Biopolymers phase separation monitored by a ...

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Biopolymers phase separation monitored by a plasmonic sensorw Suzanna Akil-Jradi,*a Safi Jradi,a Je´roˆme Plain,*a Jean-Louis Bijeon,a Christian Sanchez,b Renaud Bachelota and Pascal Royera Received 26th November 2010, Accepted 2nd December 2010 DOI: 10.1039/c0cc05212d We report here a real-time study of interactions induced phase separation between b-lactoglobulin (BLG) and Acacia gum (AG) by analyzing the localized surface plasmon resonance of silver nanoparticles. We showed that the binding of BLG to AG is accompanied by refractive index changes, in relation with optical properties and structural changes of the complexes formed. Over the past several years, biosensors based on the extraordinary optical properties of noble metal nanoparticles (MNPs) have been used to monitor interactions in a number of biomolecular systems such as antigen–antibody, DNA– protein, aptamer–protein, etc.1–6 The sensing feature of MNPs results from their collective charge density oscillations and is known as localized surface plasmon resonance (LSPR).7 This phenomenon is dependent on the dielectric environment of the MNP2,4,7–9 and any change at the metal–solution interface will be immediately detected.10–15 On this basis, the LSPR turns out to be a promising tool to study interactions between molecules in solution. Herein we report the interactions between biopolymers giving rise to phase separation, which is still lacking about the nature of interactions, kinetics and dynamics.16 We focus especially on the binding of b-lactoglobulin (BLG) to Acacia gum (AG), which constitute a protein–polysaccharide system with potential use in a variety of applications such as pharmaceutics, cosmetics, design of new food and non-food a

Laboratoire de Nanotechnologie et d’Instrumentation Optique, Institut Charles Delaunay, Universite´ de technologie de Troyes, CNRS FRE 2848, 12, rue Marie Curie BP-2060, F-10010 Troyes Cedex, France. E-mail: [email protected], [email protected] b UMR des Agro-Polyme`res et Technologies Emergentes, UM2, INRA, CIRAD, Montpellier SupAgro, 2 place Viala, F-34060 Montpellier Cedex 1, Montpellier, France w Experimental. Most BLG/AG mixed dispersions were prepared by adding AG to BLG dispersion at a protein (Pr) : polysaccharide (Pol) weight ratio of 2 : 1, the ratio corresponding to the maximum strength of interactions between BLG and AG.24 The blend was gently stirred for 30 min at room temperature. The pH of the blend was at 7.30 at the end of the stirring period. Mixed dispersions were filtered through a 0.22 mm syringe filter (Millipore, Bedford, USA). 0.11 wt% of glucono-d-lactone (GDL) was then added to the mixed dispersion and dissolved under stirring for 5 min. In the aqueous medium, GDL is hydrolyzed until equilibrium is reached between gluconic acid and GDL. Gluconic acid releases protons and gluconate in the medium. This proton release leads to a slow medium acidification. At a given pH, aliquots were taken from dispersions and deposited on the surface of nanopatterned silver nanoparticles in order to study the binding between BLG and AG.

