text:
# Electronic structure and dynamics of optically excited single-wall carbon nanotubes

## 1 Introduction

The electronic properties of single-wall carbon nanotubes (SWNTs) and specifically of semiconducting SWNTs hold promise for application in a variety of optical devices such as light-emitting- Misewich03  and photo-sensitive diodes Freitag03 , for nonlinear optical- or light harvesting materials and more. The electronic structure and the dynamics of optically excited states in SWNTs naturally plays a decisive role for the operation of such devices. In particular the scattering times of photoexcited carriers with electrons, phonons and defects as well as the competition of radiative with non-radiative decay processes are of great interest for a better understanding of electronic transport phenomena in optical devices Hertel00 ; Hertel00b ; Mele00 ; Sun00 ; Hertel02 ; Vivien02 ; Jeon03 ; Kane03 ; Lauret03 ; Lebedkin03 ; Hagen03 ; Moos03 .

Here, we make use of the recent development of techniques for the exfoliation of SWNTs from nanotube-ropes which provides the basis for a detailed investigation of optical properties and excitations in individual SWNTs using near-infrared (NIR) absorption spectroscopy or spectrofluorimetric techniques Connell02 ; Bachilo02 . These advances also allow to address questions of fundamental interest such as the character of optical excitations in systems with reduced dimensionality. Binding energies of Wannier-type excitons, for example, are predicted to depend strongly on the dimensionality and the lowest state is expected to diverge towards infinite binding energy as the system becomes truly one-dimensional Loudon59 ; He91 . It has previously been speculated that some optical transitions in individual SWNTs are excitonic, but so far, the reported evidence is not conclusive. Here, we will discuss the character of optical excitations in individual SWNTs in further detail. The corresponding transition energies are analyzed using tight-binding (TB) calculations which show that the observed variation of transition energies with tube diameter and chirality can be identified with variations in the TB band-gaps. The decay of optically excited states is determined by the competition of non-radiative and radiative decay processes. These are investigated experimentally using picosecond time-resolved photoluminescence (PL) spectroscopy.

We have furthermore studied carrier dynamics in SWNT ropes using femtosecond time-resolved photoemission. A detailed analysis of these experiments allows to determine characteristic timescales of fundamental scattering processes such as electron-electron (e-e) or electron-phonon (e-ph) scattering. We find that e-e interactions in bundles of SWNTs give rise to e-e scattering times which strongly increase as the carrier energy approaches the Fermi level. The internal thermalization of the photo-excited electron gas is facilitated by such e-e scattering processes and is characterized by a time constant of 200 fs. The electron-phonon (e-ph) coupling in metallic tubes finally is found to be extraordinarily weak with a mass enhancement of only 0.0004. The latter suggests that room temperature e-ph scattering times are on the order of 15 ps. These results are discussed with respect to the influence of radiative and non-radiative decay processes on the carrier dynamics in individual tubes and tube ropes.

## 2 Experimental

Ultraviolet to visible to near-infrared (UV-VIS-NIR) absorption spectra and time-resolved PL studies were obtained from samples synthesized by the high pressure CO decomposition technique Nikolaev99 . So called HiPCO material has been purchased from CNI Houston. For absorption experiments, individual SWNTs were isolated from HiPCO material in sodiumdodecylsulfate (SDS) micelles by the technique described in ref. Connell02 . At wavelengths below 1350 nm the well resolved spectrum from micelles in $`\mathrm{H}_2\mathrm{O}`$ was extended by results from measurements in $`\mathrm{D}_2\mathrm{O}`$. For PL measurements the samples were dispersed using sodium cholate. Time-resolved PL from SWNT cholate micelles is performed using 300 mW of output from a Tsunami Ti:sapphire with sub-100 fs excitation pulses at a repetition rate of 82 MHz and a wavelength of 795 nm. Detection is done by a Hamamatsu synchroscan streak camera with an IR-enhanced cathode. The time resolution is here found to be slightly better than 10 ps. The laser spot size in the 2 mm long quartz cuvette was $`0.5`$mm in diameter. This allowed to keep pulse fluences at a very low level of $`10^{13}`$ photons per $`\mathrm{cm}^2`$ or less.

Time-resolved photoemission experiments were here performed with samples consisting of purified nanotube ropes that were produced by the laser vaporization technique (tubes@rice). A Ti:sapphire laser pumped optical parametric amplifier (Coherent) generates tunable femtosecond pulses with wavelengths between 470 nm and 730 nm. Fundamental and second harmonic beams generated from the OPA output are used as visible pump and UV probe respectively. The fluence for the visible pump and UV probe beams is typically 50 $`\mu `$J cm-2 and 5 $`\mu `$J cm-2, respectively, which corresponds to a pump power of $`10^9`$W cm-2. For more details see reference Hertel02 .

## 3 Results and discussion

In Fig. 1 we compare the absorption spectrum from colloidal graphite with spectra from SWNT-ropes as well as from individual exfoliated SWNT-micelles in aqueous solution. The optical density of colloidal graphite increases continuously from the near infrared to the UV range due to the logarithmic singularity in the joint density of states near 5 eV for excitation of free carriers from $`\pi `$ to $`\pi ^{}`$ bands Daniels70 ; Ahuja97 . The optical density in SWNT ropes follows a similar general trend as seen for graphite illustrating the intimate relationship of the electronic structure of these two materials. However, the middle spectrum in Fig. 1 from SWNT ropes also exhibits some pronounced fine structure on top of the continuously rising background. These features can be attributed to optical transitions between different sub-bands of semiconducting and metallic SWNTs in the suspension. For tubes with a diameter of approximately 1 nm, absorption features in the range from 0.8 eV to 1.3 eV and features in-between about 1.4 eV and 2.5 eV are associated with $`E_{11}^S`$ and $`E_{22}^S`$ transitions between the first and second $`\pi `$ sub-bands in semiconduction tubes, respectively. Broad and not clearly resolved features between about 2.0 eV to 3.5 eV are due to $`E_{11}^M`$ transitions between the first sub-bands in metallic tubes. The nomenclature used here for optical transitions is $`E_{kl}^X`$, where $`k`$ and $`l`$ refer to the valence and conduction sub-band index, respectively and $`X=S,M`$ refers to transitions in semiconducting or metallic tubes, respectively. Transitions with $`k=l`$ are dipole-allowed for light polarized parallel to the tube axis Lin00 .

If individual SWNTs are combined into ropes one finds that $`E_{11}^S`$ transitions become substantially broadened. The corresponding 100 meV increase of the FWHM of absorption features is indicative for the large contribution of tube-tube interactions to the width of transitions in spectra from SWNT ropes Hagen03 . Higher lying $`E_{22}^S`$ and $`E_{11}^M`$ transitions are broad both in the exfoliated and crystalline phase which suggests that intratube relaxation from higher energy states provides an efficient decay channel for tubes in all environments. In the following we will begin the discussion with a detailed analysis of $`E_{11}^S`$ transitions within the first absorption cluster.

### 3.1 Analysis of absorption spectra from SWNT-micelles

In Fig. 2 we have plotted a background corrected absorption spectrum with well-resolved spectral features in the range of the first absorption cluster. To analyze the optical transitions in this energy range in further detail we performed a multi-peak fit with a maximum of 24 independent Voigt profiles. The resulting fit is shown in Fig. 2a) as thin line superimposed on the experimental data (dots). The excellent agreement between fit and experiment can be appreciated from the nearly vanishing residuals in the upper part of Fig. 2a). Due to the excellent signal to noise ratio, errors of peak positions are on average only about 1 meV. The experimental peak energies obtained here can be found in Table 1 together with transitions obtained from PL measurements Bachilo02 . The distribution of peak widths with its mean at 25 meV is shown in Fig. 2b). The contribution of Gaussian and Lorentzian components to the Voigt profiles is about equal. Similar average widths of 23 meV and 25 meV were recently also observed for band-gap PL from single SWNTs dispersed on a surface or SWNT micelle ensembles in solution, respectively Bachilo02 ; Hartschuh03 .

A comparison of peak positions obtained here with those reported by Bachilo et al. for PL measurements from SWNT-micelles dispersed in D2O Bachilo02  yields extraordinary agreement if a 3 meV blue-shift of the fluorimetric data is accounted for. The previously observed redshift of luminescence data of a few meV Connell02  here appears to be overcompensated by differences in transition energies in different environments, i.e. in heavy and light water. The peak assignment adopted here is practically identical to that previously discussed in ref. Hagen03  as ’scenario I’ and is guided by the energetic order of peaks in fluorimetric data of Bachilo et al. Bachilo02 . In cases where no peaks are reported from PL data, our assignment was guided by the order of peaks expected from the semi-empirical TB band-structure model discussed below. If the spectral weight of individual peaks in the absorption spectrum is combined with the assignment to specific tube types, i.e. tube diameters, we can also calculate the diameter distribution of semiconducting tubes in the present sample which is reproduced in Fig. 2c). The mean diameter of 0.98 nm obtained here is in good agreement with that recently obtained by a luminescence study 0.96 nm Bachilo02  and Raman studies, with 1.13 nm Gregan02  and 0.98 nm Kukovecz02 . The diameter distribution obtained in ref. Hagen03  is at variance with that obtained here, apparently because the statistical weight of individual tube types varies if the JDOS — as in a previous study Hagen03  — or Voigt profiles are used to fit experimental spectra. The probability distribution of the chiral vector map resulting from the above assignment and fit shows a slight preference for armchair tubes.

