FIELD OF INVENTION:
This invention relates to novel diamond like nanocomposite (DIAMAS) material used for
biocompatible coating application. Further, this invention also relates to the process for
synthesize the novel diamond like nanocomposite (DIAMAS).
BACKGROUND OF THE INVENTION:
Diamond-like films have been a subject of intensive research owing to their excellent mechanical
strength and chemical stability and have been widely used as protective coatings for cutting edges
and bioimplants. They possess a gamut of attractive properties that includes extremely high
hardness, atomic-level smoothness, very low friction coefficients, excellent abrasion resistance,
negative electron affinity, chemical inertness, and tailorable optical transparency 2-3. Diamond
films can be coated on a substrate to modify its surface properties and thereby, enhance the
substrate behavior pertaining to friction, wear, corrosion, fretting fatigue and biocompatibility .
Some of the potential applications that have been developed for Diamond like films encompass
the field of hard transparent optical coatings, wear-resistant films in precision machining and
prosthetic implants4, abrasion-resistant5, high thermal conductivity and low thermal expansion
coefficient6 films in microelectromechanical (MEMS) systems. However, conventional Diamond
like films suffers an inherent problem of high compressive stress, leading to eventual
delamination of their coatings from the substrate. This also creates restriction on the thickness of
the film coatings and as well as limits their life and performance values.
Several approaches have been developed in the past to decrease the residual stress and augment
the adhesion strength of Diamond like films. All these approaches are based on creating
nanocomposites of Diamond like films by doping it with either metals, inorganic compounds or
ceramics in different ways to reduce the internal stress of the native diamond like film.

The oldest method was developed by Dorfman et al where they introduce an interpenetrating
network of quartz like amorphous Silicon Oxide (a: SiO) in the native carbon matrix of the
diamond films during the process of deposition. This leads to a two level stabilization of the
matrix - one due to the chemical stabilization of the strong amorphous diamond like carbon
network (a: CH) and amorphous quartz like silicon oxide network (a: SiO) and the second at the
structural level due to the interpenetrating nature of the a:CH and a:SiO networks9. The same
research group also doped the above Nanocomposite with metals from Group 1-7b and 8b of the
periodic table to develop metallic Nanocomposites of Diamond like films. On the other hand
Seiger et al7 have discovered that postannealing can be used to reduce the compressive stress in
amorphous tetrahedral Diamond like films. The effect is due to the alteration of the carbon-
carbon bonding in the film leading to the formation of carbon nanoclusters. Amaratunga et al8
have introduced fullerene like carbon onions into amorphous tetrahedral Diamond like
Nanocomposites using a local high pressure carbon arc method and synthesized films with high
hardness and elastic recovery properties. In spite of such sustained improvement of the field, the
wide applicability of Diamond like films in material science and biomedical engineering has been
severely hindered owing to some of the intrinsic limitations such as high compressive stress
(hindering good adhesion)2-3 that are yet to be surmounted.
In the present scheme of work, inventors have utilized the fundamental science composite
stabilization through nanoparticle dispersion through a unique composition of precursors to
obtain a unique set of matrix characteristics and material properties. A successful attempt has also
been made to exercise control on the nanoparticle size and it's implications on biocompatibility
has been established. As a result, Diamond like films have not only been modified to lower
residual stress and enhance its adhesion properties but also to improve its biocompatibility for
applications in health related technologies.
In the present work, two representative nanocomposites of Diamond films viz. DLN (Diamond-
like Nanocomposite) 0502 and DLN 704 have been synthesized using Plasma Assisted Ion Beam
Technique through a unique combination of HMDSN and HMDSO as precursors with a variation
range of 30-50% each under different plasma conditions. The plasma conditions have been tuned

