Smooth and rough Proteus mirabilis lipopolysaccharides studied by total internal reflection ellipsometry

Thin Solid Films 548 (2013) 343–348

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Smooth and rough Proteus mirabilis lipopolysaccharides studied by total internal reflection ellipsometry J. Gleńska-Olender a,b,⁎, K. Dworecki c, S. Sęk d, M. Kwinkowski a, W. Kaca a a

Institute of Biology, Jan Kochanowski University, 25-406 Kielce, Poland Świętokrzyski Biobank, Regional Science and Technology Center, 26-060 Chęciny, Poland Institute of Physics, Jan Kochanowski University, 25-406 Kielce, Poland d Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland b c

a r t i c l e

i n f o

Article history: Received 25 July 2012 Received in revised form 4 October 2013 Accepted 7 October 2013 Available online 16 October 2013 Keywords: Lipopolysaccharides Total internal reflection ellipsometry Biomolecular film thickness Atomic force microscopy

a b s t r a c t Total internal reflection ellipsometry (TIRE), a label-free optical detection technique for studying interactions between biomolecules, was used to examine the adsorption of various forms of lipopolysaccharides (LPSs) isolated from Proteus mirabilis S1959, R110, and R45 strains on a gold surface. The thickness of the adsorbed layers was determined by TIRE, with the average values for S1959, R110, and R45 LPS layers being 78 ± 5, 39 ± 3, and 12 ± 2 nm, respectively. The thickness of LPS layers corresponds to the presence and length of Ospecific parts in P. mirabilis LPS molecules. Atomic force microscopy was used as a complementary technique for visualizing lipopolysaccharides on the surface. Force measurements seem to confirm the data obtained from TIRE experiments. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Lipopolysaccharides (LPSs, endotoxins) are major components of the cell walls of Gram-negative bacteria. LPSs have a strong effect on the mammalian immune system and play a significant role in the pathophysiology of such diseases as endotoxemia [1]. They are composed of a hydrophilic polysaccharide moiety that is covalently linked to a hydrophobic lipid moiety. LPSs from most bacterial species are composed of three distinct regions: i) the O-antigen region, which is highly variable and responsible for the serological specificity of LPS variants; ii) the core oligosaccharide, which is divided into an outer core and a highly conserved inner core containing unusual saccharides, such as 2-keto-3-deoxyoctulonic acid or heptoses; and iii) lipid A—an endotoxically active part of the molecule [2]. The molar mass of an endotoxin monomer ranges from 10 to 20 kDa. The identification of LPSs is important for bacteriological diagnostics, especially that they are involved in adhesion of bacterial cells [3] as well as in biofilm formation on different surfaces [4]. LPSs are usually analyzed by immunological (enzyme linked immunosorbent assay or Western Blot) or chemical (mass spectrometry, nuclear magnetic resonance) methods. All of them are sophisticated, time consuming, and require expensive reagents and equipment. On the other hand, techniques based on light reflection are relatively simple analytical

⁎ Corresponding author at: Department of Microbiology, Jan Kochanowski University, ul. Świętokrzyska 15, 25-406 Kielce, Poland. Tel./fax: +48 41 349 63 07. E-mail address: [email protected] (J. Gleńska-Olender). 0040-6090/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.tsf.2013.10.023

methods and make it possible to construct biosensors recognizing different LPSs or antibodies specific to them in biomedical applications. There exist several methods for analysis of biological molecules based on measurements of internal light reflection. The most popular is surface plasmon resonance (SPR) analysis, which is applied for the identification and measurement of interactions between molecules such as ligands and receptors or antigens and antibodies [5–7]. The possibility to use SPR for analysis of molecular matrices enabled intensive investigations of methods for the immobilization of biopolymers on gold surfaces. The objective was to develop a method for examination of LPS deposition on thin gold layers by means of total internal reflection ellipsometry (TIRE) which could be used to determine the size and structure of LPS supramolecular forms (micelles) as well as to control surface coverage in the preparation of sensors containing bacterial endotoxins. LPS molecules, like other amphipathic molecules, form various supra-molecular aggregates in aqueous solutions. These aggregates result from non-polar interactions between lipid chains and water molecules. Many studies have shown that in aqueous solutions LPSs can form a variety of shapes, such as lamellae, cubes, and hexagons [1]. Biomolecular binding is involved in biological specificity and recognition. If one of two components is adsorbed and bound to a surface, it can be studied by various label-free methods, such as SPR [5,8–10] and spectroscopic ellipsometry (SE) [13–16]. While SPR methods measure only the reflectivity of p-polarized light, SE methods use two experimental parameters (Psi, Delta) and can be useful for studying the optical properties and structure of samples. TIRE combines the analytical ability of SE with the high surface sensitivity of SPR [6,7,17]. The surface of a