Next, we will attempt a quantitative discussion of observed transition energies by comparison with energies predicted by a refined version Ding02 ; Hagen03  of the original TB band-structure model Saito92 ; Hamada92 . The TB calculations used here explicitly include the crucial diameter and chirality dependence of the TB hopping integral $`\gamma _0`$ without which any such comparison would be useless. The refined TB-model leads to more pronounced oscillations of band-gaps with tube diameter than predicted by the earlier zone folding schemes because the latter used to neglect mutual realignment of $`p_z`$ orbitals on the curved graphene sheet. However, the magnitude of $`\gamma _0^{\mathrm{}}`$, the TB hopping integral for the uncurved graphene sheet, is frequently used as a free parameter and there is no general agreement on the appropriate value that should be used for optical excitations. Scanning tunneling spectroscopy of individual SWNTs yields $`\gamma _0^{\mathrm{}}`$ values between 2.45 eV and 2.7 eV Odom98 ; Wildoer98 , while an analysis of optical experiments suggest that values between 2.65 eV and 3.0 eV are more appropriate (see, for example, refs. Hertel02  and Liu02 ). Resonant Raman spectra are frequently analyzed using a hopping integral around 3.0 eV Dresselhaus99 .

If experimental peak positions are here fit by the one-parameter TB model one obtains best agreement with calculated gaps if $`\gamma _0^{\mathrm{}}=3.450`$ eV. The resulting residuals can be characterized by a 25 meV root mean square (RMS) and are plotted as a function of tube diameter in Fig. 3a). The steep slope of the straight line fit to residuals clearly illustrates that the one-parameter TB model cannot account for the observed diameter dependence of transition energies. This deficiency can be removed by the addition of a number of empirical corrective terms which give successively better agreement with experiment and thereby also allow to discuss the possible physical origin of remaining discrepancies. Previously, a first order correction was introduced in the form of an empirical offset parameter $`\mathrm{\Omega }`$ Hagen03  which, in combination with a variable $`\gamma _0^{\mathrm{}}`$, allow to skew the diameter dependence of calculated transition energies (see Fig. 3b)). Within this two parameter model, best agreement is obtained for $`\gamma _0^{\mathrm{}}=3.059`$eV and $`\mathrm{\Omega }=0.120`$eV. However, the resulting residuals exhibit a clear correlation with the tube chiral angle $`\theta (n,m)=\mathrm{arcsin}(\sqrt{3}m/(2\sqrt{n^2+nm+m^2}))`$ as seen in Fig. 3c). The second order correction thus includes a term which varies with the chiral angle as $`A\mathrm{cos}(6\theta (n,m))`$ (see solid line in Fig. 3c). At this point the root mean square deviation of experimental and calculated transition energies is only 6 meV ($`\gamma _0^{\mathrm{}}=3.061`$eV, $`\mathrm{\Omega }=0.118`$eV, $`A=0.0154`$eV). The last corrective term $`Bd(n,m)\mathrm{cos}(6\theta (n,m))`$ is included to partially remove a combined correlation of residuals with diameter $`d`$ and chiral angle $`\theta `$. The phenomenological four parameter model describing optical transition energies $`E_{11}^S`$ in the band-gap region is finally given by:

$`E_{11}^{\mathrm{opt}\mathrm{S}}(n,m)`$ $`=`$ $`\gamma _0^{\mathrm{}}ϵ_{11}^{\mathrm{TB}}(n,m)`$
$`+`$ $`(A+Bd(n,m))\mathrm{cos}[6\theta (n,m)]`$
$`+`$ $`\mathrm{\Omega }`$
where $`ϵ_{11}^{\mathrm{TB}}`$ are the TB band-gaps as calculated using the diameter and chirality corrected nearest neighbor hopping integrals Ding02 . Best agreement with experiment is here obtained for $`\gamma _0^{\mathrm{}}=3.022`$eV, $`\mathrm{\Omega }=0.130`$eV, $`A=0.0543`$eV, and $`B=0.00395`$eV. The quality of the agreement between experiment and calculated transition energies is evident from Table 1 and Fig. 3d) where calculated gap-energies are plotted as a function of the tube diameter. Note, that the root mean square deviation is below 4 meV.

Alternatively, we have also combined diameter and chirality corrected TB calculations with a simple Wannier type exciton model. Transition energies are here calculated within the effective mass approximation allowing also for fractal dimensionality of excitons $`\alpha `$, with $`1<\alpha <3`$. The transition energy is then given by:

$$E_{11}^S(n,m)=\gamma _0^{\mathrm{}}ϵ_{11}^{\mathrm{TB}}(n,m)\frac{\mu E_H}{ϵ^2m_{el}\left(n+\frac{\alpha 3}{2}\right)^2}$$
(2)
where $`n=1,2,3,\mathrm{}`$ is the principal exciton quantum number while $`\mu `$ and $`m_{el}`$ are the effective exciton and free electron mass and $`ϵ`$ is the dielectric constant He91 . $`E_H`$ is the Rydberg constant. $`\mu (n,m)`$ is obtained from TB-valence and conduction band effective masses $`m_h`$ and $`m_e`$ using $`\mu ^1=m_e^1+m_h^1`$. For semiconducting tubes studied here $`\mu `$ is found to vary strongly with chirality and diameter and decreases from values around $`0.25m_{el}\gamma _0^{\mathrm{}}/\mathrm{eV}`$ at 0.6 nm diameter to about $`0.10m_{el}\gamma _0^{\mathrm{}}/\mathrm{eV}`$ at 1.3 nm diameter (see Fig. 4). The dielectric constant is assumed to be less dependent on chirality and diameter and the exciton binding energy should thus be most strongly influenced by its effective mass. Interestingly, a fit of experimental data to eq. 2, where $`ϵ`$ and $`\alpha `$ are treated as adjustable parameters provides no significant improvement over the zero-order one-parameter TB model described above. We also find no correlation of any of the residuals described above with $`\mu (n,m)`$.

This analysis suggests that optical transitions in SWNT micelles are not excitons of the Wannier type. However, other observations are in favor of such an interpretation as for example the large value of $`\gamma _0^{\mathrm{}}=3.438`$eV in the one-parameter fit. It can hardly be reconciled with previously reported values. In particular band-gaps observed in scanning tunneling spectroscopy are at variance with the transition energies measured here. The band-gap measured by STS is determined by the threshold for injection of free electrons or holes into the band-structure of SWNTs and Coulomb interactions play a comparatively negligible role. However, Coulomb interactions are crucial for exciton formation and lead to a renormalization of the band-structure obtained from non-interacting electron models such as the TB calculation. The general trend of Coulomb interactions would be to significantly raise the energy needed for excitation of free electron-hole (e-h) pairs. The discrepancy between transition energies observed in optical and STS experiments would thus be easily resolved if optical transitions in SWNTs were excitonic in character. A comparison of the high transition energies observed here for a specific tube type with results from STS experiments, suggests that Coulomb interactions lead to a substantial renormalization of the non-interacting electron band-structure and gap energies perhaps by as much as ten to tens of percent. Similar changes of band-gap energies as a consequence of Coulomb interactions have been predicted theoretically by Ando Ando97 .

Here, a further point of interest is the observation that the absorption spectrum of Fig. 3a) can be extremely well described by a linear combination of nearly perfectly symmetrical absorption features. Note, that absorption by generation of free electron-hole pairs would be associated with a pronounced asymmetry of absorption features. Specifically, the joint density of states (JDOS) shows such asymmetries and consequently also the absorption cross sections — even if spectral broadening is included Hagen03 ; Lin00 . The highly symmetrical shape of absorption features thus appears to be consistent with excitonic transitions.

In conclusion, we observe striking agreement of experimental transition energies and energies obtained from a semi-empirical 4 parameter model based on refined TB band-structure calculations. This strongly suggests that the observed oscillation of transition energies with diameter and chirality is dictated by corresponding oscillations of the TB band-gaps. The somewhat unexpectedly large band-gaps and the highly symmetrical shape of absorption features in NIR spectra are consistent with an excitonic character of optical transitions. We speculate, that the offset parameter $`\mathrm{\Omega }`$ provides a rough measure of the difference between band-gap widening due to Coulomb interactions and the exciton binding energy. This would also suggest that exciton binding energies are only weakly dependent on tube diameter and chirality, i.e. their effective mass, which appears to be at variance with expectations for Wannier type excitons. The nature of optical transitions in the band-gap region thus remains somewhat unclear and further experimental work seems necessary to unambiguously identify the character of optical excitations in SWNTs.

### 3.2 Charge-carrier dynamics in individual SWNTs

In the following we will discuss scattering and relaxation processes of optically excited carriers in individual SWNTs and SWNT ropes as studied by time-resolved photoluminescence (PL) and photoelectron (PE) spectroscopy.