to achieve a multilevel interpenetrating network synthesis in the film with the formation of
nanoparticles embedded in the matrix. The different factors to control the plasma conditions are
RF frequency (13.56 Mhz), a DC bias voltage of 500-700V and a precursor flow rate value of
0.1-1ml/min.
The network properties of the films were studied using FTIR and Grazing Incidence X- Ray
Diffraction (GIXRD) and the nanoparticle characteristics have been revealed by Atomic Force
Microscopy (AFM). Subsequently, Bovine Serum Albumin (BSA) has been used as a model
protein for protein interaction studies through Atomic Force Microscopy , Bradford Assay and
SDS- Poly-Acrylamide Gel Electrophoresis. BSA is a representative serum protein and serum
proteins come in contact with any prosthetic implanted in the body and bind to its surface thus
modifying its surface properties. Its adsorption conditions a medical equipment to a great extent
for further responses from the body and needs to be addressed as the first stage of
biocompatibility testing. Further, for cell adhesion studies, L929 mouse fibroblast cells have been
used as the model cells. These cells are anchorage dependant cells and require substrate adhesion
for survival. Cytocompatibility studies have been made through the morphological and spreading
analysis of fibroblasts employing Scanning Electron Microscope (SEM).
OBJECTS OF THE INVENTION:
An object of this invention is to propose a novel diamond like nano-composite (DIAMAS)
material.
Another object of this invention is to propose a novel diamond like nano composite (DIAMAS)
material which is stable and adhere well to novel elements like gold and silver.
Still another object of this invention is to propose a novel diamond like nano composite
(DIAMAS) which is bio compatible.

DETAILED DESCRIPTION OF THE INVENTION:
According to this invention there is provided a novel diamond- like nano composite (DIAMAS)
for bio compatible coating application.
In accordance with this invention there is also provided a process for preparing a novel diamond-
like nano composite (DIAMAS) for bio compatible coating application.
SYNTHESIS
Two samples viz. DLN 0502 and 704 were synthesized using Plasma Assisted Electron Beam
technique using a unique combination of cosmetic grade HMDSN and HMDSO as precursors
with a variation range of 30-50% each under different plasma conditions. The plasma conditions
have been tuned to achieve a multilevel interpenetrating network synthesis in the film with the
formation of nanoparticles embedded in the matrix. The different factors to control the plasma
conditions are RF frequency (13.56 Mhz), a DC bias voltage of 500-700V and a precursor flow
rate value of 0.1-1ml/min.
FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR)
The matrix properties of the individual samples were studied using FTIR using a Thermo Nicolet
machine (NEXUS - 870). The IR range used was 400 cm-1 to 4000 cm"1 with a resolution of 4 cm"
'. The samples are prepared for FTIR scans by treating them with Piranha solution for up to 5
minutes to remove any surface impurities. The FTIR scans were then taken in the Variable-
Attenuated Total Reflection (Variable-ATR) mode at an incidence angle of 60°. The FTIR
spectrum of the ambient surrounding was subtracted from that of sample to eliminate the
background noise. The substrate effect due to the glass substrate was removed by subtracting the
sample spectra against that of uncoated pyrex glass spectra, using a proprietary software.