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glass plate is coated with a layer of metal segment elevation (in this case gold). A beam of light falling at an angle greater than critical is internally reflected. When the incidence angle is above a specific resonance angle, it results in the dispersion and absorption of light energy by plasmons in the layer of gold. We combined both techniques in order to enhance ellipsometric sensitivity. For this purpose, we used evanescent field ellipsometry [5,7,16], a method where light is totally internally reflected. Full information about changes in light properties is given by two ellipsometric quantities: Psi and Delta, which depend on the angle of incidence and optical wavelength. In the reflection mode, ellipsometry parameters Ψ, Δ are defined by the ratio ρ of the complex-valued reflection coefficients Rp and Rs for polarization parallel (p) and perpendicular (s) to the plane of incidence, respectively [13,17], ρ ¼ Rp =Rs ¼ tanðΨÞ expðiΔÞ; Δ ¼ δp −δs where δp and δs are the phases of Rp and Rs. The amplitude ratio ρ is thus given by tan Ψ and Δ. Δ is the phase difference between the reflection coefficients for the p and s polarizations induced by reflection. To determine the thickness of a LPS film adsorbed on a gold surface, the film was modeled as Cauchy layers of thickness d with an index of refraction n(λ) in the form: nðλÞ ¼ no þ n1 λ

−2

þ n2 λ

−4

where n0, n1, and n2 are Cauchy coefficient parameters and λ is the light wavelength. Fitting was performed by solving Fresnel equations multiple times for different values of n and d, and subsequently minimizing the error function of the experimental and theoretical (calculated) values, and using one of the least-square fitting techniques. As the process of creating micellar systems on a gold surface is not known, we examined this issue [18,19]. In the presented experiment, LPSs isolated from the smooth Proteus mirabilis S1959 strain and its two mutants, R110 and R45, were used. The R110 mutant lacks the O-specific polysaccharide, but has a complete core oligosaccharide, while R45 has its core reduced to two 2-keto-3-deoxyoctonic acid (Kdo) residues and one 4-aminoarabinose (Ara4N) residue [20,21]. Previously, we analyzed S1959, R110 and R45 LPSs by laser interferometry methods [22], showing that the binding of the antibiotic colistin and saponins varied depending on the polysaccharide content of the LPS used [22]. In this work, we apply a different physical technique—total internal reflection ellipsometry for monitoring the adsorption process of P. mirabilis LPSs that differ in their polysaccharide content. In this work, we used atomic force microscopy (AFM) as a complementary method to visualize LPS buildup on the surface, and to reveal differences between the three LPS structures. 2. Experiments 2.1. Materials The following LPSs from three P. mirabilis strains were used: S1959 (wild type [23]), R110 (Ra mutant of S1959 without the O-antigen part [24]), and R45 (Re deep mutant having only a fragment of the inner core [25]) at a concentration of 0.5 mg/mL in phosphate buffered saline (PBS) buffer. 2.2. Adsorption between gold surface and LPS types At the beginning, PBS buffer was injected into a flow cell for 5–10min. Then, an R45/R110/S1959 LPS (0.5 mg/mL) in PBS buffer was injected at 30 μL/min through a syringe pump. Measurements were made every