If SWNTs are exfoliated from ropes and dispersed in aqueous solution one finds that the PL quantum yield increases by orders of magnitude reaching values on the order of $`10^3`$.Connell02  The associated PL spectra are characterized by a very small Stokes shift of about 5 meV. In Fig. 5 a) we have plotted a PL spectrum from water suspended HiPCO micelles in the wavelength range from 850 nm to 1200 nm. The pump power in these experiments corresponds to a pulse fluence of below $`10^{13}`$ photons per cm2 such that nonlinear effects are not expected. The major features can be linked to absorption features in the same energy range corresponding to transitions in tubes with small diameters between 9.1 Åand 7.6 Å. We have marked the peak positions and diameters of a few tubes in this energy range. The energy-integrated PL signal in Fig. 5b) decays with a time-constant of about 15 ps. Deviations from a simple exponential decay may be indicative of a distribution of relaxation times in these samples, i.e. heterogeneous broadening of the corresponding transitions. Such heterogeneities may arise, for example, from a distribution of tube lengths. In combination with the recently reported PL quantum yield of $`10^3`$ Connell02 . This indicates that the radiative lifetime of the luminescent states is on the order of 10 ns. The fast decay observed here thus underlines the importance of non-radiative decay processes for the PL lifetime. The detailed mechanism of such non-radiative decay processes, however, needs to be explored in further detail.

### 3.3 Charge-carrier dynamics in SWNT ropes

As mentioned above, the luminescence quantum yield is reduced severely if tubes are agglomerated in tube ropes were tube-tube interactions lead to a rapid decay of excited states. This is also supported by the $`100`$meV increase of linewidths in absorption spectra from crystalline SWNT samples Hagen03 . The fast decay of excited states can be clearly seen in femtosecond time-resolved photoelectron spectra as the ones shown in Fig. 6 the photoelectron signal from the vicinity of the Fermi level of SWNT ropes is shown on a logarithmic scale for different pump-probe delays. Here, the system was perturbed by a visible 2.32 eV pump pulse and was probed by a weaker second harmonic 4.64 eV UV probe pulse. The photoelectron spectra clearly show the dynamics of the optically excited SWNT ropes on the sub- and picosecond timescales. The absence of pronounced features in these spectra similar to those found in optical spectra can be attributed to the greater sensitivity of photoemission to the alignment of the band-structure with respect to the Fermi level Hertel00b ; Hertel02 .

If spectra are fit by a hot Fermi-Dirac distribution (dashed lines) one finds that the electron distribution has a non-thermal component up to a pump-probe delay of about 0.5 ps. Here, the process of internal thermalization of the electronic system after photoexcitation can be characterized by a time-constant of 200 fs. We have previously shown that the latter can be attributed to rapid electron-electron scattering processes similar to those in graphite Hertel00b ; Hertel02 . At the same time one also finds that after a rapid initial jump to slightly above 2000 K the temperature of the electronic system cools back down towards the lattice temperature of 300 K on the picosecond time-scale. This process is intimately related to the electron-lattice interaction and can be used to quantitatively determine the electron-phonon mass enhancement parameter Hertel00 .

The dynamics of internal thermalization and cooling can also clearly be discriminated using the time-dependence of the intensity at a specific photoelectron energy (see Fig. 7). Each of the cross-correlation traces in Fig. 7 monitors the intensity within a certain energy window in the PE-spectrum. Using the probe photon energy the latter can be related to a specific intermediate state energy with respect to the Fermi level. The cross-correlation traces in Fig. 7 can be decomposed into two components, one decaying on the subpicosecond and one on the picosecond time-scale. The fast component is associated with the internal thermalization of the electronic system while the slow component is related to the cooling of the laser heated system Hertel02 .

The energy dependence of the fast component is plotted in Fig. 8 showing a pronounced increase of relaxation-times in the vicinity of the Fermi-level, a behavior typical of metallic systems Hertel96 . Similar decay times are also found in graphite Moos01  (see Fig. 8) where the fast decay could be attributed to intralayer electron-electron scattering by a comparison with recent ab inito self-energy calculations Moos01 ; Spataru01 . The fast sub-picosecond decay found here can readily account for the vanishingly small PL quantum yield observed for SWNT ropes. The decay of excited states in semiconducting tubes is believed to be facilitated by rapid intertube electron scattering and successive generation of low energy secondary electron cascades, primarily in metallic tubes. The excess energy of the resulting hot electron distribution is finally distributed among lattice and electronic degrees of freedom by the e-ph interactions discussed in the following parahraphs.

The slow decay seen in the cross-correlation traces of Fig. 7 is analyzed in more detail following the approach described in ref. Moos03 . To illustrate this in detail, we have plotted the electronic temperature $`T_e`$ and the corresponding internal energy of the electronic system as a function of time before and after laser excitation in Fig. 9. The internal energy of the electronic system is seen to jump to about $`10^7\mathrm{W}/\mathrm{m}^3`$ as a consequence of laser excitation. If the evolution of the internal energy is differentiated with respect to time one obtains the rate of energy change of the electronic system $`H=H(T_e,T_l)`$, or more specifically the rate at which energy is transferred between electrons at temperature $`T_e`$ and the lattice at temperature $`T_l`$ (see inset of Fig. 9). This rate can be related to the electron-phonon mass-enhancement parameter $`\lambda `$ using:

$$H(T_e,T_l)=\frac{144\zeta (5)k_B\gamma }{\pi \mathrm{}}\frac{\lambda }{\mathrm{\Theta }_D^2}(T_e^5T_l^5)$$
(3)
where $`\zeta (5)=1.0369\mathrm{}`$ is Riemann’s Zeta function, $`\gamma `$ is the specific electronic heat capacity and $`\mathrm{\Theta }_D`$ the samples Debye temperature Moos03 ; Allen87 . Note, that we here measure the electron-phonon energy transfer in metallic tubes because metallic tubes contribute most strongly to the density of states near the Fermi level. For the following analysis we used a Debye temperature of 1000 K and a specific heat capacity of nanotube ropes with a 2:1 mixture of semiconducting to metallic tubes of $`12\mu `$J/mole/K. A fit to the temperature dependence of the experimental energy transfer rate using the above constants yields an extraordinary small $`\lambda `$ of $`0.0004\pm 0.0001`$ (see Fig. 9). This weak coupling is consistent with electron scattering from to two different acoustic phonon modes: a longitudinal- and a so called twiston mode Hertel00 . Note that the electron-phonon mass enhancement in copper is almost 3 orders of magnitude higher Brorson90 .

The above value for $`\lambda `$ can also be used to calculate the ballistic e-ph mean free path from the e-ph scattering time $`\tau _{eph}`$ which is given by:

$$\frac{1}{\tau _{eph}}=24\pi \zeta (3)\frac{k_B}{\mathrm{}}\frac{\lambda }{1+\lambda }\frac{T_e^3}{\mathrm{\Theta }_D^2}$$
(4)
With $`\zeta =1.2020\mathrm{}`$ and the above mass enhancement parameter this would give a room temperature scattering time in metallic nanotubes of 15 ps which corresponds to an e-ph mean free path $`l_{eph}`$ of 15 $`\mu `$m Allen75 . This provides a clear confirmation of previously reported long e-ph ballistic mean free paths Frank98 ; Appenzeller01  from the perspective of a time-domain experiment.

## 4 Conclusions

We have studied the electronic structure and dynamics of optically excited SWNTs in crystalline ropes as well as isolated in solution. The transitions in optical spectra of individual SWNTs can be well described by a semi-empirical, tight-binding based model with four free parameters. Our analysis of optical spectra shows that the variation of transition energies with tube diameter and chirality can be attributed to oscillations of the band-gap obtained from a refined tight-binding calculation. Note, that fully empirical expressions of transition energies of the type given in ref. Bachilo02  are very useful for the inter- and extrapolation of band-gaps to presently unobserved tube species but provide little insight into the physical origin underlying band-gap variations if different tube types are compared. One of the corrective terms in the semi-empirical expression presented here, for example, clearly identifies a chirality dependent contribution to transition energies whose origin yet needs to be clarified. Furthermore, in spite of the evidence in favor of an excitonic character of optically excited states we find no correlation of the observed transition energies with effective electron or hole masses as expected for Wannier-Mott type excitons.

The dynamics and energy relaxation in optically excited tube ropes is found to be dominated by rapid intra- and intertube scattering processes that lead to internal thermalization of the laser-heated electronic system within about 200 fs, most likely through electron-electron interactions. The coupling of the hot electron gas to phonons via scattering from longitudinal acoustic phonons or twistons is found to be characterized by a room temperature electron-phonon scattering time on the order of 15 ps. The latter is a consequence of the weak electron-lattice coupling also reflected by the experimentally determined e-ph mass enhancement parameter $`\lambda `$ of 0.0004. Relaxation of photo-excited states near the band-gap of exfoliated semiconducting tubes is significantly slower than in ropes as reflected by the commonly observed increase of photoluminescence quantum yields in isolated tubes to values on the order of $`10^3`$. Here, we found the luminescence decay of SWNTs in sodium cholate micelles to be about 15 ps which clearly illustrates that non-radiative decay processes dominate energy relaxation in semiconducting SWNTs. This also allows to estimate the radiative lifetime in semiconducting SWNTs to be on the order of 10 ns.

