Diffractive optical element spectroscopy of biomaterial

Diffractive optical element spectroscopy of biomaterial surface Vladimir Vetterl1,2, Stanislav Hasoň1, Heikki Tuononen3, Martti Silvennoinen3, Kari Myller4, Jiri Vaněk2, Raimo Silvennoinen∗3 1

Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i. Kralovopolská 135, CZ-612 65 Brno, Czech Republic 2 Stomatological Research Center, Faculty of Medicine, Masaryk University, Komenského nám. 2. CZ-662 43 Brno, Czech Republic 3 University of Joensuu, Department of Physics and Mathematics, P.O. Box 111, FI-80101 Joensuu, Finland 4 MGM-Devices Ltd, Länsikatu 15, FI-80110 Joensuu, Finland ABSTRACT Optical properties of different type of surface treatments of titanium biomaterial as polishing, grinding, and chemical etching are investigated in details. The main aim of this study is in sensing the organisation of nano-scale fibrinogen and oligonugleotides adhered on biomaterial surface. Thus permittivity change and the fluctuation in optical roughness of treated titanium surface, when titanium surface is subjected to the contamination of buffer fractions as well as to the contamination of human plasma fibrinogen fraction, are investigated through optical window of a cuvette by using diffractive optical element based sensor. During the progress of this work also optical ellipsometry as a corroborative method was used to verify the attachment of the molecules on the biomaterial surface. Key words: Diffractive optical element, complex refractive index, optical roughness, specular gloss, titanium, biomaterial, ellipsometry

1. INTRODUCTION The investigation of the interactions of biopolymers - especially nucleic acids and proteins – at surfaces is a challenging task in biomedicine. Advances in nucleotide sequencing of genomes of different organisms (including the human genome) stimulated interest in DNA hybridization sensors, which commonly rely on the immobilization of a single-stranded oligodeoxynucleotide (ODN) probe onto a transducer surface to recognize (by hybridization - formation of a DNA duplex) its complementary sequence in the target DNA with unknown sequence. Sequence-specific DNA detection is a topic of great interest because it can be used for the screening of genetic and infectious diseases and identifying decease before any symptoms appear. A number of conceptions of electrochemical sensor were available based on carbon, gold or ITO electrodes, differing in the ways of DNA immobilization at the electrode surface and methods of hybridization detection [1]. Immobilization of the probe oligonucleotide at the electrode surface can be performed covalently using thiolated oligonucleotides which are bound to the electrode. Another method of immobilization use the self-assembled layers of thiols to fasten the oligonucleotide probe [2]. Immobilization is affected by the physical properties of the electrode surface (etching etc.). One of the promising method of hybridization detection is electrochemical impedance spectroscopy [3] and optical methods. Titanium is frequently used as a biomaterial for hard tissue replacement, such as dental and orthopaedic implants. Biomaterial devices made of titanium give a satisfactory performance. The surface morphology, which can be varied by

∗

[email protected] Advanced Laser Technologies 2007, edited by Risto Myllylä, Alexander V. Priezzhev, Matti Kinnunen, Vladimir I. Pustovoy, Mikhail Yu. Kirillin, Alexey P. Popov, Proc. of SPIE Vol. 7022, 702203, (2008) · 0277-786X/08/$18 · doi: 10.1117/12.803903 Proc. of SPIE Vol. 7022 702203-1