30 min for 4 h. Subsequently, the thickness of the adsorbed biomolecular film was determined. We also optimized the coverage time of LPS on the substrate of gold. 2.3. TIRE experiments The TIRE experimental set-up consisted of a SE 800 SENTECH spectroscopic ellipsometer operating in the spectral range of 280– 850 nm and a home-made flow cell connected to a syringe pump to control the flow rate. Experiments were carried out on a glass slide support covered by evaporation with 2 nm of chromium, followed by 28 nm of gold, which was adjacent to the optical prism. The presence of a chromium layer improved the adhesion of the gold film to the glass substrate [5]. We selected a gold surface due to the fact it is a model for ellipsometric studies of bioadsorption as its surface is chemically very stable in most liquids. The prism and glass slide were made of BK7 glass. An index matching oil of n = 1,515 was used to minimize the reflection of light between the prism and the glass slide. The response of TIRE to changes in biomolecular film thickness and refractive index was modeled using Spectra Ray ellipsometer software (SENTECH Instruments, GmbH) [26]. In TIRE, light enters the system from the glass side, and the layers are named as follows: (0) substrate (water), (1) dielectric film (or Cauchy layer), (2) thin chromium-gold film, and (3) ambient (glass), as shown in Fig. 1 [11,12]. The dielectric layer is transparent (k = 0), and the refractive index is described by the Cauchy dispersion formula [13]. The angle of incidence should be close to the angle of total internal reflection, which dictates the choice of prism. A 68° prism was used for TIRE measurements in aqueous solutions. The presence of a thin metal film is vital for TIRE because of the phenomenon of plasmon resonance in the interaction of an evanescent field with the surface plasmons of the metal film. 2.4. Atomic force microscopy (AFM) experiments Atomic force microscopy experiments were carried out using a 5500 AFM (Agilent Technologies, Santa Clara, CA, USA). Images were recorded using the Magnetic AC mode with type VI MAC levers (Agilent Technologies, Santa Clara, CA, USA). The resonance frequency of the cantilevers was in the range of 50–60 kHz in air, and was suppressed to 18–24 kHz in solution. Images were acquired at 20 °C in PBS aqueous solution on the same samples which were previously used for ellipsometric experiments. The root-mean-square (RMS) surface roughness for LPS films was determined for 1.2 × 1.2 μm2 images of five different

Layer 1. 2. 3.

4.

Materials Comments BK7 (ambient) glass Fixed during fitting n=1,515; k=0 at 633 nm Shott index (data sheet) Cr/Au 30 nm From Palik [11] LPS layers: Cauchy model with LPS S1959 n0=1,394 n1=0,01 LPS R110 n2=0 LPS R45 n and k are fixed thickness variable Buffer substrate n=1,33; k=0 at 633 nm (water) Fixed during fitting From Palik [12 ] Fig. 1. Four-layer model for TIRE data fitting.

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spots for each sample. RMS surface roughness (Sq) was calculated as a standard deviation of height distribution according to the equation:

Sq ¼

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 XN−1 XM−1 2 z x¼0 y¼0 x;y NM

where N is the number of points in a scan line, M is the number of scan lines, and zx,y is the difference in height at a given point (x,y). Force– distance curves were determined using PNP-DB silicon nitride cantilevers (NanoWorld AG) with a nominal spring constant of 0.06 N/m. 3. Results and discussion TIRE spectra were obtained at 69°, 71°, 73°, and 75° following the adsorption of S1959, R110, and R45 LPS, respectively. Figs. 2 and 3 show the Psi and Delta results for the different LPSs studied. Analysis of these results indicates that the extreme values of resonance shift towards shorter wavelengths. Resonance occurs at different wavelengths, depending on the angle of incidence. If the angle increases, the resonance point moves towards shorter wavelengths. Changes were observed for

Fig. 3. Spectral response of the ellipsometry parameter Δ(λ) for P. mirabilis LPS structures measured at angles of incidence of 69°—square, 71°—circle, 73°—up triangles, and 75°— diamonds; the symbols represent experimental data while continuous represent the fit of the Cauchy model; (A) S1959 LPS, (B) R110 LPS, (C) R45 LPS.

Fig. 2. Ellipsometric angle Ψ as a function of wavelength for P. mirabilis LPS structures measured at angles of incidence of 69°—square, 71°—circle, 73°—up triangles, and 75°— diamonds; (A) S1959 LPS, (B) R110 LPS, (C) R45 LPS.

modified LPSs. Values of the Psi and Delta surface resonance of plasmons depend on the molecular structure of the LPSs. Shifts indicate different values of collapse and thickness. The value of shifts in terms of resonance is proportional to thickness and the refractive index. Fitting the theoretical results corresponding to the model adopted in the Cauchy system stratified into four experimental results made it possible to determine the thickness of the biomolecular film for different LPS structures. The results of this fitting are included in Fig. 3. The thickness of LPS films obtained by the Bruggeman effective medium approximation model are much smaller than the film thickness assessed by the Cauchy model. In the stream of flowing solution, LPS multilayers formed micelles. After some time, the biomolecular film was found to stabilize the micelle monolayer adsorbed on the Au surface. Changes in layer thickness were observed over time. Fig. 4 shows that after 1 h S1959, LPS R110, and R45 LPSs formed 78 ± 5, 39 ± 3, and 12 ± 2 nm thick biomolecular films, respectively.