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surface-active or texturing ingredients, etc.17 The thermodynamic compatibility between these macromolecules results in complex coacervation, an associative liquid–liquid phase separation mainly induced by electrostatic interactions between oppositely charged biopolymers.16–25 Unfortunately, the structures of BLG/AG complexes formed upon phase separation and the dynamics of the mechanism remain poorly known.25 We report on a careful study of the complexation/ coarcervation of the BLG/AG system using a LSPR biosensor. Particularly, we show the potentiality of LSPR-based biosensors to study complex biosystems with various structural transitions upon interaction. Moreover, we emphasize the possibility to quantify the refractive index changes. Our approach consists in depositing a slightly acidified mixed dispersion of BLG/AG on silver nanoparticles prepared by electronic beam lithography (diameter: 100 nm and height = 50 nm) and observed by SEM (Scheme 1) and then to follow their extinction spectra as a function of interaction between biopolymers adsorbed, as shown in Scheme 1. First, we performed a static study of the complexation using our sensor. We observe a red shift of the extinction spectra of Ag nanoparticles with decreasing pH, which indicates the occurrence of complexation as shown in Fig. 1d. We note that Ag nanoparticles had a 610 nm of maximum extinction wavelength (MEW) in air (Fig. 1a). In water, the MEW of Ag particles is measured to be B660 nm (refractive index of 1.33, Fig. 1b). In Fig. 1c, no change appears for the blanks, BLG and AG. From the beginning of acidification until pH 5.41, the BLG/AG mixture displayed a maximum extinction wavelength (lmax) of B660 nm (Fig. 1d), which corresponds to the deionized water response. From pH 5.41, lmax continues to increase and then its shift (Dl) gradually increases with decreasing pH until a maximum value (Dlmax) of 25 nm was

Scheme 1 SEM image of our plasmonic sensor. Schematic view of the experimental approach used in this work. The color variation of metallic nanoparticles highlights the change in the refractive index of their surrounding medium and indicates then the binding onset as the pH decreases.

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The Royal Society of Chemistry 2011

Fig. 1 Shift of LSPR spectra of silver nanoparticles illustrating the binding between BLG and AG. (a) Ag nanoparticles in air, lmax = 610 nm. (b) Ag nanoparticles in water, lmax = 660 nm. (c) Ag nanoparticles after depositing either BLG or AG blank dispersion at pH 5.41, lmax E 660 nm. (d) Ag nanoparticles after depositing BLG/AG mixed dispersion at pH 5.41 (lmax E 660 nm) and pH 3.75 (lmax = 685 nm). Each point is the average of three independent experiments.

reached at pH 3.75 as shown in Fig. 1d. This shift corresponds to the increase of the refractive index around Ag nanoparticles, in relation with optical changes involving the formation and structuring of complexes. We plotted in Fig. 2, the maximum extinction wavelength collected from LSPR spectra as a function of pH in order to define the structural transitions. Thus, we observed two zones of pH-delimited optical/structural transitions, which can be described as follows. From pH 5.41 until pH B5.25, the Dl remains constant at B662 nm. From pH 5.25, Dl gradually increases until pH B4.04. From this pH, the increase of Dl was stronger until pH 3.75 from which Dl becomes constant at 685 nm. Between pH 5.25 and pH 4.04, the corresponding curve exhibits three transition pHs. Before discussing these results, we summarize the current state in the literature about the pH-induced structural transitions in our system. The relevant studies reveal that the strength of interaction between BLG and AG is largely influenced by the net electrical charge on the individual biopolymer that is directly dependent on the pH of dispersion. In the abundant literature, two characteristic pH-delimited structural and morphological changes were identified, pHc and pHf.16–24 The critical pH, pHc, reflects a phase transition on the molecular scale, which corresponds to the formation of

