6. EXPERIMENTAL METHODS

6.1 Synthesis and main features of detonation nanodiamonds

Detonation nanodiamonds (catalogue reference: PL-Nanopure-G-50 m) were purchased from

PlasmaChem GmbH (Rudower Chaussee 29, D-12489, Berlin). The nanodiamonds were in a 4%

water suspension and were stored in the fridge until they were about to be used.

6.1.1 Synthesis of detonation nanodiamonds

This type of nanomaterial is obtained from the explosion of carbon-containing explosives (e.g.

trinitrotoluene, cyclotrimethylenetrinitramine). Three major steps in the conversion of carbon-

containing explosives to modern detonation nanodiamond products include synthesis, post-

synthesis processing, and modification (see figure 4).

Synthesis Processing Modification

•Composition of explosives•Cooling media

•Removal of metal impurities•Removal of non-diamond carbon

•Deep purification•Size fractionation•Deagglomeration•Alteration surfacegroups•Bonding withmacromolecules•Shell formation•Intrinsic defectsformation

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Figure 4. Schematic representation of the three major stages, and their main steps, in the

synthesis of modern detonation nanodiamonds from carbon-containing explosives.

The processing step included purification of detonation soot synthesis at the vendor’s

laboratories. The result of the processing was detonation nanodiamonds with a purity >87% and

an average size of 5 nm. Additional information of these nanoparticles is included in Table 1. The

explosion method for diamond production results in diamond clusters that are formed from

carbon atoms contained within the explosive molecules; thus, only the explosive is used as a

precursor material. The explosion takes place in a non-oxidizing cooling medium of CO2 gas or

water (ice), so-called ‘dry’ or ‘wet’ synthesis, respectively. To prevent the detonation

nanodiamond formed in the detonation wave from transforming into graphite at the high

temperature generated by the detonation, the cooling rate of the reaction products should be no

less than 3000 K/min62,63. The initial shock from a detonator compresses the high-explosive

material, heating it and causing chemical decomposition, which releases enormous amounts of

energy in a fraction of a microsecond. As the detonation wave propagates through the material, it

generates high temperatures (3500 to 4000 K) and high pressures (20 to 30 GPa) that correspond

to the phase region for thermodynamically stable diamond64. During detonation, the free carbon

coagulates into small clusters, which might grow larger by diffusion65. The product of detonation

synthesis, called “detonation soot” or “diamond blend” contains 40 to 80 wt % of the diamond

phase depending on the detonation conditions66,67. The carbon yield is 4 to 10% of the explosive

weight.

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There are two major technical requirements for detonation nanodiamond synthesis using

explosives; the composition of the explosives must provide the thermodynamic conditions for

diamond formation, and the composition of gas atmosphere must provide the necessary

quenching rate (by appropriate thermal capacity) to prevent diamond transformation to graphite.

The diamond yield depends to a large extent on the explosive mixture and cooling media 66. The

shape of the explosive also influences the yield; the ideal shape is spherical but for convenience a

cylindrical charge is regularly used. The relationship between the mass of the explosives and the

mass of the surrounding media also influences the yield. Thus, 5 kg of explosive requires ∼11 m3

of detonation chamber with gas media at ambient pressure to provide the necessary quenching

rate62,63. The mass of the charge influences the average size of the primary particles, although not

significantly68,69. For example, for charges with a mass of 0.2 to 2 kg, the average primary

particle size is 4 to 5 nm70 as is the case of the detonation nanodiamonds used in this study

Table 1. Additional information of the dry form of the detonation nanodiamonds used in the

development of this investigation. The actual sample was received in a 4% water suspension

directly from the vendor.

Product name Nanodiamond powderCatalogue number PL-D-GAverage particle size 4-6 nmStabilizer NonePurity >87%Ash content <6%

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Impurities, wt % Fe<1.2; Ca+Zn+Cr+Ni+Cu+Mn<2Non-diamond carbón content, wt %

<6

Specific surface area 290 m2/gZeta potential -50 ±5 eV

Biomedical applications set high standards on nanomaterial purity, so the development of

detonation nanodiamond products of ultra-high purity remains an important goal. In addition to

the diamond phase, the detonation soot contains both graphite-like structures (25 to 45 wt %) and

incombustible impurities (metals and their oxides–1 to 8 wt %). The metal impurities originate

from a detonator and from the walls of the detonation chamber. The impurity content of

nanodiamonds produced by detonation synthesis is higher when compared with other artificial

diamonds (for instance, HPHT diamonds contain no less than 96% carbon). After typical

purification steps, powders of detonation nanodiamonds can be considered a composite

consisting of different forms of carbon ( 80% to 89%), nitrogen ( 2% to 3%), hydrogen ( 0.5%∼ ∼ ∼

to 1.5%), oxygen (up to 10%) and an incombustible residue ( 0.5% to 8%). The carbon phase∼ ∼

consists of a mixture of diamond (90% to 99%) and non-diamond carbon (1% to 10%)71 (see

figure ).