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Fig. 4. Time evolution of biomolecular film thickness observed for LPS with modified structure: S1959 LPS—up triangles, R110 LPS—circles, and R45 LPS—squares. Symbols represent experimental data while continuous represent a linear fit.

The LPS film evolution trend showed different values depending on the inclination of the tested substance: S1959 B =2.05± 0.34, R110 B = 1.64 ± 0.45, and R45 B = 0.73 ± 0.33. Over time, LPS biomolecular film reduced in thickness, which may indicate that LPSs detached from the gold plate, suggesting that the energy of the flowing liquid was greater than the energy of LPS molecules binding to each other and to the gold surface. This behavior of different bimolecular films can be explained by the action of electrostatic and Van der Walls forces arising between supramolecular structures and the Au surface and resulting in different shapes of levels. It is most likely that LPSs form micelles membrane like gold surface. The reduction in the thickness of bimolecular LPS films on a gold surface during the experiment may have been caused by micellar fusion and lateral diffusion [18]. It was previously found that LPS micelles were bound to surfaces via electrostatic rather than hydrophobic interactions [27]. Some experiments have emphasized the role of surface energy in the adhesion of LPSs on different surfaces [28].

(A)

12

In order to evaluate the structures of LPS films, AFM examinations were performed on samples previously tested by ellipsometry. Fig. 5 shows images of freshly prepared gold substrates of either bare or modified with LPS layers recorded in PBS buffer. The immobilization of LPS molecules results in the formation of amorphous films. However, the topography of R45 and R110 LPS films is noticeably different from that observed for S1959 LPS films. R45 LPS molecules have a tendency to form large patches of aggregated material coexisting with smaller spherical objects and relatively smooth domains (see Fig. 5B). The features of the underlying substrate can also be distinguished as grooves forming triangular shapes. The latter is most probably related to the existence of Au (111) terraces on the substrate (see Fig. 5A). This observation would suggest that the R45 LPS film is relatively thin as compared to the other two adlayers. The depth of the said grooves ranges from 8 to 12 nm, which roughly corresponds to the thickness of the R45 LPS film as measured by ellipsometry, which indicates that the continuity of the adlayer is disturbed at the edges of the gold terraces. RMS surface roughness measured for R45 LPS is 3.8 nm on average. Probably the defects significantly contribute to this value. The value of RMS surface roughness determined for bare gold substrate was 0.6 nm indicating that indeed major contribution to the roughness comes from the LPS film. R110 LPS shows an even stronger tendency to aggregate (see Fig. 5C). In this case, patches of up to 100 nm in diameter and 20 nm in height can be seen. This is also reflected by slightly higher roughness, which was found to be 4.4 nm. No substrate-related features are present in the R110 LPS film, so it can be inferred that it is thicker than that formed by R45 LPS. In this case the gold surface is covered with irregular features with sizes ranging from 20 nm up to 100 nm. A distinctly different topography is characteristic of the S1959 LPS film (Fig. 5D). In this case, spherical features can be seen and the diameter of individual spots varies between 40 and 50 nm. The RMS surface roughness of the film is 3.8 nm, which is the same as that observed for R45 LPS. Since the thickness of thin films can also be measured by AFM-based methods, additional experiments were performed to verify ellipsometric data. For this purpose, nanoindentation measurements were carried out [29]. In this method, the tip approaches the surface modified with the adlayer and the force is measured as a function of the distance separating the tip from the substrate. As can be seen in

(B)

10

25 20

8 15 6 10 4 5

2

1,2×1,2 µm2

(C)

0 nm

25

1,2×1,2 µm2

(D)

0 nm 20

15

20 15

10

10 5 5

1,2×1,2 µm2

0 nm

1,2×1,2 µm2

0 nm

Fig. 5. AFM images obtained for bare gold (A) and the substrates modified with R45 LPS (B); R110 LPS (C) and S1959LPS (D). All images were obtained in PBS solution using MAC mode.