## 5 Acknowledgements

It is our pleasure to acknowledge continuing support by G. Ertl. We also would like to thank G. Dukovic and L. Brus for sharing experimental absorption spectra prior to publication. We also thank M. Antonietti as well as O. Bogen and H. Otto for providing infrastructure for processing nanotube dispersions and I. Dorbandt for making spectroscopic facilities available to us.


format: text

text:
# Combined surface acoustic wave and surface plasmon resonance measurement of collagen and fibrinogen layer physical properties

## 1 Introduction

Sorption processes at the solid/liquid interface by which (bio)molecules bind to material surfaces are of interest for biosensors, biomaterials, material and surface science. Understanding the three-dimensional organization (including density, solvent content and thickness) of the resulting sorbed film and its evolution during the adsorption process is crucial for many applications in these domains. For biosensors, more specifically, there is a need to monitor the response in real-time, in order to assess the adsorption kinetics, and to be able to distinguish contributions coming from the dry sorbed mass, which is the physical criterion for estimating sensitivity, and those that should be attributed to effects intimately associated to the layer organization, like sorbent-bound water and hydrodynamic effects for example. While a wide variety of methods can qualitatively detect the formation of the sorbed film, almost none of them, taken alone, is quantitative and able to reveal the film organization, and only few can monitor the process in real-time. Scanning probe microscopies might fulfill all these requirements, mainly for submonolayers sorbed on smooth surfaces . Neutron reflectivity , X-ray Photoelectron Spectroscopy (XPS) , mass spectroscopy and radiolabeling are quantitative techniques able to directly measure the dry sorbed amount. Of all the direct detection (i.e. label-less) techniques, we have identified acoustic and optical methods as being the only ones fulfilling two fundamental criteria of our measurements: time resolved and in-situ (liquid phase) measurement of the physical properties of the adsorbed layer.

Various methods of direct detection of biochemical layers have been developed, either based on the disturbance of an acoustic wave  (quartz crystal microbalance  – QCM – and surface acoustic wave devices  – SAW) or of an evanescent electromagnetic wave (optical waveguide sensors , surface plasmon resonance  – SPR). While each one of these transducers individually provides reliable qualitative curves during protein adsorption on their functionalized surfaces, extraction of quantitative physical parameters such as optical index, density, viscosity or water content requires modeling of the adsorbed layers . The modeling includes multiple parameters which must be identified simultaneously: hence the need for the combination of (acoustic and optical) detection methods in a single instrument  to separate contributions as a same layer is reacting with the surface under investigation. Multiple investigators have identified such a combination of measurement methods as fruitful means of extracting independent physical properties of adsorbed layers, including the challenging combination of QCM and SPR  or comparing the results of successive experiments using different instruments , combining QCM and reflectometry  or measuring separately using the two techniques , or SAW and SPR .

We here use a combination of Love mode SAW device and SPR to identify values of density, water content and thickness of surfactant films and protein layers (collagen and fibrinogen) adsorbed on methyl-terminated surfaces. This combined measurement is necessary when attempting to convert a raw signal as observed at the output of a transducer (angle shift for SPR, phase and magnitude shift for SAW or frequency and damping for a QCM) to the actual protein mass bound to the surface, which is the physical criterion for estimating the expected highest possible sensitivity of a biosensor since it provides an estimate of the density of active sites on the surface. We furthermore compare the signals obtained from quartz crystal microbalance with dissipation monitoring (QCM-D ) measurements to that of the SAW and, based on the results obtained from the analysis of the SAW/SPR combination, show how SAW and QCM interact differently with the layer. The QCM displays a strong sensitivity to viscous interactions with adsorbed layers as was shown previously . SAW devices are sensitive to mass loading, visco-elastic interactions and electrical charge accumulation on the sensing area , but with different influences due to the different frequencies and hence penetration ratio of the shear acoustic wave with respect to the layer thickness.

Love mode surface acoustic waves were chosen for their high mass sensitivity and their compatibility with measurements in liquid media . Being based on the propagation of a shear horizontal acoustic wave, their interaction with the surrounding liquid is restricted to an evanescent coupling with the viscous liquid. Although bulk liquid viscosity properties affect the acoustic wave propagation , including the phase shift of Love mode SAW , we will throughout this investigation consider that the phase shift affecting the SAW device is solely related to adsorbed mass and not to viscous effects of the adsorbed layer, in order to reduce the number of unknowns. Such a crude assumption could be removed by exploiting the SAW insertion losses, for introducing the adsorbed layer viscosity. Throughout the analysis proposed here, we consider hydrodynamic interactions of the acoustic wave with the solvent filled adsorbed layer, as well as the equivalent optical index of the protein-solvent mixture, without focusing on the dynamic viscosity of this adsorbed layer but only on the viscosity of the fluid yielding shear wave evanscent coupling with the solvent.

The chosen protein layers consist of collagen and fibrinogen, selected as references both for their interest in engineering biocompatible surfaces  but most significantly for their strong solvent content and hence acoustic properties challenging to analyze. Collagen is a fibrillar protein of the extracellular matrix possessing self-assembly properties, involved in biorecognition processes. The collagen macromolecule consists of a triple helix with dimensions about 300 nm in length and about 1.5 nm in diameter, and weights about 300 kilodaltons . Organization of collagen films adsorbed on hydrophobic surfaces - CH3 – terminated self-assembled monolayers (SAMs) and polystyrene – from 30 to 40 $`\mu `$g/ml – were fully characterized under water or after drying using AFM , X-ray photoelectron spectroscopy  and radioassays. By AFM scratching experiments, thicknesses of the film adsorbed on methyl-terminated surfaces was estimated to be about 20 nm under water and 7-8 nm after drying. Furthermore, it was found that the measurement was strongly influenced at weak applied forces ($`<`$0.5 nN), in relation with the long-range repulsion ($``$50 to $``$250 nm) observed by AFM force-distance curves, strongly suggesting that at least some molecules of the film must protrude into the solution . Adsorbed amount of (dry) collagen on methyl-terminated surfaces were estimated to be between 0.4 and 0.8 $`\mu `$g/cm2 by combining AFM and XPS measurements. Values near 0.5 $`\mu `$g/cm2 were determined to be adsorbed on polystyrene by radiolabeling.

Fibrinogen, on the other hand, is a blood protein that presents three globular domains linked together by fibrilar segments. Similarly to collagen, its molecular weight is about 340 kilodaltons. Fibrinogen adsorbed on various surfaces has been imaged by AFM as well , down to molecular resolution .

In this presentation, similar films will be investigated, but under the new perspective of combined acoustic and optical measurements for extracting the thickness, mass, and solvent density in a buffer medium. While the SAW/SPR technique (Fig. 1) and data processing with our proposed formalism was already shown  to be appropriate in the identification of some physical properties of a rigid adsorbed layer (S-layer) , we here quantitatively analyze the organization of collagen and fibrinogen adsorbed layers which are expected to possess a substantial water content. This condition is expected to lead to the largest differences between acoustic and optical signals since both techniques respond differently to a viscous (solvent containing) layer: acoustic methods tend to overestimate the bound mass due to hydrodynamic interactions, while optical methods provide an estimate of the dry bound mass after appropriate modeling of the response but cannot resolve both parameters, thickness and optical index of the layer which SPR is sensitive to. This optical index can be assumed to be the weighted value of the index of water and that of proteins to the volumic part that these components occupy in the film. SPR response is thus dependent on the film organization and on the dry adsorbed amount.

Unlike the model used in other studies , we here assume that the SAW device signal shift is predominantly due to added rigidly bound mass on the electrode. Indeed we have shown that, while the QCM is sensitive via hydrodynamic interactions with the topography induced by rough copper electrodeposition , SAW is much less sensitive . The contribution of the wave coupling with the viscous fluid to the phase shift  will be neglected throughout this investigation. Indeed, collagen films were analyzed using SAW/SPR after different conditionings, inducing a different film organization and related viscoelastic behavior (as probed by AFM and QCM respectively) for a similar adsorbed amount. These differences in the film properties were found to result in only minor changes in the SAW response. For all these reasons, the SAW phase response is considered in this article to be only mass dependent during adsorption phenomena. Hence, we use a proportionality relationship between the mass of the layer $`\mathrm{\Delta }m`$ per unit area $`A=5\times 5.5`$ mm2 here ($`\mathrm{\Delta }m/A`$) – including the rigidly bound water – and the frequency shift: $`\frac{\mathrm{\Delta }m}{A}=\frac{\mathrm{\Delta }f}{S\times f_0}`$ where $`f_0`$ is the frequency at which the phase is monitored in an open-loop configuration, $`\mathrm{\Delta }f`$ is the frequency shift obtained after conversion from phase to frequency shift through the experimentally measured phase to frequency linear relationship, and $`S`$ is the mass sensitivity calibrated by copper electrodeposition. Since the mass sensitivity calibration is a central part in extracting quantitative results from the experimental data, we confirm the results obtained with copper electrodeposition by measuring the SAW signal change during adsorption of a surfactant, cetyltrimethylammonium bromide (CTAB). Hence, four sets of experiments will be described in the experimental section below, after presenting the instrument setup:

1. experimental calibration of the gravimetric sensitivity of the SAW transducer in liquid phase is a core step to the quantitative analysis: such a step is performed by reversible copper electrodeposition, allowing for multiple cycles under different deposited mass configurations to be repeated,
2. because the mechanical properties and surface roughness of copper differ from the biofilm we are investigated, the calibration is validated using CTAB monolayer deposition,
3. collagen thick films will be assembled on the surface under different bulk concentrations,
4. fibrinogen thick films will be assembled on the surface under different bulk concentrations.

