Enhancement of Raman light scattering in dye-labeled cell membrane on metal-containing conducting polymer film

An enhanced Raman spectroscopy method based on a plasmon resonance in ultrathin metal-containing LB-film deposited on nanoporous anodic alumina supports has been proposed. This material has been utilized to enhance Raman scattering of light in fluorescent-labeled subcellular membrane structures. It has been shown that the plasmon resonance between vibrational modes of the organometallic complexes monolayers and dye-labeled subcellular structures happens. It makes possible to detect interactions between living cell monolayers and an extracellular matrix.


Introduction
At present, it has been found out that an interaction of biological cells with any extracellular-matrix occurs through subcellular structures formed by integrins and called focal adhesions. 1,2 The binding of focal adhesions with surface of substrate can be affected by globular glycoproteins, for example, a plasma lactoferrin. 3 Lactoferrins bind preferably to double-stranded (ds) than to single-stranded DNAs. Because of this, one can assume that the ds-DNA is a regulatory molecule in the process of cell attachment/detachment during active adhesion of focal contacts. This is possible if the cytoplasmic membrane (cm) DNA exists. 4 The presence of nucleic acids in the plasma membrane is provided by both an intercellular transport by gap junction channels 5,6 and microtubules 7 and an extracellular transport of apoptotic DNA fragments. 8 But, it has been shown that the plasma membrane contains small molecules of a specific cmDNA, sequence of which is identical to telomere DNA-sequence. 9 To date, there are no experimental facts concerning the role of the specific cmDNA. A presence of this cmDNA has to affect the process of active adhesion. However, the detection of such interaction is hampered due to the nanosize of the focal adhesions with height about 10-15 nm 1 and due to the dependence of DNA Raman spectra on the DNA protonation. 10 A sensitive method of nanosizestructure visualization is the surface-enhanced Raman spectroscopy (SERS). Owing to the closeness of metallic nanoparticles, oppositely charged parts of particle surfaces produce electric dipoles, quadrupoles and so on, which oscillate in resonance, and a phenomenon of giant Raman scattering is observed. 11 Due to the high intensity of this giant resonance, the intensities of Raman spectrum bands depend on random distribution of nanoparticles and, because of this, change from one experiment to another in a random manner. The variability of SERS spectrum intensity is a serious disadvantage of SERS-method based on metallic nanoparticles, because it demands to create an ideal layer of periodically arranged nanoparticles.
In this paper, we use high-ordered metal-containing LB-films of organometallic complexes from a conducting oligomer of thiophene-pyrrole series and conducting multi-walled carbon nanotubes (MWCNT) to enhance the Raman scattering. We will demonstrate that these graphene-like materials can screen the electric field of electrically charged double layer on the interface.
The goal of the paper is to establish both a screening effect of conducting metal-containing LB-film deposited on nanoporous anodic alumina (AOA) and a phenomenon of plasmon resonance in the oscillating electric field of screened electrically charged double layer on the interface water/condensed state, and to develop a SERS-method based on these effects and to apply it in Raman spectroscopy of adhered living cell monolayers.
MWCNTs with diameters ranging from 2.0 to 5 nm and length of ∼5-10 μm were obtained by the method of chemical vapor deposition (CVD-method). 13 MWCNTs were covalently modified by carboxyl groups and non-covalently functionalized by stearic acid molecules.
RNA and protein fractions in high-purity ds-DNA (1.03 mg/ml in 10 −5 M Na 2 CO 3 buffer medium (TE-buffer)) were less than 0.1% (optical density ratio C6 rat glioma cells obtained from culture collection of Institute of Epidemiology and Microbiology (Minsk, Belarus) were grown on Fe-containing LB-DTPfilms with or without aligned MWCNT arrays in a Dulbecco's modified Eagle medium (DMEM) (Sigma, USA) supplemented with 10% fetal bovine serum and 1 × 10 −4 g/ml gentamycin at 37 • C in a humidified 5% CO 2 atmosphere. A cellular monolayer has been stained with 1 μM propidium iodide (PI, Sigma, USA) for five minutes before spectral studies were carried out.
Membranes of porous AOA have been used as a support for LB-films. Fabrication of AOA films membranes (thickness of 20.0 μm) was carried out by means of two-stage anodic treatment in 10% sulfuric acid solution at voltage of 10 V and temperature of 2 • C. The diameter of AOA pore was equal to 10 nm.
All used materials belong to class of analytical pure reagents.