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Figure 5. Tentative scheme of major structural components of detonation soot (left) and

commercial DND (detonation nanodiamonds) product (right)69.

For DND purification from detonation soot, mechanical and chemical methods are used. After

mechanically removing process admixtures, the diamond-carbon powder is subjected, for

example, to thermal oxidation with nitric acid under pressure to separate the diamond phase66. In

this method, metals are dissolved and non-diamond carbon is oxidized simultaneously. Other

‘classical’ purification methods, based upon the use of liquid oxidizers for the removal of

metallic impurities, include sulfuric acid, mixture of sulfuric and nitric acids, hydrochloric acid,

potassium dichromate in sulfuric acid, as well as other schemes72.

6.1.2 Miscellaneous optical properties of detonation nanodiamonds

Raman spectrum of detonation nanodiamonds used in this analysis shows a print more like a

disorder amorphous carbon content (peak at approximately 1400 cm-1). The characteristic

shoulder of nanodiamonds at 1332 cm-1 is also visible in the Raman spectrum (see Figure 6).

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Photon correlation spectroscopy is based on dynamic light scattering of the sample. The

time decay of the near-order of the nanodiamonds caused by their Brownian motion was used to

evaluate the size of the nanoparticles via the Stokes-Einstein relation (see figure 7).

Further analysis of the X-Ray diffraction spectrum suggests that the aggregates formed by

detonation nanodiamonds have an extended spatial structure composed of nine or ten clusters,

each involving four or five crystallites with a diamond-like crystal lattice (see figure 8).

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Figure 6. Raman spectrum of detonation nanodiamonds. The peak at A position indicates the

presence of disorder amorphous carbon while the one at B position is a print related to

nanodiamonds.

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Figure 7. PCS (photon correlation spectroscopy) of detonation nanodiamonds. The figure shows

that most of the particles in the sample have an average radius of around 100 nm, indicating a

degree of aggregation.

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Figure 8. X-ray diffraction of detonation nanodiamonds. The equation written on the graph is

Scherrer equation used to obtain the size of the nanodiamonds’ crystallites.

6.2 Fluorescence experiments

In order to accomplish the aforementioned objectives, a laser scanning confocal fluorescence

microscope is used. Confocal microscopy (CM) is a major advance upon conventional light

microscopy since it allows us to create a three-dimensional image of the specimen under

examination. There are many attributes to a confocal microscope that make it the ideal

instrument for biological imaging and particularly for live samples.

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Laser scanning confocal fluorescence microscopy (LSCFM) is built around a conventional light

microscope (see figure 9) and uses laser rather than a lamp as a light source, sensitive

photomultiplier tube detectors (PMTs) and a computer to control the scanning mirrors and to

facilitate the collection and display of the images. The images are subsequently stored using

computer media and analyzed by means of a plethora of computer software either using the

computer of the confocal system or a second computer73. Figure 10 is a schematic representation

of the way LSCFM operates. An overview of the disposition of the main elements that compose

a LSCFM is represented in figure 11. The parts, as they are depicted, are the most commonly

found in a conventional CM system and are briefly discussed in the following paragraphs.

Lasers are used as the light source in confocal microscopy because they provide images

from samples at a single, specific excitation wavelength, and also because the high intensity and

directionality of the laser thus producing high-quality images. In fact, lasers provide sufficient

intensity to compensate for the light lost after passage through different pinholes, thereby

producing a distinct and spatially constrained light point74. Typical lasers area Ar (as the one used

in this work) or ArKr lasers that operate with low tube current and provide a set of lines thar can

be chosen depending on the fluorescent dye that is being excited.

In conventional microscopy, much of the depth or volume of the specimen is uniformly

and simultaneously illuminated in addition to the plane in which the objective lense is focused

(see figure 12A). This leads to out-of-focus blur from areas above and below the focal plane of

interest. Out-of-focus light reduces contrast and spatial resolution, making it difficult to discern

various cellular structures. In contrast, the configuration of a confocal microscope (pinhole +

geometry) avoids out-of-focus signal and so we can get images with improved contrast and

spatial resolution75,76,77.

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Figure 9. A generic LSCM hardware. 1.On/off switch. 2. Fluorescent light on/off. 3. Halogen

on/off. 4. Zero key. 5. Focus key. 6. Focusing drive coarse/fine. 7. Objective toggle. 8. Reflector

toggle. 9. Toggle switch for illumination intensity. 10. Binocular tube component. 11. Eyepiece.