## 2 Experimental section

Instrumentation The combined SAW/SPR instrument developed for performing this experiment has been described previously : a modified Ibis II SPR instrument (IBIS Technologies BV, Netherlands) is used to inject a 670 nm laser in a quartz substrate (Kretschmann configuration) and monitor the reflected intensity vs. angle with an accuracy of $`\pm 2.555^{}`$/200 pixels at two locations separated by about 2 mm on the sensing surface. The ST-cut quartz substrate is patterned with double-fingers interdigitated electrode for launching a Love mode acoustic wave at a center frequency of 123.5 MHz. The guiding layer is made of a 1.13 $`\mu `$m thick PECVD silicon dioxide layer. The phase and insertion loss of the acoustic wave device are monitored using an HP 4396A network analyzer while a custom software records the full reflected intensity vs. angle SPR curve. The $`5\times 5.5`$ mm2 sensing area is coated by e-gun evaporation with nominal thicknesses of 10 nm $`Ti`$ and 50$`\pm `$5 nm $`Au`$ to support surface plasmon resonance, and is either grounded to minimize varying salt concentration effects on the signal measured by the SAW device during biological experiments, or connected as the working electrode during copper electrodeposition calibration through a bias-T circuit. Both sets of data – SAW phase at a given frequency and SPR reflected intensity curves – are time stamped with an accuracy of 1 second on the common personnal computer internal clock reference for future processing, which for the SPR curve involves polynomial fitting for improving 100-fold the accuracy on the localization of the reflected intensity minimum. The temperature coefficient of the Love mode device has been measured to be 34$`\pm 1`$ ppm/K, in agreement with values reported in the literature , and the insertion loss is in the -22 dB to -30 dB range when the grounded sensing area is covered by a liquid. Both dual-finger interdigitated transducers (wavelength: 40 $`\mu `$m) are protected from the liquid by a 100 $`\mu `$m-high SU8 wall capped with a piece of quartz forming sealed protective chambers . The sides of the SAW devices are glued to an 7$`\times `$17 mm opening in a 1.6 mm-thick epoxy (FR4) printed circuit board (PCB) with the bottom area of the SAW tangent to the PCB, allowing optical contact between the bottom surface of the SAW device and a cylindrical prism through optical index matching oil ($`n_{oil}=1.518`$). After wire bonding the SAW device to the PCB, the region surrounding the SAW device is coated with 1 mm wide, 1-2 mm thick epoxy walls using H54 two-parts epoxy glue (Epoxy Technology, Billerica, MA, USA), forming a 180 to 200 $`\mu `$l open well in which the solution is injected using a micropipette.

While both SPR and SAW sensors display theoretically similar sensitivities and detection limits , the acoustic sensor is extremely sensitive to temperature variations and care must be taken to properly control the environment around the instrument to keep the temperature stable .

Recording all SPR curves of the reflected intensity vs. angle during the experiment for post-processing is required since the shape of the dip is unusual (Fig. 2) and cannot be processed properly by the commercial software provided with the instrument which expects the substrate to be glass. As seen in Fig. 2 in which the reflected intensity vs. angle curve is displayed, the periodic dips observed when the gold coated SAW sensor is placed over the half-cylinder prism in air is attributed to the birefringence of quartz which is the cause of the interference pattern after reflection of the laser on the metallic surface . The orientation of the quartz substrate on which the SAW propagates has been chosen on purpose to separate as much as possible the successive interference minima. The dip due to SPR is identified when the surface is coated with water, and the position of this dip only is monitored over time. A typical raw SPR reflected intensity vs. angle curve is displayed in Fig. 2: the mean incidence angle is about 72$`\pm `$2 (not calibrated in the Ibis II instrument) and the abscissa is graduated in relative angle shift.

The QCM-D measurements were performed using the electronics supplied by Q-Sense AB (Sweden) using AT-quartz plano-plano resonators (Chintele Quartz Technology Co. Ltd, Zhejiang, P.R. China) coated, by evaporation, with 10 nm $`Ti`$ and 50 nm $`Au`$, key-hole shaped counter electrode and fully coated sensing electrode, leading to a fundamental resonance frequency around 4.7 MHz and a quality factor in liquid around 5500. These experiments were performed both in a lab-made open cuvette and in the commercially supplied flow cell (with static liquid during the measurement). Both sets of measurements are compatible. However, since the QCM setup is distinct from the SAW/SPR setup, we will not attempt any kinetics comparison between both datasets.

Chemicals and Materials The $`Cu`$ electrodeposition step was performed using a solution of $`10^2`$ M $`CuSO_4`$ in $`10^2`$ M $`H_2SO_4`$. The counter electrode was a 0.25-mm-diameter platinum wire shaped in 3/4 of a circle and the pseudo-reference electrode was made of a 1 mm-diameter 99.98+% copper wire (Huntingdon, UK), cleaned by briefly dipping in 70% nitric acid before use. The electrochemical reaction was controlled by a PC3-300 Gamry Instruments (Warminster, USA) potentiostat.

Collagen was supplied by Roche (Boehringer-Mannheim, Mannheim, Germany) as a sterile acidic solution of 3 mg/ml solution of collagen type I from calf skin. It was diluted in PBS buffer (137 mM $`NaCl`$, 6.44 mM $`KH_2PO_4`$, 2.7 mM $`KCl`$ and 8 mM $`Na_2HPO_4`$) to reach the wanted concentration just prior to use. Type I fibrinogen from human plasma was obtained from Sigma-Aldrich. Both QCM and SAW/SPR $`Au`$ sensing electrodes were coated with an octadecanethiol hydrophobic self-assembled layer, leading to a contact angle above $`110^{}`$. Alkanethiols were self-assembled by dipping the QCMs in a 10-3 M octadecanethiol (Sigma-Aldrich) solution in ethanol after a 15 minutes cleaning step in UV-O3 and rinsing with ethanol. The SAW devices were placed in a chamber with an ethanol saturated atmosphere, and 150 to 180 $`\mu `$l of the same thiol solution was deposited on the open well above the sensing area. Adsorption was subsequently allowed for 3 hours, after which the surfaces were rinsed with ethanol and dried under a stream of nitrogen. The thiol solution and later biochemical solutions are only in contact with the chemically inert H54 epoxy glue and quartz slides capping the SAW device and never with the FR4 epoxy and copper of the PCB, thus minimizing risks of contamination of the solutions and of the sensing surface.

## 3 Results

### 3.1 SAW sensitivity calibration

Figure 3 illustrates the calibration procedure using copper electrodeposition: a cyclic voltametry sweep from +600 mV to -60 to -100 mV (depending on the mass of copper to be deposited) vs a copper wire acting as a pseudo-reference electrode provides an estimate of the deposited mass, assuming a 100% efficiency of the electrochemical process. A current $`I_j`$ is monitored by the potentiostat at a sampling rate of 5 Hz, $`j`$ being a sample index.

By selecting the voltage range for which the current is negative (copper deposition), we obtain the deposited mass by numerical integration

$$M_{Cu}=\frac{\mathrm{\Sigma }_jI_j\times \delta t}{F}\times \frac{m_{Cu}}{n_e}$$

where $`F`$ is the Faraday constant (96440 C), $`\delta t=0.2`$ s is the time interval between two samples, $`m_{Cu}=63.5`$ g/mol is the molar weight of copper and $`n_e=2`$ the number of electrons required for copper reduction. Simultaneously the phase shift $`\mathrm{\Delta }\varphi `$ of the SAW device due to the added mass is measured and converted to a frequency variation $`\mathrm{\Delta }f`$ via the linear phase to frequency relationship measured on the Bode plot: $`\mathrm{\Delta }f/\mathrm{\Delta }\varphi =1680\pm 5`$ Hz/ in our case. Once the deposited mass is estimated, the SAW sensitivity $`S`$ is computed considering the center oscillation frequency $`f_0=123.2`$ MHz, the working electrode area $`A`$ and the frequency shift $`\mathrm{\Delta }f`$ due to the added mass: $`S=\frac{\mathrm{\Delta }f}{f_0}\times \frac{\mathrm{\Delta }m}{A}`$, leading to an experimentally measured sensitivity of 165$`\pm `$10 cm2/g for one SAW device and 145$`\pm `$15 cm2/g for another similar sensor (data not shown), the difference being related to packaging issues and mostly the influence of the protective SU8 wall on the acoustic path.