2008 SPIE Digital Library -- Subscriber Archive Copy

different processing methods influence the final interactions of the implant with the surrounding environment. Rough surfaces promote better oseointegration than smooth surfaces [4-7]. Soon after implantation – within a few seconds - the biomaterial surface becomes coated with a film of adsorbed proteins, which mediate the interaction between the implant and the body environment. Since most implants are exposed to blood during implantation, the initial protein film is mainly composed of plasma proteins. Human plasma fibrinogen (HPF) is one of the most relevant proteins that are adsorbed on biomaterial surfaces. HPF takes part in blood coagulation, facilitates adhesion and aggregation of platelets [8]. The structure and composition of the adsorbed protein layer determine the type and extent of the subsequent biological reactions, such as activation of coagulation and immune response and oseointegration [9]. Thus the initially adsorbed protein layer is a factor conditioning the biocompatibility [10-12]. The mechanisms and the factors important for protein adsorption and desorption are still subject of scientific research and not very well understood. Therefore it is important to investigate how different titanium surfaces influence the formation and properties of adsorbed protein layers. The need to develop novel methods for the investigation of biopolymer adsorption at surfaces is arisen from the observation that conventional surface characterization methods and tools (as Scanning Electron Microscopy - SEM, Transmission Electron Microscopy – TEM, Environmental Scanning Electron Microscopy – ESEM, Atomic Force Microscopy – AFM, diamond stylus and optical profilometer) appears to be un-effective when a need is to make observation from a sample in immersion or in buffer liquid. Thus an interesting attempt is the integration of optical methods together with electrochemical methods (as voltammetry, and electrochemical impedance spectroscopy (EIS)) for detecting of organization of different type of layers or molecule volumes on metal surface. Previously we have applied diffractive optical element (DOE) based sensor for inspection of different type of bulk and fragile materials [13], and later the same principle is applied to investigate quality of electrode surface used in electrochemistry [14,15], and we have started a new direction of application of DOE separating the non-coherent and the coherent response of the DOE based sensor. This procedure will be described in details in the book [16]. The DOE procedure is utilized together with electrochemical methods (as voltammetry, and electrochemical impedance spectroscopy (EIS)) in aim to investigate permittivity change and fluctuation in optical roughness (Ropt) of an electrode surface, as titanium, when electrode surface is located in the immersion or buffer liquid and the protein molecules attach on this biomaterial. Thereafter we have extended our investigations to adsorption of biopolymers (human blood plasma proteins and nucleic acids) on the titanium surface. We have started with the investigation of adsorption of homopurinic (AAG)12 and homopyrimidinic (TTC)12 oligodeoxynucleotides at a mechanically polished titanium surfaces. During our research we have made observations that with the aid of the DOE based sensor we are able to detect permittivity changes (refractive index changes) up to level of 0.1 per mille even higher and optical roughness changes up to level of 0.1nanometer. In the context of the knowledge that the probe laser beam is possible to focus up to waist diameter respecting the diffraction limit, allow these findings the DOE based spectroscopy of biomaterial surfaces as titanium with high accuracy. Recently we have disseminated our tentative results in organization of nano-scale synthetic oligonugloetides on immersed electrode surface [17], in DOE sensor detection of oligonucleotides on chromium, amalgam and titanium surfaces [18-20]. In this paper we report, according to our knowledge for first time, results from our recent investigations, which are extended to sense organization of nano-scale fibrinogen adhered on test surface. The permittivity change and the fluctuations in optical roughness (Ropt) of an test surface is investigated through an optical window of an cuvette by using the DOE sensor. We have studied the effect of the physical properties of titanium surface (roughness, surface treatment acid etching) on the adsorption of oligodeoxynucleotides and fibrinogen.

2. EXPERIMENTAL 2.1. A diffractive optical based sensor setup We used a diffractive optical element based sensor (DOE) for a detection of permittivity change and a fluctuation in optical roughness (Ropt) of a titanium surface without and with adsorbed protein and oligonucleotide (ODN) molecules. The permittivity and optical roughness changes were investigated through an optical window of a cuvette by using the DOE sensor as is shown in Fig. 1. The DOE procedure utilizes the focused (by lenses L1 and L2) laser wavefront, which hits the test surface (denoted by a refractive index n4), and scatters via beam splitter (BS) through the DOE aperture to perform a 4 × 4 bright light spot matrix on the 2D array of CCD-camera. In experiments it was used a small tilts (∼ 5º) from optical axis to cancel out optical feedbacks to laser and to DOE signals. If the permittivity of the test surface is

Proc. of SPIE Vol. 7022 702203-2

changed as a consequence of ODN molecules adsorption, the DOE image will become distorted. By a numerical analysis of the grabbed images in a PC it is expected to gain separable information from permittivity change, which relates to change of refractive index and reflectance of the test surface, and from optical roughness. The changes in permittivity and in optical roughness can be understood also as porosity change of adsorbed molecule volume, when the molecules (denoted by the refractive index n3 in Fig. 1) are adsorbed from their liquid solution on test surface. The permittivity is assumed to relate to non-coherent response of DOE signal, and the optical roughness to coherent response of DOE signal.