GRAZING INCIDENCE XRD AND HIGH RESOL UTION TEM
The Grazing Incidence X-Ray diffraction spectra were acquired by Philips PW 1710
diffractometer at an incidence angle of 2 ° using Al-Cu Ka radiation with a scanning range of 10°
to 80°. High resolution TEM was done by placing a thin film on a copper grid in a C M 12
PHILIPS TEM.
ATOMIC FORCE MICROSCOPY (AFM)
The surface topography was analyzed using Atomic Force Microscopy in the Tapping mode with
the cantilever tip radius of less than 10 nm. Imaging was done on bare sample surfaces as well as
that with protein adsorbed. For protein adsorption, an aqueous solution of 2% Bovine Serum
Albumin (BSA) was incubated for 1.5 hrs at 37 °C. This was followed by vigorous washing in
PBS for 30 mins. Vigorous washing with PBS was done to ensure only protein molecules with
strong surface interaction were remaining on the DLN surface and are observed under AFM. The
samples were lyophilized to avoid any image artifacts due to tip-water interaction, since the AFM
imaging is done in the air for better resolution of adsorption pattern.
CELL CULTURE
Mouse fibroblast cell line L929 was obtained from National Center for Cell Sciences, India, was
maintained as a monolayer culture in Dulbecco's modified Eagle's medium supplemented with 2
mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (GIBCO), and 10% heat-inactivated
fetal calf serum and incubated in a CO2 incubator (Heraeus) at 37 °C and 5% CO2.
CELL ADHESION
Cell Attachment
L929 Fibroblast cells were harvested by trypsinization and seeded at a concentration of 105
cells/ml on DLN 704, DLN 0502 and glass. Cells were allowed to adhere to the substrate by
incubating at 37 °C and 5% CO2 for 1 h. They were then rinsed with DMEM media and observed
under the optical microscope after an incubation of 1 day.

Scanning Electron Microscopy
Cells were allowed to adhere to DLN 704, DLN 0502 and Glass after incubating at 37 °C and 5%
CO2 for 48 hrs. Then the samples were rinsed thoroughly (3 times) with sterile PBS and cells
were treated with 25% Gluteraldehyde (in PBS buffer). The cells were rinsed, with 50%, 70%,
90%, 95%, 100% Ethanol, progressively two times for 20 mins each with each progressive
concentration of Ethanol. The cells were then finally treated with Isoamyl Alcohol for 5 mins and
observed under SEM after gold deposition, using a JEOL JSM-5800 Scanning Microscope. A
quantitative estimation of the cell spreading was done by estimating the average cell surface area
using NIH Image processing software ImageJ.
PROTEIN ADSORPTION
BSA Study
The samples were incubated with 2% BSA for 1 hr at 37 °C. They were then washed with PBS
for 30 mins and then incubated with 1% SDS for 1 hr. The protein estimation was done using
Bradford Assay.
SDS PAGE
The DLN coated sample were incubated with 2% FBS for 2 h at 37°C. FBS was removed and
samples were transferred to 35 mm disposable petriplates (Tarson),washed with PBS for 30 min,
with gentle agitation. The samples were transferred to new petriplates and incubated with
Laemmli sample buffer for 1 h at 37°C. The sample buffer was removed from each of the
samples, and subsequently stored in different mini-eppendorf tubes at 4°C. The samples were
washed thrice with PBS and transferred again to new petriplates. Samples were incubated with
1% trichloroacetic acid (TCA) at 37°C for 1 h. The TCA was removed from each sample
individually and the recovered TCA was stored in individual mini eppendorf tubes at 4°C. A 10%
separating gel and an upper stacking SDS PAGE gel were prepared. 20 μl of each sample was
loaded into individual wells in the gel, and the gel was run at 120 V for 120 min. FBS sample was
run along side, with 20 μl was added to 5 μl of sample buffer. BSA was run along side as the
molecular weight marker. After completion of the electrophoresis, gel was fixed using a fixative
solution (50% Methanol, 12% Acetic Acid, 37% Formaldehyde 0.05%) for minimum 1 h with
gentle agitation. Fixed gel was stained by Silver-staining method.

RESULTS
ATOMIC FORCE MICROSCOPY
The surface profile of the DLN surfaces reveals several individual peaks which are believed to be
made up of the nanoparticles dispersed in the Diamond-like Nanocomposite matrix. Further
Analysis of the Root Mean Squared Average roughness can be used to predict the average size of
the nanoparticles which has been listed in Table 1. DLN 704 surface also reveals a highly
undulated surface for probably due to the clustering of nanoparticles which has been later
confirmed in the HRTEM studies.