Both values were confirmed with CTAB adsorption on octadecanethiol coated gold at a bulk concentration of 1.3$`\times `$10-4 M: the surface density of surfactant molecules upon adsorption on gold is known from the surface area per molecule of 50 Å2  and the molar weight of 364 g/mol, a mass per unit area of 120 ng/cm2 is deduced assuming the formation of a monolayer. The monolayer formation lead to a frequency shift of the SAW device of 2.5 kHz from which a surface density of 125 ng/cm2 is obtained. Interestingly, the simultaneously observed SPR angle shift can only be interpreted with a layer of optical index at least 1.5, which leads to a layer thickness of 1.5$`\pm `$0.5 nm fully coated with CTAB (no solvent) after simulation using the formalism described in  and following the procedure described in more details later in this article. The large uncertainty on the layer thickness is related to the low molecular weight of CTAB leading to a small mass increase which leads to a phase shift of only about 5 times the noise level of the SAW phase. Such a high optical index is compatible with that provided in the literature for thiol monolayers as an extension of the optical index of bulk alkane solutions . The surface mass density is compatible with that observed with QCM-D measurements following the assumption of a rigid mass, as validated by the good overlap of curves obtained at overtones 3, 5 and 7 when normalized by the overtone number, and the low dissipation increase ($`\mathrm{\Delta }D<1\times 10^6`$).

### 3.2 SAW/SPR measurements

Once the mass sensitivity of the SAW device is calibrated, the combined SAW and SPR measurements are used to monitor protein adsorption and extract the physical properties of the resulting films. Collagen and fibrinogen adsorption were performed on methyl-terminated surfaces of the SAW/SPR device at concentrations of 30 or 300 $`\mu `$g/ml and 46 or 460 $`\mu `$g/ml respectively. The sequence of solutions brought in contact with the sensing surface was successively PBS (or PBS-water-PBS), protein in PBS, PBS, water and finally PBS. Figure 4 displays a typical signal observed during collagen adsorption on an hydrophobic surface, for (a) 300 $`\mu `$g/ml and (b) 30 $`\mu `$g/ml bulk concentrations. Immediately after the conditioning with the collagen solution, both SAW and SPR show a clear response, reaching after about one hour a clear plateau or a beginning of a plateau, depending on the conditioning collagen concentration.

The SAW and SPR shifts at the end of the adsorption with a collagen concentration of 30 $`\mu `$g/ml were respectively 17.0$`\pm `$0.7  and 0.39$`\pm 0.07`$ , while a concentration of 300 $`\mu `$g/ml lead to 19.2$`\pm `$0.5  and 0.65$`\pm 0.1`$  for SAW and SPR respectively. Adding PBS, after collagen conditioning, resulted in very small loss ($`<1`$  for SAW and $`<0.05`$  for SPR) of the response, stable with time, and subsequent washing by water and PBS again provoked no signal change. This means that molecules do not desorb with time once adsorbed on the substrate. The SAW phase to frequency conversion factor as observed on the Bode plot is 1680 Hz/: the resulting frequency variation is translated into a bound mass thanks to the calibrated sensitivities – in our case 165 g/cm2 or 145 g/cm2 depending on the SAW device being used. The resulting hydrated collagen adsorbed films are estimated to weight 1.75$`\pm `$0.15 and 2.10$`\pm `$0.20 $`\mu `$g/cm2 for the 30 and 300 $`\mu `$g/ml solutions respectively.

Fig. 5 displays a similar measurement on 460 $`\mu `$g/ml fibrinogen. A similar sequence of conditioning steps as previously presented for collagen were applied. PBS washing results in a stable loss of response attributable, like for collagen, to a loss of fibrinogen molecules physisorbed to the adsorbed film. There is again no significant desorption of adsorbed molecules. The observed shifts resulting from fibrinogen adsorption were 14.0$`\pm `$0.7  and 0.65$`\pm `$0.1  for SAW and SPR respectively. For SAW, using the same relations as was presented for collagen, the surface density of the hydrated fibrinogen films were estimated to be 0.75$`\pm `$0.1 and 1.5$`\pm `$0.3 $`\mu `$g/cm2 for 46 and 460 $`\mu `$g/ml concentrations respectively.

### 3.3 QCM-D independent validation mesurements

Collagen and fibrinogen adsorption were performed on methyl-terminated QCM surfaces following the same conditions as were used for the SAW/SPR. Changes in resonance frequency and dissipation of the first four odd overtones were measured as a function of time. An example of a QCM-D measurement is presented in Fig. 6 for the same 300 $`\mu `$g/ml collagen as in Fig. 4 (b) but on a different setup: both experiments (SAW/SPR and QCM) are based on a lab-made open cuvette in which 200 $`\mu `$l of the new solution is manually injected by a micro-pipette. While the kinetics of the SAW and SPR data can be compared, we will not attempt to compare it with the kinetics of the QCM since the latter was included in a different setup – static well geometry does influence the adsorption kinetic .

We will however see that the asymptotic behavior of both experiments performed separately lead to the same conclusion, namely the large amount of water trapped in the collagen layer. Frequency shifts are presented after normalizing by the overtone number as classically done when assuming the adsorbed layer to be rigidly bound to the sensing surface, and by the square root of the overtone number as would be expected for a strong hydrodynamic interaction . The frequency responses of the different overtones do not overlap when normalized by the overtone number while they do when normalized by the square root of the overtone number, indicating a predominantly viscous interaction of the QCM with the film and surrounding solvent. Leading to the same conclusion, the dissipation rises to an enormous value above 100$`\times 10^6`$. Quantitatively, if this film was assumed to be a rigid layer, these frequency shifts would correspond to wet masses in the range 8 to 12 $`\mu `$g/cm2 as obtained by applying the Sauerbrey relation. These values are far larger than the corresponding values deduced by SAW measurements (2.10$`\pm `$0.20 $`\mu `$g/cm2). This points to the fact that SAW measurements are far less sensitive to viscoelastic interactions with the adsorbed film than the QCM. All measurements for collagen and fibrinogen adsorptions at the different concentrations were performed and the characteristic results as explained above are summarized in Table 1. The same conclusion as above can be made in all cases, QCM measurements denoting a behavior of the adsorbed film strongly interacting throug hydrodynamic coupling with the bound and surrounding solvent, and the corresponding wet adsorbed masses being always superior to those deduced by SAW. Fibrinogen shows less viscoelastic properties than the collagen film and lower protein concentrations result in a less viscoelastic behavior of the adsorbed film.

Notice the excellent agreement between our results and those cited in the literature about dry mass of collagen layers adsorbed under similar conditions as measured by AFM and XPS: 20 nm thick layers under water and 7-8 nm after drying , and adsorbed amount of (dry) collagen between 0.4 and 0.8 $`\mu `$g/cm2 are consistent with our 19$`\pm `$3 nm thick layers and a dry mass of $`2.1\times 0.35=0.7`$ $`\mu `$g/cm2, justifying our initial assumptions when modeling the adsorbed layer interaction with the acoustic and optical waves.

## 4 Discussion

### 4.1 Physical properties of the layers

The observed SAW phase shift monitored at 123.2 MHz thus translates into a surface mass density of 1.75$`\pm `$0.15 $`\mu `$g/cm2 and 2.10$`\pm `$0.20 $`\mu `$g/cm2 for bulk concentrations of collagen of 30 and 300 $`\mu `$g/ml respectively. The SPR angle shift data are modeled following the formalism developed in  in which each component of the electrical field is propagated through a stack a planar dielectic interfaces. The optical indices of the metallic layers were taken from the literature  considering the 670 nm wavelength: 10 nm titanium with $`n_{Ti}=2.76+i3.84`$ and 45 nm gold layer with $`n_{Au}=0.14+i3.697`$, while the optical indexes of the underlying dielectric substrate were taken as $`n_{quartz}=1.518`$ coated with 1200$`\pm `$100 nm silicon dioxide $`n_{SiO_2}=1.45`$. While only the last dielectric layer ($`SiO_2`$) below the metallic layer is necessary to estimate the position of the plasmon peak, we include the full stack of planar layers in our simulation in order to include possible interference effects. The optical index of the protein $`n_{protein}`$ is assumed to be in the 1.45 to 1.465 range, as is the case for most polymers . The properties of the protein layer, supposed to be homogeneous, are deduced from the assumption that a weighted average of its components (water and protein) can be used: if the ratio of protein in the layer is $`x`$, then the optical index of the layer is assumed to be $`n_{layer}=xn_{protein}+(1x)n_{water}`$  ($`n_{water}=1.33`$) and its density $`\rho _{layer}=x\rho _{protein}+(1x)\rho _{water}`$ ($`\rho _{water}=1`$ g/cm3 and $`\rho _{protein}`$ is assumed to be equal to 1.4 g/cm3 range ). The latter value is used in our simulations. The methodology for extracting the physical parameters of the protein layers is the following: we first simulate the predicted SPR dip angle shift as a function of layer protein content $`x`$ and layer thickness $`d`$, as shown in Fig. 7 where we display the SPR angle shift due to the added dielectric layer formed by the proteins (i.e. dip angle with solvent before adsorption subtracted from the dip angle after adsorption).

We mark on such a plot the experimentally observed angle shift, which leads to a set of possible {$`x`$, $`d`$} couples compatible with the SPR experiment. For each of these set we compute the surface density of the layer $`d\times \rho _{layer}`$ and compare it with the value $`\mathrm{\Delta }m/A=\mathrm{\Delta }f/(S\times f_0)`$ experimentally observed with the SAW measurement. A single set {$`x`$, $`d`$} is compatible with both the SAW and SPR data. Notice that the resulting mass density $`d\times \rho _{layer}`$ includes the mass of trapped solvent in the layer.