CCD PC DOE

n2

n1

n4

n3

Laser L L 1

BS

2

Cuvette Fig. 1. DOE sensor geometry, where laser beam is expanded and focused by lens system L1 – L2 to hit test surface n4 located in liquid cuvette via beam splitter BS and cuvette window n1. Backwards scattered light is directed by BS on DOE aperture, which analyses if the wavefront is distorted by contaminated fractions n3 from cuvette liquid n2 on test surface. Distorted 4×4 light spot DOE image is grabbed from 2D photoarray of CCD camera and analysed by a PC.

2.2. System function of DOE sensor DOE sensor is capable to sense reflectance from air-test surface as well as from liquid-test surface interface. If a test surface is located in air, we first measure the reference base line from a known reflectance reference, whose complex refractive index is known, and thereafter the reflectance from the test surface as shown in Fig. 1. The analysis of a DOE image is based on the calculation of the total irradiance and irradiance distribution of the image pattern. We calculate the total irradiance of a DOE image in respect to non-coherent response as follows

I NC =

1 n SW m SW

nSW , mSW

∑ I iSW , jSW −

iSW =1, j SW =1

1 n pks m pks

n pks , m pks

∑I

i pks , j pks i pks =1, j pks =1

.

(1)

where the irradiance of peaks (coherent response) are subtracted from the total irradiance (first term of Eq. (1)) of that DOE image. The non-coherent response of DOE sensor image relates to the reflectance of reference or test surface because the both surfaces are assumed to be optically smooth, and the angle of incidence of the laser probe beam coincides the normal incidence of the surface. The reflectance from air-test surface interface, in normal direction, can be calculated from the following formula:

R1 = where

1 − N n1k1 1 + N n1k1

2

=

n12 + k12 − 2n1 + 1 , n12 + k12 + 2n1 + 1

(2)

N n1k1 = n1 + ik1 is the complex refractive index of reference or test surface. When measurements are required to

perform in liquid we use the same principle, as it was the case with the reflectance from air-test surface interface. The reflectance from liquid-test surface interface, in normal direction, can be calculated from the following formula:


R12 =

N n1k1 − N n2 k 2 N n1k1 + N n2 k2

2

=

n12 + n22 + k12 + k 22 − 2n1n2 − 2k1k 2 , n12 + n22 + k12 + k 22 + 2n1n2 + 2k1k 2

(3)

where N n1k1 = n1 + ik1 denotes complex refractive index of liquid and N n 2 k 2 = n2 + ik 2 is the complex refractive index of a test surface. However, in the investigations of the temporal evolution of liquid-test surface interface, when the fragments from a buffer solution or from a HPF solution are contaminated on the test surface, we use the laser probe beam reflectance from the test surface as a base line reflectance when test surface locates in water. By using the measured reflectance data from a liquid – test surface interface it is possible to gain information from the effective complex refractive index of the interface. In aim to gain information from the uniformity of the liquid-test surface interface volume we utilise the coherent response of our DOE sensor by such a way that we calculate the peak irradiance of a DOE image in respect as follows: n

,m

pks pks 1 IC = Ii , j . ∑ n pks m pks i pks =1, j pks =pks1 pks

(4)

After that we calculate the peaks irradiance ratio from the reference surface and surface interface under test. This data is possible to convert to the length of the optical path difference [16], which relates to optical roughness and porosity of that interface layer or volume. 2.3. Ellipsometry of biomaterial surfaces We used ellipsometry as a corroporative method to inspect the contamination of the fragments of buffer, HPF fraction and oligonucleotide fraction on the test surface. All these measurements were performed in dry environment by using the incidence angle of 80o for probe beam to avoid the harmful effects caused by the possible appearance of surface roughness [21, 22]. 2.4. Chemicals Human plasma fibrinogen (HPF), fraction I, type III was purchased from Sigma. In all experiments the HPF was dissolved in phosphate buffer solution (PBS) + 0.136 M sodium citrate at a concentration of 500 nM. Oligodeoxynucleotides (ODNs), 36-mers 5´-(AAG)12-3´, 5´-(CTT)12-3´ were purchased from Thermo Electron (Ulm, Germany). The ODNs concentrations (5 µM) were determined spectrophotometrically using a Libra S22 spectrophotometer. In all experiments the ODNs were dissolved in 0.3 M NaCl + 50 mM Na2HPO4 (pH ∼ 8.3) solution. To prepare a (AAG)12-(TTC)12 duplex, a mixture of both 36-mer ODNs, each at a concentration of 5 µM in 0.3 M NaCl + 50 mM Na2HPO4, was heated to 95°C in a water bath. After 1 min the sample was allowed to slowly cool down at room temperature. Measurements were performed at room temperature.