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Fig. 6 (A–C), at large separations the interactions between the tip and the sample are absent, but when the tip is close to the surface the force increases, reflecting electrostatic repulsion. Subsequently, the tip comes into contact with the sample and the film becomes elastically deformed. This causes a further increase in the measured force until a characteristic discontinuity is observed followed by a sharp increase in the repulsive force. The latter results from the direct interaction of the tip with the underlying substrate while the observed discontinuity of the force–distance curve corresponds to the penetration of the tip through the film [30]. By determining the distance at which the said discontinuity occurs, it is possible to estimate the thickness of the film. However, it should be noted that the thickness measured in this way is usually underestimated. This is related to the fact that indentation occurs when the film is already elastically deformed [31]. Fig. 6 shows sample force–distance curves obtained for R45, R110, and S1959 LPS samples. It is clear that mutant strains exhibit slightly different behavior from that of native LPS. At distances of below 100 nm, there are noticeable electrostatic repulsions between the tip and the sample. This is followed by a gradual increase in force and an indentation event. Analysis of indentation depth allowed us to determine the thickness of R45 and R110 LPS films, with the mean values being 12 nm and 33 nm, respectively (see Fig. 6D). Although elastic deformation was not taken into account, these values are in quite good agreement with the ellipsometric data. A distinctly different behavior was observed for the S1959 LPS film. In this case, most of the force– distance curves revealed multiple jump-in events reflecting a more complex structure of the film. This most probably results from the presence of the O-specific part in S1959 LPS, which consists of long flexible polymeric chains. The latter may form a polymer brush, which also contributes to the measured force at relatively large distances.

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Here, the thickness of the LPS film was estimated as the distance from the first jump-in event to the sharp increase in the force when the tip comes into contact with the underlying substrate. The resulting mean thickness was 70 nm (see Fig. 6D). Thus, force measurements seem to confirm the data obtained from the TIRE experiments as the thickness determined for LPS films follows the same trend. The thickness of biomolecular films on a gold surface determined using TIRE corresponds well to the results of AFM experiments. In both methods, the trends of film thickness change for S1959, R110, and R45 LPS were the same. This is in accordance with the properties of these molecules described previously using chemical methods. R45 LPS is the smallest molecule and has only some hydrophilic and charged saccharide residues. R110 LPS is larger and contains several additional charged saccharide residues. S1959 looks like R110 connected to a long O-antigen “tail” with many charged saccharide and amino acid residues. Additionally, samples of P. mirabilis S1959 LPSs consist of both LPS types: long with an O-antigen “tail” and short ones of the R110 type. This results in a different form of micelles in the S1959 sample, as they are composed of both short and long LPS types. For this reason, AFM analysis of the films produced by all three LPS types (Fig. 6D) indicates that size dispersion is the smallest for R45 and the largest for S1959 micelles. TIRE results (Fig. 4) provide only average film thickness values, but are in good correlation with AFM results. The adsorption of different LPS micelles on the surface changed during the TIRE experiments (Fig. 4). The interaction of micelles with a strongly charged gold surface may lead to modification of their structure. A similar effect was observed for interactions of LPSs with metal-oxide surfaces [28] or mica surfaces coated with positively charged amine residues [27].

Fig. 6. Examples of force–distance curves recorded during the tip approach for R45 LPS (A); R110 LPS (B) and S1959 LPS (C). Panel D shows histograms illustrating the spread of the measured thickness for each sample. Red curves are given as the references and correspond to the forces measured on bare gold surfaces.

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4. Conclusions We studied the optical properties of three P. mirabilis lipopolysaccharide structures and examined their adsorption process using TIRE and, additionally, AFM. Measurements of the thickness of biomolecular films formed on gold surface indicates that LPSs are adsorbed as supramolecular aggregates (micelles) creating a “single-micellar” film, which was confirmed by AFM results. Time was found to be an important factor in the evolution of biomolecular film thickness on gold surface. The Psi and Delta parameters changed significantly when a monolayer was formed. The film thickness of each LPS was in agreement with the estimated size of micelles formed by them [18]. This may be applied as a tool for controlling the size of LPS molecules isolated from several sources as well as for selecting the properties of solvents used for the control of supramolecular LPS structures. It was also shown that total internal reflection ellipsometry could be very useful for real time monitoring of the adsorption process of LPSs or whole Gram-negative bacterial cells. Such investigations could be applied in analysis of LPS interactions or in determination of biofilm formation, which are important issues in biomedical research.

Acknowledgments This work was supported by the EU program Human Capital (POKL/ 2009/8.2.1) and grant no. NCN-NN 304 275540. Some of the experiments were run on apparatus purchased with EU grant 2.2 Innovation Industry. The study was partially carried out at the Biological and Chemical Research Centre, University of Warsaw, established within the project co-financed by the European Union from the European Regional Development Fund under the Operational Programme Innovative Economy, 2007–2013.

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