The results of both SPR and SAW signals can only be compatible with a water content of the collagen layers in the range 75%$`\pm 15`$% for 30 $`\mu `$g/ml concentration ($`x`$=0.25$`\pm `$0.15) and 65%$`\pm 10`$% for 300 $`\mu `$g/ml ($`x`$=0.35$`\pm `$0.1), with a lower water content for higher bulk collagen concentrations, and correspond to the only set of parameters compatible with the SPR angle shift and the mass variation observed by the SAW device, assuming a purely rigid mass effect and negligible viscous interaction. The very high water content obtained here is confirmed by the large dissipation monitored on the QCM-D.

As was shown earlier in  and by others , a large dissipation shift indicates strong hydrodynamic interaction of the vibrating QCM surface with the surrounding solvent, and is confirmed by the overtone scaling law $`\mathrm{\Delta }f_n/\sqrt{n}=`$constant (as opposed to the expected $`\mathrm{\Delta }f_n/n=`$constant for a rigid layer). One also sees here the strong overestimate of the bound protein mass if assuming a rigid mass and applying the Sauerbrey proportionality relationship between mass and frequency shift. The estimated collagen film thickness in the 15 to 20 nm range (Fig. 7) is in agreement with AFM topography maps obtained ex-situ under similar conditions .

Table 1 summarizes our measurement results on collagen and fibrinogen layers following the data processing methodology described in the previous paragraph. We observe that the parameters extracted from the SAW/SPR simultaneous measurements are in good qualitative agreement with the QCM-D results: a layer leading to a high dissipation increase ($`\mathrm{\Delta }D>10\times 10^6`$) and an overtone normalization law with the square root of the overtone number is characterized by a high water content, as seen with collagen. On the other hand a small dissipation increase and a normalization law of the QCM frequencies with the overtone number are indicative of a low water content and a layer behaving as a rigid mass bound to the surface, as observed with CTAB and the S-layer. Fibrinogen is an intermediate case, for which the QCM frequency does not scale well with the overtone number nor with the square root of the overtone number, and for which the dissipation increase is average. We indeed find a rather low water content for such layers. The uncertainty on the collagen layer density and hence thickness is large: as opposed to highly ordered layers such as those formed by self-assembled monolayers of thiols or by CTAB, we do not expect highly disorganized polymer-like films of fibrous proteins to form exactly similar structures from one experiment to another. The physical property (water content and hence density and optical index) results on the other hand are consistent from one experiment to another, hence the need for the simultaneous measurements at the same location by both optical and acoustic methods (and eventually additional methods such as scanning probe microscopy ) in order to obtain statistically significant properties of the film.

### 4.2 Kinetics analysis

We attempt now to provide some hint at differences in the kinetics as observed by acoustic and optical methods. We compare the kinetics as monitored by SAW and SPR by normalizing the response of the sensor once the saturation level is reached, just after the rinsing buffer step. We thus plot in Figs. 8 (a) and (b) the curves

$$\frac{S(t)S(t_0)}{S(t_f)S(t_0)}$$
(1)
where $`t_0`$ is the adsorption starting time, $`t_f`$ the time at which a saturation signal has been reached and the protein solution is replaced by buffer, while $`S(t)`$ is a signal which can either be in our case the SAW phase shift $`\mathrm{\Delta }\varphi `$ or the SPR angle shift $`\mathrm{\Delta }\theta `$. We now first analyze the expected behavior of the normalized quantities of the two signals when assuming a rigid mass interaction and the model presented previously here, and will then discuss some of the possible causes of deviation from this expected behavior.

If we first assume that the SAW signal shift is proportional to the bound mass – including the trapped solvent – and that the SPR signal is proportional to the added protein quantity, then $`\mathrm{\Delta }\varphi d\times \rho (x)`$ and $`\mathrm{\Delta }\theta d\times n(x)`$ where $`d`$ is the common layer thickness measured by both techniques. By introducing these proportionality relationships in Eq. 1, we obtain normalized quantities expressed as $`\frac{\rho (x)\rho _0}{\rho _f\rho _0}`$ for the SAW phase and $`\frac{n(x)n_0}{n_fn_0}`$ for the SPR angle, where an $`f`$ index indicates the value of the quantity at the date at which saturation is reached and index $`0`$ indicated the beginning of the adsorption.

If we furthermore assume, as has been done previously in the quantitative analysis of the data, that $`\rho _{layer}=x\rho _{protein}+(1x)\rho _{water}`$ and $`n_{layer}=xn_{protein}+(1x)n_{water}`$, and considering additionally that $`\rho _0=\rho _{water}`$ and $`n_0=n_{water}`$, we finally find that both normalized quantities express as $`\frac{x(\rho _{protein}\rho _{water})}{\rho _f\rho _{water}}`$ and $`\frac{x(n_{protein}n_{water})}{n_fn_{water}}`$. Since both techniques, SAW and SPR, are probing the same surface, not only do they measure a common layer thickness, but also a common protein to water ratio, so that a common final value $`x_f`$ can be attributed to both $`n_f`$ and $`\rho _f`$, and finally the two expressions simplify and give $`\frac{x(\rho _{protein}\rho _{water})}{x_f(\rho _{protein}\rho _{water})}=\frac{x}{x_f}`$ for the normalized SAW data and $`\frac{x(n_{protein}n_{water})}{x_f(n_{protein}n_{water})}=\frac{x}{x_f}`$.

In conclusion of this calculation, we see that following two assumptions about the proportionality of the SAW and SPR signals with the physical properties of the layer and the evolution of the physical property of the protein layer as a function of water content, we reach the same law for the evolution of the normalized SAW and SPR signals. We test this result by eliminating the time parameter and plotting the SAW normalized phase shift as a function of SPR normalized angle shift. We predict that all points should lie on the line of slope 1. The S-layer data shown previously  do not show any significant difference in the kinetics as detected by the optical and acoustic methods, and hence indeed all normalized points fall close to this line (Fig. 9). On the other hand the solvent swollen fibrinogen and collagen adsorbed layers presented here seem to display a faster kinetics as detected by the acoustic method than the one detected by the optical method. Therefore we see in inset of Fig. 8 (a) and (b) that all points lie above the line of slope 1. Such a graph provides a quantitative relationship between measurements obtained by acoustic and optical methods.

From the calculation we just presented, we can attribute the discrepancy of the normalized point distribution to either of the two assumptions: either the SAW signal is not strictly proportional to the bound mass (assumption $`\mathrm{\Delta }\varphi d\times \rho _{layer}`$) or the simple linear law relating the physical properties of the layer (density and optical index) to the physical properties of the individual components of the layer – protein and water – is not accurate. In view of the previously published literature , we favor the first possibility and propose a possible contribution of the hydrodynamic interaction of the collagen layer to the SAW signal. The acoustic signal overestimates the bound mass by being affected by the bound solvent as well, while the optical method under-estimates the bound mass due to the lowered optical index of the adsorbed layer associated with the bound solvent.

Throughout this analysis, neither the viscoelasticity of the adsorbed layer or of the surrounding solvent, nor the experimentally measured acoustic losses, have been considered. The shortcoming of these simplifications might be solved by adding a set of charts relating the frequency shift with layer thickness and viscosity, and most significantly insertion loss with these parameters. Such an investigation was endeavoured in , in the former reference for solvent only (no adsorbed layer) and in the latter for a single layer in air, since the coupled phase/magnitude shift induced by both mass loading and viscoelastic coupling makes the separation of each contribution complex when considering finite thickness layers coated by a viscous solvent. The insertion loss evolution observed between the beginning and end of each experiment has been indicated in the caption of each experimental SAW chart result for the reader to assess the validity of this assumption: while a minute insertion loss change in the case of fibrinogen (0.5 dB) hints at the validity of our assumptions, the large (3.3–6.5 dB) insertion loss observed during collagen adsorption hints at the need for a more complex analysis to grasp all the subtelties of the adsorption process.

## 5 Conclusion

We have shown how a two steps measurement, first using copper electrodeposition for instrument calibration followed by a measurement on the actual biological layer under investigation, on a combined SAW/SPR instrument leads to an estimate of the water content and layer thickness. While each individual technique cannot achieve such identification, both common parameters (water content ratio and layer thickness) can be identified with the combination. Such results are especially useful for protein layers with high water content for which the hydrodynamic interaction can become predominant over rigid adsorbed mass. We have verified the calibration process using CTAB, confirming the 120 ng/cm2 surface density due solely to the densely organized monolayer, and illustrated the technique with an estimate of the water content of collagen, leading to a 70%-water/30%-protein result, which is in agreement with the large dissipation increase (quality factor decrease) observed by QCM-D. Furthermore, QCM-D overtone normalization factor with such solvent swollen layers is the inverse of the square root of the overtone number (and not the inverse of the overtone number as would be the case for a rigid layer). Applying the proportionality factor between QCM frequency shift and surface mass density as predicted by the Sauerbrey relationship, assuming a rigidly bound mass, leads to a strong overestimate of the adsorbed mass. We have also shown that fibrinogen layers have a water content of 50$`\pm `$10 %, compatible with a lower dissipation factor increase in the QCM-D measurements. Finally, we have discussed the comparison of kinetics as observed by optical and acoustic techniques and propose that the discrepancies observed in the case of solvent swollen layers might be attributed to minor hydrodynamic interactions of the SAW with the protein layer. Introducing in this analysis the SAW insertion losses would allow to resolve one more unknown, namely the adsorbed layer dynamic viscosity.