3. RESULTS AND DISCUSSION The DOE sensor measurements from the grinded (N=10), chemically etched (N=8) and polished (N=12) titanium samples were performed in a cuvette. The DOE measurements were made through the cuvette window according to the principle shown in Fig. 1. In the beginning of all measurements the DOE sensor images for reference signal level from test surface were made in water for 200 seconds, and during that time frame 2000 reference samples were grabbed. After that the water was removed by syringe from cuvette and the buffer solution, in turn, was injected in the cuvette. Immediately after injection of buffer, grabbing of the DOE images was started, and the image grabbing was repeated after two minutes interval. Before human plasma fibrinogen (HPF) measurement, the cuvette was washed, and after washing the new titanium sample was installed in the sample holder inside the cuvette. The water was injected in the cuvette, and the DOE image references from the new sample surface were taken. Before adding the HPF solution in the cuvette, the immersion water was removed, and grabbing process of DOE images was started. The image grabbing was repeated three times consecutively after two minutes interval as shown in Fig. 2, where also the normalized temporal reflectance and the optical roughness responses are shown for one measurement. Immediately after the DOE sensor measurements in buffer and HPF solutions the samples were subjected for ellipsometric measurements, which were made during a half hour time frame when surface sample is taken out from the solution


-8

1.1

5

(a)

x 10 (b)

4.5

1.05

4 1

Ropt

3.5

RN

0.95

3

2.5

0.9

2 1.5

0.85

1 0.8 0.75 0

0.5 500

t

0 0

1000

500

1000

t

Fig. 2. (a) Normalized reflectance RN and (b) optical roughness Ropt in meter as a function of time in seconds calculated from the DOE sensor data, which is measured through the optical window of cuvette from grinded titanium (TiGr) when TiGr surface is in buffer or in 500 nM human plasma fibrinogen (HPF) solution. The two first graphs denote the effective changes of the buffer - TiGr interface for reflectance and for optical roughness when buffer fractions contaminate on the TiGr surface, whereas the last three graphs denote respective effective changes when the HPF molecules in buffer solution acts as immersion solution. Normalization of reflectance and optical roughness shown in the graphs is made against the respective reflectance and optical roughness of TiGr surface in water.

The averaged normalised reflectance (RN) and optical roughness (Ropt) values for grinded titanium surface are listed in the Table 1. The results indicate that the reflectance of the HPF-titanium interface is lower compared with the buffertitanium interface, whereas the average optical roughness of that volume remains rather constant. However the uniformity of that layer or volume is rather low as one can conclude from the Fig. 2(b). The averaged complex refractive index values, drawn out by ellipsometer, indicate great variance for the surfaces, which are contaminated by the HPF fractions compared with the pure grinded titanium surface and surface contaminated by the buffer fractions, whereas the reflectance values R (0o) indicates rather low variance as shown in Table 2. Please also observe the correlation of the real part versus imaginary part of complex refractive index (Fig. 4(a)). This may indicate that the HPF molecules are attached on the grinded titanium surface. Table 1. Normalized reflectance RN and optical roughness Ropt (λ=632.8nm) with their standard deviations (∆) of grinded titanium (TiGr) surface measured by DOE sensor through optical window of liquid cuvette, when TiGr surface is in buffer or in 500 nM human plasma fibrinogen (HPF) solution.

Surface Buffer on TiGr surface HPF on TiGr surface

(1)

RN (1) 0.987 0.920

∆RN 0.007 0.066

Ropt (nm) (1) 19.4 18.7

∆Ropt (nm) 1.2 7.2

Normalization of reflectance and optical roughness is made against the respective reflectance and optical roughness of TiGr surface in water. Table 2. Complex refractive index (Nnk = n + ik) and reflectance R (λ=632.8nm) with their standard deviations (∆) of grinded titanium (TiGr) surface measured by ellipsometer in dry environment during a half hour, when TiGr surface is taken out from the buffer or from 500 nM HPF solution after DOE measurement.