Methods
A Langmuir-Blodgett (LB) monolayer formation was carried out on an automated hand-made Langmuir trough with controlled deposition on a substrate, and with computer user interface working under Microsoft Windows operational system. Control of the surface tension was performed by a highly sensitive resonant inductive sensor. The Y-type transposition of monolayers on supports was performed by their vertical dipping. The complexes Fe(II)DTP 3 of high-spin Fe(II) with thiophene-pyrrole ligands were synthesized by LB-technique at compression of H-dithionilepyrrole molecules on the surface of subphase with salts of three valence Fe. 12 Horizontally and vertically arranged LB-MWCNT-bundles can be fabricated from the multi-walled CNTs. 14 We use the Langmuir-Blodgett technique to fabricate new biocompatible layered nanoheterostructures consisting of two MWCNT LB-monolayers and/or 5-, 7-monolayer LB-film of the organometallic complexes. Spectral studies in visible range were carried out using a confocal micro-Raman spectrometer Nanofinder HE ("LOTIS-TII", Tokyo, Japan-Belarus) by laser excitation at wavelength 532 nm and vertical spatial resolution 150 nm.

Screening Depending on Charge State of Mixed-Valence Complexes
Coordination complexes of Fe are shown in Fig. 1(a) and 1(b). Metal atoms in the LB-monolayer of the iron complexes FeDTP 3 in charge state "3+" are placed in the monolayer plane. Since high-spin iron complexes are dome-shaped, the LBmonolayer of the iron complexes in charge state "2+" represents itself as a graphenelike material decorated by a monolayer of adhesive iron atoms (adatoms), as shown in Fig. 1  Let us consider the screening in true two-dimensional (2D) semimetals. As known, [15][16][17] free charge carriers at the Fermi level in these materials are absent. In single-pole approximation for small momenta p the condition of emerging of plasmon resonance can be found from the following dependency of dielectric permeability (p, ω) on a frequency ω: where r D = ( a (e 2 a n a / 0 k B T )) −1/2 is the Debye screening radius, ω plasmon = ( a (e 2 a n a / 0 m a )) 1/2 is a plasmon frequency, a is a type of the charge carriers, n a , m a and e a are a density, a mass and a charge of a-th type, respectively; 0 is the dielectric constant, T is the temperature, k B is the Boltzmann constant. Pseudofermion charge carriers in bipolar graphene-like two-dimensional material are massless: m c = 0 and their charge density is vanishing: n c → 0 in Dirac point K(K ) of a Brillouin zone. Due to the fact that r D → ∞ and plasmon frequency gets a finite value ω 2 plasmon = 2(e 2 / 0 ), the right side of the condition (1) is always positive and, respectively, plasmon resonance in 2D system is absent. Now, one can examine decorated 2D semimetals. In Coulomb field of adatoms, a band structure of the monolayer acquires the energy gap E g and on Fermi level there appears non-zero charge-carrier density n c = δn F < 1. Therefore, the dielectric permeability LB (p, ω) of the decorated system is determined by the following expression The expression (2) can acquire zero and negative values, corresponding to plasmon resonance. Figures 2(a) and 2(b) demonstrate the absence of screening and, respectively, plasmon oscillations for non-decorated 2D Dirac metals. An enhancement of Raman light scattering in the organometallic LB-film, on whose surface the MWCNT-containing monolayers have been deposited, is shown in Fig. 3(a). This is possible, if MWCNTs are decorated by the monolayer of high-spin Fe-adatoms, that makes MWCNTs to be able to screen an electromagnetic field.  These experimental results prove that one of Fe charge state in FeDTP 3 is a state "+2". A Raman spectrum of FeDTP 3 in Table 1 has oscillations 1342, 1348, 1410 and 1432 cm −1 , which are absent in the original H-dithionilepyrrole spectrum. These oscillations arise from Fe atom periodically leaving the ligand monolayer plane and coming back when changing the valence state of Fe atom from coordinate state II to III and vice versa. The assumption, that these oscillations are due to the mixedvalence state of the metal center is confirmed by the presence of more high-frequency oscillation 1432 cm −1 of thiophene ring in the Raman spectrum. Because of that, we call this oscillation as the oscillation of metal-center valence. Protonation of the LBfilms, placed in water, leads to shift of Raman bands into high-frequency region, for example, for 2-subst pyrrole Quadrant stretch from 1462 to 1472 cm −1 , according Table 1. A laser plasmon mode for the LB-film is represented by plasmon oscillations in the frequency regions 1650-2800 cm −1 and 1575-2300 cm −1 for samples placed in air and water, respectively, in Fig. 3. The frequencies of charge-density oscillations for free charge carriers in the LB-film are shown in Table 2. Thus, an enhancement of Raman light scattering by the Fe-containing organometallic LB-film is stipulated by both the plasmon and metal-center-valence oscillations. Further, we will demonstrate results in Raman light scattering enhancement in sites of biological-cell adhesion to support.