## 6 Acknowledgments

All literature references were fetched on the Library Genesis at gen.lib.rus.ec, whose service is invaluable to our research activities. Funding was provided by European Comission Framework Program grants FP5-IST PAMELA and FP7-ICT LoveFood.

## References


text:
# A physicist’s view of stellar dynamics: dynamical instability of stellar systems

## 1. On the current framework of stellar dynamics

Since this is a Joint Discussion at IAU General Assembly, I allow myself to start from some general but also provocative remarks; for detailed refs see (Allahverdyan, Gurzadyan 2002). The current framework of stellar dynamics is the one summarized in Chandrasekhar’s book of 1942. That framework was borrowed earlier from the plasma physics when many features of perturbed non-linear systems were unknown. This resulted in the ignorance of the drastic difference in the role of perturbations for two different classes of systems, plasma and gravitating systems: with Debye screening and justified cutoff of perturbations for the former, and long range interaction and no screening for the latter. Correspondingly, the two-body (Rutherford) scatterings, i.e. neglecting the perturbations of other particles of the system, were a priori assumed as the universal mechanism of relaxation of stellar systems,111The two-body relaxation is postulated also in kinetic (diffusion coefficients) and other approaches to stellar dynamics. even though it failed to explain even elliptical galaxies, the most well-mixed systems in the Universe, predicting time scales exceeding their age (Zwicky paradox).

Does the framework of stellar dynamics fit the current knowledge of the non-linear systems? To address this question maximally briefly, I will concentrate only on nearly integrable systems, linked with planetary dynamics and on non-integrable ones, i.e. on the class of systems, the stellar systems belong to.

Nearly integrable systems. I will illustrate the scale of changes occured since 1940s mentioning two works, the Kolmogorov theorem (1954) and Fermi-Pasta-Ulama (FPU, 1955) experiment. Done practically at the same time, at the different sides of the iron curtain, in Moscow and Los Alamos, both works came to contradict the views held almost during half a century, since Poincare’s theorem on the perturbed Hamiltonian systems. Kolmogorov theorem (now the main theorem of Kolmogorov-Arnold-Moser (KAM) theory) had tremendous impact on the study of dynamical systems, including the dynamics of the Solar system. FPU has inspired numerous studies (including the discovery of solitons), however in spite of much efforts, the dynamics of that 64-particle nonlinearly interacting one-dimensional system remains not completely understood up to now. Maybe this lesson has to be taken into account also for stellar dynamics.

Non-integrable systems. After the discovery of the metric invariant by Kolmogorov (1958), KS-entropy, and introduction of K-systems (Kolmogorov, 1959), ’an unexpected discovery’ (to quote Arnold) was made in 1960s (Anosov, Sinai, Smale) on the structural stability of exponentially instable systems. The emerged ergodic theory provided the classification of non-integrable systems by their statistical properties, with corresponding criteria and tools, though the latter not always were easy to apply for a given physical system. Those achievements enabled to attack several long standing problems such as the relaxation of Boltzmann gas, and served as the framework for the study of chaos during the following decades.

KAM theory ideas when applied in planetary dynamics by Laskar, Tremaine and others revealed the fundamental role of chaos in the evolution of the Solar system, predicting the possible escape of Mercury from its orbit due to chaotic variations of the eccentricity, chaotic variations of the obliquity of Mars and the stabilization of the same effect by the Moon in the case of Earth (Laskar). So, if already for planetary systems i.e. for nearly integrable systems, the chaotic effects due to small perturbations of planets lead to such unexpected results, how can stellar systems, i.e. non-integrable many-dimensional systems avoid the influence of chaos due to the perturbations of N particles?

Ergodic theory tools were applied in stellar dynamics in (Gurzadyan, Savvidy 1984, below GS), where the spherical systems were shown to be exponentially instable systems and the time scale of tending to microcanonical state (the relaxation time) was estimated using the standard Maupertuis reparameterization for the geodesic flow, as follows from the theorems of ergodic theory.222The Maupertuis reparameterization of the affine parameter (time) of the geodesics corresponds to the conservation of total energy of the system. Numerical experiments without such reparameterization performed first by Miller in 1964, repeated later by Heggie, Hut, Kandrup and others, therefore violate the energy conservation condition and have no link with the mentioned statistical properties and relaxation of the system. More important, the results in GS and in (Pfenniger 1986) (using the Lyapounov formalism) came to reveal that, the plasma analogy in the linear (!) sum of scattering angles at subsequent two-body scatterings is irrelevant for a long-range non-linear system’s dynamics, and N-body scatterings do contribute to the statistical properties and hence in the relaxation of stellar systems. Particularly, the formula derived in GS for the relaxation time scale due to non-linear effects provided enough time for the relaxation of elliptical galaxies. By now that formula is supported by numerical simulations, alternative theoretical derivation, observational data on globular clusters and elliptical galaxies; see refs in (Allahverdyan, Gurzadyan 2002).

There are preliminary indications from deep surveys on the existence of elliptical galaxies at redshifts $`z>4`$, i.e. of 10 per cent of their present age. If confirmed, this fact would moreover require more rapid mechanism of relaxation than the two-body one.

The chaotic effects are not only responsible for the relaxation and evolution of globular clusters and elliptical galaxies, but also they are indicators of the morphological type and other properties of galaxies.

How many decades are needed to realize the necessity of replacement of the ’plasma’ framework of stellar dynamics and abandoning of the two-body relaxation myth?

## 2. Relative instability of stellar systems

I will now briefly discuss the problem of relative instability of stellar systems, concentrating particularly on the role of central massive objects, in view of recent progress in their studies in the cores of galaxies and globular clusters. The results are obtained by means of the above mentioned ergodic theory formalism.

In accord to the criterion introduced in (Gurzadyan, Kocharyan 1988) among two systems the one with smaller negative Ricci curvature $`r_u`$ has to be considered as more instable. For N-body gravitating systems the Ricci curvature equals

$$r_u(s)=\frac{(3N2)}{2}\frac{W_{ik}u^iu^k}{W}+\frac{3}{4}(3N2)\frac{(W_iu^i)^2}{W^2}\frac{(3N4)}{4}\frac{|W|^2}{W^3},$$
(1)
where

$$W=EV,V=G\underset{i<j}{\overset{N}{}}m_im_j/r_{ij},$$

and

$$W_i=\frac{W}{r^i},W_{ik}=\frac{^2W}{r^ir^k},|W|^2=\underset{i}{}(\frac{W}{r_i})^2,$$

$`m_i`$ denote the masses, $`u`$ is the velocity of geodesics with affine parameter $`s`$ in the configuration space. The minimal values of the Ricci curvature have to be compared within given interval of the affine parameter. The contribution of direct impacts of stars, i.e. when two stars get the same coordinates, is neglected in Eq.(1), as they are rare events for real stellar systems.

The idea is based on the average description of the exponential deviation of geodesics in the configuration space with sign-indefinite curvature tensor. The latter condition appears to be fulfilled for N-body gravitating systems thus indicating the diversity of possible configurations with very different properties, from semi-regular planetary systems to mixing spherical systems. The $`r_u(s)`$ is related with the Ricci tensor $`Ric`$ by the following expression

$$r_u(s)=\frac{\mathrm{𝑅𝑖𝑐}(u,u)}{u^2}.$$

This criterion has a principal difference from that of Lyapounov exponents, since provides local in time characteristics of the system and hence does not require ’infinite’ iterated computations.

Numerical experiments using this criterion have been performed for various N-body configurations by Bekarian, El-Zant, Melkonian, Kocharyan and others. It is natural to see that, for example, disk configurations with rotational momentum are more regular than spherical ones. More rigorous consideration based on Arnold’s theorem (1966) on one-parametric groups of manifolds with right-invariant Riemannian metric enables one to conclude that the Galactic disk does not possess the property of mixing (and hence the corresponding relaxation time scale), as spherical stellar systems do.

The following classification for the systems of our interest by the increase of statistical properties is emerging from numerical experiments:

1. Spherical systems;

2. Systems with a massive central object (nucleus);

3. Systems with double nuclei.

The role of a massive center, with similar conclusions, has been studied using other methods by van Albada, Norman, Rauch, Tremaine and others. We now see that double (or binary) massive objects, like those apparently observed in galaxies Markarian 273, Arp 220, have to make the system even more chaotic, i.e. with further increase in the rate of evolution driving effects (Bekarian, Melkonian 2000).

The topics mentioned above on the role of non-linear effects in stellar dynamics gain more importance in view of ever increasing possibilities of numerical experiments (Makino 2003).

## References

Allahverdyan A.E., Gurzadyan V.G.: 2002, Nuovo Cimento, 117B, 947

Bekarian K.M., Melkonian A.A.: 2000, Astron.Lett. 26, 1

Gurzadyan V.G., Savvidy G.K.: 1984, Sov.Phys.Doklady, 29, 520 (GS)

Gurzadyan V.G., Kocharyan A.A.: 1988, Sov.Phys.Doklady, 33, 494

Makino J.: 2003, talk at this Joint Discussion

Pfenniger D.: 1986, A & A, 165, 74