Surface TiGr Buffer on TiGr surface(1) HPF on TiGr surface(2)

(1)

n 2.111 2.063 0.818

∆n 0.123 0.101 0.361

k 2.667 2.627 1.701

∆k 0.044 0.042 0.325

R (0o) 0.497 0.494 0.497

∆R (0o) 0.001 0.006 0.042

TiGr surface was immersed in buffer solution for 10 min, (2)TiGr surface was immersed in HPF solution for 10 min.


A temporal normalized reflectance (RN) and optical roughness (Ropt) values for chemically etched titanium surface are shown in Fig. 3(a). The graph indicates that the reflectance of the HPF-etched titanium interface is lower and rather constant compared with the buffer-etched titanium interface, whereas the average optical roughness of that volume (Fig. 3(b)) shows increasing roughness evolution as a function of time with lower uniformity compared with HPF– TiGr interface (cf. Fig. 2(b)). The averaged normalised reflectance (RN) and optical roughness (Ropt) values for chemically etched titanium surface are listed in the Table 3. -8

1.2

8

(a)

x 10 (b)

7

1.1

6 1

Ropt

RN

5

0.9

4 3

0.8

2 0.7

0.6 0

1 500

t

1000

0 0

500

t

1000

Fig. 3. (a) Normalized reflectance RN and (b) optical roughness Ropt in meter as a function of time in second calculated from the DOE sensor data, which is measured through the optical window of cuvette from chemically etched titanium (TiEt), when TiEt surface is in buffer or in 500 nM HPF solution. The two first graphs denote the effective changes of the buffer - TiEt interface on reflectance and optical roughness when buffer fractions contaminate on TiEt surface, whereas the last three graphs denote respective effective changes when the HPF molecules are injected in immersion solution. Normalization of reflectance and optical roughness is made to the respective reflectance and optical roughness of TiEt surface in water. Table 3. Normalized reflectance RN and optical roughness Ropt (λ=632.8nm) with their standard deviations (∆) of chemically etched titanium (TiEt) surface measured by DOE sensor through optical window of liquid cuvette, when TiEt surface is in buffer or in 500 nM HPF solution.

Surface Buffer on TiEt surface HPF on TiEt surface (1)

RN (1) 0.986 0.878

∆RN 0.004 0.005

Ropt (nm)(1) 25.8 30.5

∆Ropt (nm) 0.1 5.2

Normalization of reflectance and optical roughness is made to the respective reflectance and optical roughness of TiEt surface in water.

The results indicate that the reflectance of the HPF -TiEt interface is lower compared with the buffer - TiEt interface, whereas the average optical roughness of that volume goes vice versa. The averaged complex refractive index values, drawn out by ellipsometer, indicate great variance for the surfaces, which are contaminated by the HPF fractions compared with the pure grinded titanium surface and surface contaminated only by the buffer fractions. However the reflectance values indicate also variance as shown in Table 4. Please also observe the correlation of the real part versus imaginary part of complex refractive index (Fig. 4(b)). This finding reinforces the conclusion that the HPF molecules are attached on the chemically etched titanium surface.


Table 4. Refractive index and reflectance (λ=632.8nm) of chemically etched titanium (TiEt) surface measured by ellipsometer in dry environment during a half hour when the TiEt surface was taken out from buffer or from 500 nM HPF solution after DOE measurement.

(1)

Surface TiEt Buffer on TiEt surface(1) HPF on TiEt surface(2), (3)

n 1.489 1.507 0.13-0.85

∆n 0.157 0.127 -

k 2.150 2.179 1.14-1.85

∆k 0.076 0.268 -

R (0o) 0.450 0.452 0.50-0.80

∆R (0o) 0.002 0.045 -

TiEt surface was immersed in buffer solution for 10 min, (2) TiEt surface was immersed in buffer solution for 10 min, (3) Contamination of HPF showed also appearance of crystallization, where Nnk =1.43+i×0.28 with R = 0.044 was observed. 3 Ti

BTi

Ti

Gr

PBTi

2.5

Gr

2

2

1.5

1.5

1

1

0.5

0.5

0 0

0.5

(b) Et

BTi

k

k

2.5

3

(a) Gr

1

n

1.5

2

2.5

0 0

Et

PBTi

Et

0.5

1

n

1.5

2

2.5

Fig. 4. Correlation of the real part versus imaginary part of complex refractive index (Nnk = n + ik) at λ=632.8nm of (a) grinded titanium and (b) chemically etched titanium surface measured by ellipsometer in dry environment during a half hour, when TiGr or TiEt surface is taken out from the buffer solution (BTi) or from 500 nM HPF solution (HPFTi) after DOE measurement.