HIGH RESOLUTION TRANSMISSION ELECTRON MICROSCOPY (HRTEM)
A cross sectional view of DLN 704 film at 8K magnification reveals clusters of nanosized
particles. The size of these nanoparticles varies from 50-100 nm. A higher magnification reveals
that the clusters have been formed due to the merging of smaller sized nanoparticles (50-100 nm)
and vary in sizes.

SELECTED AREA ELECTRON DIFFRACTION (SAD)
SAD was taken for regions containing the nanosized particles and areas devoid of them. The
former showed potential debye rings while the latter lacked it, suggesting the polycrystalline
nature of the nanoparticles. The potential Debye rings found in the SAD when matched against
the JCPDF database reveal the d-spacing corresponding to cubic (beta) SiC phase.
FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR)
An FTIR spectrum of DLN 704 shows a strong peak at 1100 cm-1 corresponding to Si-O
stretching mode 17-20. Another peak overlapping with the Si-O peak is seen at 946cm-1 which
possibly corresponds to the amorphous Si-N stretching mode21,33-35. This is different from the
usual Si-N stretching mode peaks32 seen at 844 cm-1 and the shift is due to the interpenetrating
and amorphous nature of the Si3N4 network. This interpenetrating network facilitates cross links
between the amorphous Si-0 network and presence of more electronegative elements like O in
the neighborhood of Si-N bonds, leading to the drift of the Si-N peak to higher wave numbers 22-
23. The density of Si3N4 network, which is signified by the integrated area under the absorption
peak, is lower than that of the Si-O network21. The peak at 795 cm-1 is attributed to the transverse
optical phonon vibrations of Si-C bonds in crystalline  Si-C 24. DLN 0502 also exhibits an
amorphous Si-N peak at 920 cm-1 which implicates SiN matrix characteristics similar to that
observed in DLN 704. The Si-O peak in the FTIR spectrum of DLN 0502 appears at 1070 cm-1.
The 780 cm-1 peak in DLN 0502 indicates presence of amorphous SiC, unlike the crystalline SiC
phase exhibited by DLN 704 25-26. The density of the interpenetrating Si-N network is comparable
to that of Si-0 network due to the comparable magnitude of the integrated areas under their
respective peaks21.
GRAZING INCIDENCE X-RAY DIFFRACTION (GLXRD)
Grazing Incidence XRD has been used to investigate the bulk crystalline nature of the films. The
spectra of the samples show a broad hump and the absence of any sharp peaks. This suggests a
bulk amorphous nature of the chemical network in the samples.

PROTEIN ADSORPTION
Bradford Assay and SDS-PAGE
Table 2. BSA adsorption data after elution with TCA
BSA 2% - Elution with 1%TCA
Sample At
HDLN01
HDLN02
HDLN03
HDLN04
DLN0502 0.018 37.89473684 least hydrophillic
DLN704 0.005 10.52631579 ???
Glass 0.083 174.7368421 slightly hydrophillic
Table 3. BSA adsorption data after elution with SDS
BSA 2% - Elution
with 1% SDS
Concentration of Protein
Sample Absorbance (ug/ml) Nature of the surface
PDLN01 0.293 308.4 Hydrophobic
PDLN02 0.360 378.9 Hydrophyllic - best
PDLN03 0.297 312.6 Hydrophobic
PDLN04 0.337 357.7 Low hydrophillic
PDLN05 0.479 504.2 High hydrophillic
DLN0502 0.338 355.7 least hydrophillic
DLN704 0.311 327.3 ???