Metal-containing LB-film enhancement of Raman light scattering in hydrated complexes
An electrically charged double layer arises on an interphase boundary which includes the ultrathin LB-film. This layer is similar to a plane capacitor. Since a distance between plates of such capacitor is very small, its electric field strength is large. A high value of low-frequency dielectric permeability of water is due to the water ionization in this double layer with following formation of hydrated complexes of ions OH − , H + and impurity ions. The existence of such complexes impedes the ion recombination. A resonance of the free-charge density oscillations in the LBfilm at the eigenfrequencies of the hydration water oscillations enhances a process of hydrated complexes decay and, respectively, released ions recombine to neutral molecules H 2 O that results in decreasing of dielectric permeability of the medium. Fluorescence of hydrated PI molecules is quenched by hydration water molecules. The resonance of oscillations of free-charge-carrier density in the metalcontaining LB-films at eigenfrequencies of hydrated shells leads to decay of complexes of PI with water and, respectively, to suppression of PI fluorescence quenching, as shown by comparison of curves in Fig. 3(b) and comparison of luminescence intensity of images of PI stained cells, which were grown on a glass and the conducting organometallic LB-film deposited on AOA in Fig. 4. Thus, LB-film screens the electric field of electrically charged double layer on the interphase boundary.

Metal-containing LB-film enhancement of Raman light scattering in living cell monolayers
Let us consider the screening effects of electrically charged double layer on the interface living-cell monolayer physiological medium. The ds-DNA inside the stearic acid micelles is similar to cmDNA. Therefore, to find a Raman spectrum of cmDNA for the cell under investigation, we carried out a comparison of characteristic frequencies of the Raman spectra for the living cell monolayers with a Raman spectrum of the ds-DNA inside stearic acid micelles in Fig. 5. According to Table 3, the spectrum of model micellar ds-DNA shifts relative to the Raman spectrum for DNA being at condition of 92% humidity, because, as it is known, 10 the protonation of DNA causes a shift of DNA characteristic frequencies into high-frequency region. However, this shift is negligible compared to the shift of the corresponding bands of a Raman spectrum for water solutions of the ds-DNA 24 due to deprotonation of screened double layer at the condition of plasmon   resonance. According to Table 3, the characteristic frequencies of the Raman spectra of the cellular monolayer and the ds-DNA inside the micelle are close to each other. Moreover, the addition of PI into medium enhances the luminescence of the cellular monolayer. As known, 25 besides PI molecules intercalation into double helix of DNA, PI molecules can complexify with DNA being situated in DNA minor groove. Fluorescence of hydrated complexes DNA/PI in minor groove is quenched by hydrate water molecules. Plasmon resonance at eigenfrequencies of hydrate shells leads to these complexes decay and, respectively, to suppression of fluorescence quenching of DNA/PI in water. Thus, the observed enhancement of fluorescence for the cell monolayer stained with PI is the luminescence of such DNA/PI complexes in Fig. 4. Moreover, by virtue of the effective adhesion of DNA on a hydrophobic surface of graphene and graphene-like materials due to stacking interaction of their π-electrons, 26 with DNA frequencies is also explained by the hydrophobicity of LB-film surface repulsing hydrophilic proteins, such as bovine serum albumin, that results in the small intensity of characteristic bands Amide-I (carbonyl group C=O, 1650-1690 cm −1 ) and Amide-II (group N=C, 1450 cm −1 ) 30 in the Raman spectrum. The fact, that we observe the enhanced Raman scattering of light in the cell cmDNA, is also proved by the absence of band Amide-I with maximum 1665 cm −1 for DNA, 31 and the band (C=O, 1615-1635 cm −1 ) of linker histone H1 30 in their Raman spectrum.

Conclusion
So, a SERS in the living cell monolayer grown on ultrathin organometallic LBfilms is observed, in preference, on the characteristic frequencies of DNA. This is due to the facts that LB-film surface is hydrophobic and repulses the hydrophilic proteins, and cmDNA adheres effectively on the hydrophobic surfaces of graphene and graphene-like materials owing to the stacking interaction of their π-electrons.