A temporal normalised reflectance (RN) and optical roughness (Ropt) values for polished titanium surface without and with contamination of oligodeoxynucleotides (ODNs) are shown in Fig. 5. The graph indicates that the reflectance of the homopurinic ODN (AAG)12-polished titanium interface (Fig.5A(a)) and homopyrimidinic ODN (TTC)12-polished titanium interface (Fig.5B(a)) is lower and rather constant compared with the buffer-polished titanium interface, whereas the temporal optical roughness of those volumes shows clear threshold in evolution of optical roughness (ca. 15 nm) with decreasing evolution as a function of time for the (AAG)12–TiPo interface (Fig. 5A(b)) and clear threshold in evolution of optical roughness (ca. 10 nm) with increasing evolution for (TTC)12–TiPo interface (Fig. 5B(b)). Also the averaged normalised reflectance (RN) gained from DOE sensor data (cf. Table 5) and reflectance R (0o) gained from ellipsometer data (cf. Table 6) reinforces the findings that homopurinic and homopyrimidinic ODN molecules are attached on the polished titanium surface. The temporal normalised reflectance (RN) and optical roughness (Ropt) values of the (AAG)12(TTC)12 duplex (dsDNA) do not differ significantly from the respective values of the polished titanium (TiPo) or from the buffer-TiPo interface (Fig. 5C). Also averaged normalised reflectance (RN) gained from DOE sensor data (cf. Table 5) and reflectance R (0o) gained from ellipsometer data (cf. Table 6) may indicate that the (AAG)12-(TTC)12 duplex (dsDNA) has weak attraction to polished titanium surface, and when the sample is removed from the cuvette the dsDNA molecules are remained in the solution.


-8

1.2

8

(a)

x 10 (b)

7

1.1

6

1

5

RN

Ropt

0.9

4

0.8 3 0.7

2

0.6

1

0.5 0

500

t

0 0

1000

500

t

1000

Fig. 5A. (a) Normalized reflectance RN and (b) optical roughness Ropt in meter as a function of time in second calculated from the DOE sensor data, which is measured through the optical window of cuvette from polished titanium (TiPo), when TiPo surface is in buffer or in 5µM homopurinic (AAG)12 ODN solution. The two first graphs denote the effective changes of the buffer - TiPo interface on reflectance and optical roughness when buffer fractions contaminate on TiPo surface, whereas the last three graphs denote respective effective changes when the (AAG)12 molecules are injected in immersion solution. Normalization of reflectance and optical roughness is made to the respective reflectance and optical roughness of TiPo surface in water. -8

1.2

8

(a)

x 10 (b)

7

1.1

6

1

5

RN

Ropt

0.9

4

0.8 3 0.7

2

0.6 0.5 0

1 500

t

1000

0 0

500

t

1000

Fig. 5B. (a) Normalized reflectance RN and (b) optical roughness Ropt in meter as a function of time in second calculated from the DOE sensor data, which is measured through the optical window of cuvette from polished titanium (TiPo), when TiPo surface is in buffer or in 5µM homopyrimidinic (TTC)12 ODN solution. The two first graphs denote the effective changes of the buffer - TiPo interface on reflectance and optical roughness when buffer fractions contaminate on TiPo surface, whereas the last three graphs denote respective effective changes when the (TTC)12 molecules are injected in immersion solution. Normalization of reflectance and optical roughness is made to the respective reflectance and optical roughness of TiPo surface in water.


-8

1.3

5

x 10

4.5 1.2

4

(a)

3.5

Ropt

RN

1.1

1

3

2.5 2

0.9

1.5 1

0.8

0.5 0.7 0

500

1000

t

0 0

500

1000

t

Fig. 5C. (a) Normalized reflectance RN and (b) optical roughness Ropt in meter as a function of time in second calculated from the DOE sensor data, which is measured through the optical window of cuvette from polished titanium (TiPo), when TiPo surface is in buffer or in 5µM (AAG)12-(TTC)12 duplex (dsDNA) solution. The two first graphs denote the effective changes of the buffer - TiPo interface on reflectance and optical roughness when buffer fractions contaminate on TiPo surface, whereas the last three graphs denote respective effective changes when the dsDNA molecules are injected in immersion solution. Normalization of reflectance and optical roughness is made to the respective reflectance and optical roughness of TiPo surface in water. Table 5. Normalized reflectance RN and optical roughness Ropt (λ=632.8nm) with their standard deviations (∆) of polished titanium (TiPo) surface measured by DOE sensor through optical window of liquid cuvette, when TiPo surface is in buffer or in 5µM oligodeoxynucleotide solution.