12. Setting ring of the eyepiece. 13. Microscope stage. 14. Stage adjustment. 15. Halogen lamp.

16. HBO illuminator. 17. Scan module. 18. LCD display. 19. Motorized condenser.

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Figure 10. Information flow in a generic confocal microscope. The laser is used as excitation

source, it is filtered and the focused on the sample. The x-y unit scans in one plane. The signal is

then directed to the detector through a pinhole.

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Figure 11. General overview of a typical confocal microscope (Picture courtesy of Carl Zeiss

Inc.).

The illumination is focused as a spot on one volume element of the specimen at a time (see

figure 12B). Depending on the specific microscope design, wavelength of light, objective lens,

and confocal microscope settings, the spot size may be as small as 0.25 μm in diameter and 0.5

μm in depth. As the illumination beam diverges above and below the plane of focus, volume

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elements away from the focal plane receive less illumination, thus reducing some of the out-of-

focus information78.

Figure 12. Comparison of specimen illumination in conventional and confocal microscopes. In

conventional microscopy (A), the whole depth of the specimen is continuously illuminated and

out-of-focus signals are detected. In contrast, with a confocal microscope (B), the specimen is

scanned with one or more finely focused spots of light that illuminate only a portion of the

specimen at a time.

The term confocal refers to the condition where two lenses are arranged to focus on the same

point, therefore, sharing the same foci79. In conventional light microscopy, two-dimensional

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images of the object are formed in the X and Y planes, generally parallel to the plane of

sectioning. This is also true of confocal microscopy, but each image represents only part of the

thickness of the sample. This is because the microscope optics excludes features outside of the

plane of focus. The major optical difference between a conventional microscope and a confocal

microscope is the confocal pinholes, which allow only light from the plane of focus to reach the

detector80 (see figure 13).

The ability of this system to discriminate between light that is not in the focal plane yields

images of higher lateral and axial resolution. Clearly, such a system is limited by its small field

of view. A larger field of view can be obtained by scanning the specimen either by moving it

whereas the microscope remains static or by moving the confocal system over the stationary

specimen81. That is the reason why this system receives the name of LSCFM.

When fluorescent molecules are exposed to a light source of one color, through a

microscope a user will usually see a light of a different color being emitted. This is the basic

principle of fluorescence. Molecules at ground state will absorb a high energy light photon

which increases the energy state of the molecules. Some of the energy from the light is lost

internally within the molecule. The molecule will then quickly move back to its ground state.

For this to occur, energy must leave the molecule, and this happens in the form of an emitted

light. This lesser energy photon being emitted from the molecules will thus lead to a different

color of emitted light in respect to that of the excitation light. The excitation laser must be

suitable to excite the molecule so that both excitation and emission wavelengths are dependent

upon the molecules being examined.

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Figure 13. Schematic representation of the confocal principle using a pinhole. The laser light is

focused at a selected depth in the specimen of interest and reflected light is then refocused onto

the detection system by the same lens. Only returning light refocused through the pinhole is

detected. The light detected and scattered at other geometric angles from the illuminated object

or refocused out of plane with the pinhole is excluded from detection.

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The experiments planned for the present work are schematically shown in figure 14. In the first

part the luminescence spectrum of detonation nanodiamonds (water solution 4%, average size 5

nm) obtained from PlasmaChem (catalog number: PL-D-G) is measured at room temperature

using a laser scanning confocal microscope() and an argon laser (488 nm) as excitation source ()

in order to localize the optical centers present in the sample. Once these color centers are well-

characterized and the optimal parameters of the microscope established, luminescence spectra

are measured at different temperatures within the physiological range (30-60°C) with the aid of a

heating plate. From the previous data and the analysis of the intensity, the area, the half-width

and energy of the emission bands and the ratio among its peaks a nanothermic scale able to

measure the internal temperature of a cancer cell indirectly and non-invasively is investigated.

Determination of the lumi-nescence spectrum of DND’s@ room temperature.

PREVIOUS PHYSICAL CHARACTERIZATION

TEMPERATURE DEPENDENCE

Determination of the lumi-nescence spectra of DND’s in the physiological range of temperatures (30-60�C).

Analysis of the emissionband (intensity, half width, area, peaks ratios…)

Obtention of an appropriatenanothermic scale.

CELL TEMPERATURE Internalization of DND’sinto HeLa cells.

Heat up the HeLa cell and measure its internal tempera-ture.

Figure 14. Three-folded diagram of experiments.

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