The BSA adsorption data from Bradford using TCA and SDS elution separately suggests that a
huge difference in protein eluted is observed in TCA elution whereas minor difference is seen for
SDS elution. This indicates that the interaction between protein and sample is stronger in DLN
0502 as compared to DLN 704. This could be attributed to the smaller nanoparticle size that is
dispersed in the Diamond Nanocomposite and creates surface features closer to the size of the
BSA protein molecule which is of the order of few nanometers. The gel data for DLN 704, DLN
0502 shows that most of the proteins in FBS are adsorbed by these samples including BSA.
Atomic Force Microscopy
The phase image of the protein adsorbed sample reveals multiple protein layers adsorbed on the
surface of DLN 0502. The phase signal is very sensitive to the electrostatic31 interactions between
the protein molecules and the AFM tip reveals the protein layer on BSA adsorbed DLN very
distinctly, when compared to image of the surface without BSA. A comparison of the surface
roughness statistics shows a discernible increase in the mean height of the surface features by a
factor of 2 (increase of approx. 80nm). This implies that a multiple layer of BSA is adsorbed on
the DLN surface (the dimension of BSA is approx 14nm * 4nm). There is also an increase in the
peak to peak height and average root mean squared roughness of DLN 0502 which is possibly
due to the granulated protein adsorption i.e. protein adsorbed as agglomerates.(Table 4)
DLN 704 doesn't reveal any direct adsorption from the phase or height image or RMS roughness
due to the prominent surface features. However, a decrease in the peak to peak height (Table 4)
was observed which suggests a majority adsorption of the BSA molecules in the valley regions of
the undulated surface terrain of DLN 704.

Table 4. Surface Profile statistics of DLN 704 and DLN 0502 before and after BSA adsorption

CYTOTOXICITY
The fibroblast adhesion study reveals good cell viability and density for DLN 0502 and DLN 704
as compared to Blank. Further morphological analysis of the filopodia shows that fibroblasts
incubated on DLN 0502 exhibit higher numbers of filopodia on an average and adopt a stellate or
spindle morphology revealing points of anchorage to the sample surface. DLN 704 and Blank on
the other hand exhibit a more rounded morphology which may have been because they were not
fully spread, or were loosely adhered to the sample surface. The histogram reveals the highest cell
surface area for DLN 0502 and therefore the highest spreading, followed by bare Glass and then
DLN 704.
CONCLUSIONS
MATRIX PROPERTIES
Dorfinan et al9 have introduced the concept of stabilizing DLN films, composed of diamond like
a:CH network, by introduction of another interpenetrating quartz like a: SiO network. Moreover,
the hybrid DLN network exhibits properties native to the constituent networks thus unfolding a
method for tailoring their properties by introducing extra interpenetrating networks. Networks
with desired properties if integrated into the DLN matrix can give rise to exotic properties,
biocompatibility and high adhesion strength being the target of study for this paper.

FTIR spectrum of DLN 704 revealed characteristic peaks corresponding to a:SiO and a: SiN
while AFM, HRTEM and SAD studies unveiled crystalline SiC nanoparticles embedded in the
DLN matrix. The peak corresponding to CH network was not prominent in the FTIR due its
weak nature but nevertheless is expected to be present in the DLN by the virtue of the synthesis
process which causes the distribution of the carbon phase into SiC and diamond like a:CH. The
overall nature of the matrix has been confirmed as amorphous by the absence of peaks in the
GIXRD spectrum. Conclusively, DLN 704 and DLN 0502 exhibit a three phase interpenetrating
network of a:CH, a:SiO and a: SiN with SiC nanoparticles dispersed in the matrix. A control on
the size of the nanoparticles could be exhibited by changing the precursor composition and
plasma parameters.
The novelty in the synthesis of the DLN films was achieved by the successful integration of
several interpenetrating networks like a:SiN, a:SiO, a: SiC and a:CH , which reduces the overall
residual stress in the DLN films9. Silicon nitride was introduced to take advantage of its superior
resistance to mechanical wear, fracture toughness and low coefficient of friction. Also, it has no
cytotoxic effects and has been considered for biomedical application50 owing to its favorable
physiochemical properties and is expected to improve the biocompatibility of DLN films.
Similarly SiC adds to the mechanical strength and cytocompatibility of DLN films51. SiC
nanoparticles observed in the DLN 704 matrix are expected to further reduce the residual stress.
The reduction in the residual stress enhances the adhesion strength of the DLN films and can have
profound implications on its applications in coating biomedical devices and implants e.g. joint
implants, stents etc.
CELL ADHESION AND A TTACHMENT
Cell adhesion results in a directed positioning of the filaments responsible for the contractile
mechanisms of the cell 36 and as such affects the cytoskeleton, cell shape and the behavior of
cells. The cell morphology can thus reflect the degree of adhesion of the cell onto the substrate
and can be used as a parameter for measurement. DLN 0502 and DLN 704 exhibits a stellate
morphology of fibroblasts and therefore believed to promote cell proliferation and colonization of
the surface42.