Surface Buffer on TiPo surface (AAG)12 on TiPo surface (TTC)12 on TiPo surface dsDNA in buffer on TiPo surface(2)

RN (1) 1.061 0.834 0.973 1.042

∆RN 0.017 0.038 0.007 0.035

Ropt (nm)(1) 22.9 27.1 34.4 23.1

∆Ropt (nm) 0.6 1.9 1.1 1.0

(1)

Normalization of reflectance and optical roughness is made to the respective reflectance and optical roughness of TiPo surface in water, (2) dsDNA is the duplex of (AAG)12-(TTC)12.

Table 6. Refractive index and reflectance (λ=632.8nm) of polished titanium (TiPo) surface measured by ellipsometer in dry environment during a half hour when the TiPo surface was taken out from buffer or buffer oligodeoxynucleotide solution after DOE measurement.

Surface TiPo Buffer on TiPo surface(1) (AAG)12 on TiPo surface(2) (TTC)12 on TiPo surface(3) dsDNA in buffer on TiPo surface(4),

n 1.853 1.778 1.907 1.909 1.883

∆n 0.199 0.249 0.138 0.015 0.107

k 2.204 2.094 2.026 2.185 2.347

∆k 0.134 0.116 0.079 0.081 0.008

R (0o) 0.430 0.413 0.393 0.423 0.455

∆R (0o) 0.016 0.008 0.021 0.016 0.005

(5) (1)

TiPo surface was immersed in buffer solution for 10 min, (2) TiPo surface was immersed in 5µM homopurinic (AAG)12 ODN, TiPo surface was immersed in 5µM homopyrimidinic (TTC)12 ODN in buffer solution for 10 min , (4) TiPo surface was immersed in 5µM duplex of (AAG)12-(TTC)12 (dsDNA) in buffer solution for 10 min, (5) dsDNA is the duplex of (AAG)12(TTC)12. (3)


2.4 Ti 2.35 2.3

Po

PuBTi PyBTi

Po

Po

dsDNABTi

Po

2.25

k

2.2 2.15 2.1 2.05 2 1.95 1.9 0.37

0.38

0.39

0.4

0.41

0.42

R

0.43

0.44

0.45

0.46

0.47

Fig. 6. Correlation of reflectance of normal incidence versus imaginary part of complex refractive index (Nnk = n + ik) at λ=632.8nm of polished titanium measured by ellipsometer in dry environment during a half hour, when TiPo surface is taken out from the 5µM homopurinic (AAG)12 ODN solution (PuBTi), from the 5µM homopyrimidinic (TTC)12 ODN solution (PyBTi) or from 5 µM duplex of (AAG)12-(TTC)12 (dsDNA) solution (dsDNABTi) after DOE measurement.

4. CONCLUSION In the progress of this work the optical properties of different type of surface treatments of titanium as polishing, grinding, and chemical etching are investigated in details. The DOE sensor was observed to be effective in detection of permittivity changes and fluctuations in optical roughness of treated titanium surface, when surface locates in dry environment as well as when it is subjected to the contamination of human plasma fibrinogen fractions in wet environment. The temporal responses in normalized reflectance revealed reasonable differences in molecule adsorption of human plasma fibrinogen and oligodeoxynucleotides between the treated surfaces under test. The results are also in accordance with the ellipsometer results, which are gained from the same surfaces in dry environment. Moreover, the observation of the magnitude of temporal optical roughness evolution, which relates to the porosity of the molecule layer or volume, are valuable and reveal novel information from the dynamic processes of organization of the molecules on the biomaterial surface.

ACKNOWLEDGEMENT This work was supported by the Ministry of Education and Sport of the Czech Republic (1M0528 to J.V.), the Academy of Sciences of the Czech Republic (KAN200040651 to S.H.), the Ministry of Education of the Czech Republic (LC06035), and by an institutional research plan (AVOZ 50040507).

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