at the specific locations on the surface of a polymer substrate, for the purpose of applying the photosensitizing properties of C 60 as the function of microfluidic and mi-croarray chips, called μ-TAS (micro total analysis systems).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 applications [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 solution 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 coworkers demonstrated "all-inkjet-printed" microfluidic multianalyte chemical sensing paper for the simultaneous determination of pH, total protein and glucose in clinically relevant concentration ranges for urine analysis [7].
In this study, by discharging a fullerene solution using the ink-jet method, crystalline C 60 particles were synthesized and immobilized at the specific locations on poly (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].

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 micropositioning 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 C 60 (Frontier carbon corporation: 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.

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.

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 dihydroethidium (DHE) with 488 nm excitation and 600 nm emission.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 × 1 t mm) with the areas spotted by the ink-jet system were immersed in the DCF-DA and DHE solutions in Quartz cells.And then green lasers (532 nm, 1 mW) were irradiated to the spotted areas on the substrates up to 4 h ("irradiation"), and fluorescence intensities were measured by using the fluorescence spectrometer (Hitachi: F-7000) at given times.As the comparison, the spotted PDMS substrates 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").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 diameter seemed to be partially embedded in a film-like residue, which might be remaining PMMA.

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 C 60 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 (I 0 ) obtained at 0 h (before the measurement) are plotted as a function of the measurement time.Concerning the crystalline C 60 particle formed PDMS substrate irradiated a green laser (irradiation) and the same PDMS substrate in the dark room (dark), the values of I/I 0 monotonously increased with an increase in the measurement time, and I/I 0 for pure water maintained a virtually constant value.In the case of "irradiation", the increasing rate of I/I 0 against the measurement time was larger than that of "dark", so that it was suggested that the crystalline C 60 particles had the ability to generate ROS, hydrogen peroxide, by green laser irradiation.As is the case in Figure 4(a), Figure 4(b) indicates the variations of I/I 0 obtained from red fluorescence emission (600 nm) by DHE as a function of the measurement time.I/I 0 of "irradiation" was higher than that of "dark", implying that the crystalline C 60 particles mediated the generation of ROS, superoxide anion, under green laser irradiation.The reason why I/I 0 of not only "irradiation" but also "dark" were  As the re rticles formed at various discharge conditions, the ratios of "irradiation" to "dark" for the increasing rates of intensities 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 fluorescent dyes, respectively.Note that an increase in the ratio means an increase in the amount of generated ROS; hydrogen 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 discharge conditions.The results revealed that the optimal discharge conditions to form the particles for generating more ROS existed.4 that needle-shaped particles with 0.5 to 1.0 μm in diameter 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 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 discharge conditions for generating more ROS, that is to say, the droplets per discharge of 10 to 20 and the total droplets of 400, the finer needle-like particles were three-dimensionally 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 of les formed in the various di particles and the ROS production, the total surface area of the crystalline C 60 particles formed on the PDMS substrate was estimated by the following calculation procedure.The total amount of the fullerene solution discharged 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 C 60 particles formed on the PDMS substrate was calculated with the total discharged amount and the concentration of the C 60 dissolved in the solution, 35 mg/ml.Here, the weight of one crystalline C 60 particle was obtained using the density of C 60 , 1.73 g/cm 3 [11], and the sizes of the crystalline C 60 particles measured from the SEM images.Assuming that all dissolved fullerene in the fullerene solution was precipitated as the crystalline C 60 particles, the number of the crystalline C 60 particles formed on the PDMS substrate was obtained from the weight of one crystalline C 60 particle and the calculated weight of all crystalline C 60 particles formed on the PDMS substrate.Multiplying the surface area of one crystalline C 60 particle, which was calculated from the size measured using the SEM observation results, by the number of the crystalline C 60 particles, the total surface area of the crystalline C 60 particles formed on the PDMS substrate was obtained.
For the crystalline C 60 partic scharge conditions, the variation of the ratio of increasing 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 C 60 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 surface led to more production of ROS.Meanwhile, for the The crystalline C 60 substrates under the various discharge conditions by the ink-jet spotting system, and investigated for the relationships among the conditions, the shape and the structure of particles, and the ROS generation.The fluorescence measurement results implied that the formed crystalline C 60 particles mediated the generation of ROS, hydrogen peroxide and superoxide anion.The optimal ink-jet discharge conditions, the droplets per discharge of 10 to 20 and the total droplets of 400, existed for synthesizing the crystalline C 60 particles to generate more ROS.It was indicated from the SEM observation results that under the optimal conditions the finer needle-like particles were three-dimensionally formed.It was found that there was a possibility that increasing the surface area of crystalline C 60 particles led to an increase in the ROS generation amount, considering that the particles formed under the optimal conditions had larger surface area.

Figure 1
Figure 1(a) shows a typical image obtained from the SEM observation of the area spotted on a PDMS substrate 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 pipetted 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 consisted of a number of particles.The results indicated that Figure 2(b) shows a perspective SEM image of same spotted surface as Figure 2(a), indicating that the film-like PMMA residue 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 Figure3(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 (Figure 2 .
Figure 2. Typical SEM images of the ink-jet spotted PDMS substrate, (a) The surface and (b) The perspective view.

Figure 3 .
Figure 3. TEM observation images of (a) Particles on a TEM grid and (b) The magnification of the edge of a particle seen in (a).

Figure 6 Figure 5 .
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.

Figure 6 .
Figure 6.Typical SEM obse ion images of the crystalline

Figure 6 (
a) shows rvat C 60 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.

Figure 7 .
Figure 7. Variation in the ratio of increasing rate as a f nc- The authors would like to e Manabu Suzuki of Research & Development Center for Advanced Materials and Technology, Nippon Institute of Technology for TEM observations and the specimen preparation.This work was supported by KAKENHI (22510125).and H. Isobe, "Functiona Water," Accounts of Chemical Research, Vol.36, No. 11, Copyright © 2012 SciRes.WJNSE