There are four factors viz. composition, surface energy, roughness and topography of a surface
that affect cellular interactions44-45. A smoother surface and topography, biocompatible
composition and possibly favorable surface energy of DLN 0502 enhance its cytocompatibility.
DLN 704 samples on the other hand exhibit high surface roughness due to larger nanoparticle
sizes, of the order of a few 100 nm, which is probably the main reason for lower fibroblast cell
spreading.
PROTEIN ADSORPTION
The protein interaction was observed to be higher in DLN 0502 which can be attributed to the
smaller size of the nanoparticles dispersed in the matrix. It can be concluded that reducing the
size of the nanoparticles enables stronger protein interaction and favorable biocompatibility of the
samples.
SURFACE PROPERTIES
The high surface roughness of DLN 704 apparent is believed to be due to the presence of
interspersed SiC nanoparticles and their clusters in the DLN matrix. This can have undesirable
effects on the frictional properties of the films although it imparts it a good adhesion property
important for sustainable coating purpose3. DLN 704 due to its prominent surface features doesn't
allow a protein layer cover to form on its surface exposing the surface to direct interaction with
cells and tissues. DLN 0502 on the other hand exhibits a multilayer of BSA molecules adsorbed
on its surface completely restricting the direct interaction of cells with the DLN surface. BSA is
known for its effect on bio-mineralization38, inhibition of bacterial adhesion39 and biofouling40-
4Ias well as plays a role in osteoblast adhesion46' 49 and eliminating non specific protein
adhesion37. Therefore its adsorption pattern can have considerable implications on the overall
biocompatibility of DLN coated implantable biomedical devices and prosthetics.

BIOCOMPATIBILITY
DLN 0502 showed good cell attachment, cell viability and lower vulnerability to biofouling
making it the ideal material among the group of DLN samples analyzed for coating cutting
instruments involving contact with living tissues. Nanoparticle stabilization can tailor the surface
roughness of the DLN due to conglomeration and higher size distribution is believed to have
adverse effects on its frictional properties as well as biocompatibility. DLN 0502 on the other
hand exhibits a smooth surface and is composed of smaller sized nanoparticles dispersed in a
biocompatible amorphous CH, SiC and SiN constituents which are seen to promote cell adhesion
and spreading, even individually. It also exhibits a strong surface adsorption of Serum Albumin
proteins (represented by BSA), believed to have important implications on its overall
biocompatibility.
The implications of different physical and chemical characteristics of these Nanocomposite films
have been studied with respect to its implications on biocompatibility and favorable results have
been obtained. This allows for an immediate application of such films in protecting the cutting
edges of surgical instruments which exhibit the need for good biocompatibility due to its
interaction with live cells and tissues and can evoke unfavorable immune response is such
conditions are failed to be met.
The novelty of the film lies in its unique composition and the biocompatibility achieved through
these compositions. The composition implies the precursor compounds used to synthesize the
films as well the chemical signature of its matrix which has been found to be unique.
The inventiveness lays in the fact that matrix properties of different ceramic and polymeric
materials have been combined in a unique way so as to achieve good adhesion strength of the
DIAMAS films combined with an excellent biocompatibility for intended applications in coating
Surgical and other cutting instruments that come in contact with live tissues.