intrapolymeric soluble complexes. On the other hand, pHf is considered as a transition on the microscopic level at which phase separation begins.16–19 In the case of BLG/AG mixed dispersions, more than two pH induced structural transitions were identified.21–28 Mekhloufi et al.25 determined six structural transitions during slow acidification of BLG/AG dispersions. These transitions were described as corresponding to (pH = 4.9) the formation of soluble complexes pHsc, (pH = 4.8) the conformational change of the protein induced by complexation pHpct, (pH = 4.7) the aggregation of complexes pHca, (pH = 4.4) the initiation of phase separation pHpsi, (pH = 4.2) the phase separation pHps and (pH = 4.0) the first appearance of coacervates, pHcoa. In the present study, we also determined six structural transitions, which correspond to pH of 5.25, 4.85, 4.51, 4.19, 4.04 and 3.75, respectively. There is some shift between our values and those of the authors mentioned (see Fig. 2) that could arise from the different analysis methods and the chemical composition of BLG and AG powders used. Among these values, pH 4.85, 4.51, 4.19 and 4.04 are in accordance with pHpct, pHpsi, pHps and pHcoa. We focus in this communication on pHc and pHf, which correspond to the formation of soluble complexes (pHsc E 4.9) and the first appearance of coacervates (pHcoa E 4.0) as reported by Mekhloufi et al.25 These results show that the interactions begin at pH 4.9 and complex coacervation takes place at pH 4. This last pH can be correlated to the inflection point at pH 4.04 observed in Fig. 2, which can be attributed to the onset of complex coacervation. On the other hand, LSPR detected a beginning of interaction and then of complexation/ aggregation at pH 5.25. This is the original result obtained by LSPR that concerns the initiation of interaction and which indicates that BLG/AG complexes were formed before pH 4.9 as shown previously. Indeed, the results highlight the higher efficiency of the LSPR-based sensor to detect binding occurring as compared to the analysis methods used by Mekhloufi et al. that do not allow us to observe the presence of these complexes. This property relies on the high sensitivity of the localized plasmon absorbance of metal nanostructures to changes in the dielectric properties of the surrounding medium.7,10 Up to date, the optical properties of complexes/coacervates based on interactions between biopolymers are unknown. Nevertheless, we have shown a clear refractive index change (Dn) in our case that gave rise to the red shift shown in Fig. 1d. In order to quantify this shift, a calibration curve of our sensor has been defined using different liquids with well-known refractive indexes. From these, the dependence of Dl with Dn for these compounds has been obtained and is shown in Fig. 3a. In order to determine the refractive index that corresponds to the measured shift in wavelength, we used the well known model first introduced by Jung and co-authors29 for surface plasmons of a thin metallic layer and also well used for LSPR,7 allowing us to correlate Dl with Dn. Dlmax = mDn [1

Fig. 2 Comparison of (lmax vs. pH) of BLG/AG interaction and blanks of BLG and AG. Each point is the average of three independent experiments.

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exp( 2d/ld)]

(1)

where Dlmax is the maximum wavelength shift as assessed above, m is the refractive index sensitivity,7 Dn is the refractive Chem. Commun., 2011, 47, 2444–2446

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techniques and by simulations. To make this possible, the complex coacervation in the BLG/AG system would be systematically studied through the modification of variables such as biopolymer concentration and temperature.

Notes and References Fig. 3 (a) (black circles) Evolution of the maxima extinction wavelength (Dl) as a function of the refractive index shift (Dn) for different liquids. (red line) Linear fit of the curve. (b) Variation of the refractive index (black circles) and size (Rh) (red circles) of BLG/AG particles with pH during biopolymers interaction.

index change, d is the effective adsorbate layer thickness and ld is the characteristic evanescent electromagnetic field decay length. As our result has been obtained in the semi-infinite layer limit (eqn (1) is simplified to Dlmax = mDn), the evolution was then fitted by a linear model that allowed us to determine the refractive index sensitivity of our sensor m = 190 2 RIU. Based on this model, we calculated the refractive index at each pH value during the binding and we plotted the results as shown in Fig. 3b. In order to make it obvious that spectral shifts correspond to a structural change, we tried to establish a relationship between the refractive index and the size of BLG/AG complexes. Therefore, we performed Dynamic Light Scattering (DLS) measurements to get information about the size evolution (hydrodynamic radius, Rh) with pH. Thus, we correlated Rh to the refractive index as shown in Fig. 3b. It is interesting to distinguish the two areas of evolution from this curve. Above pH 4, n varies rapidly as compared to Rh that remains relatively constant. Below pH 4, n vs. Rh exhibits a similar linear variation. Sanchez et al.28 attributed the phase separation in the BLG/AG system to a nucleation and growth mechanism. This indicates that above pH 4 the number of BLG/AG particles increases at a constant rate, in correlation with our results. It is worthy to note that Dn may result from a variation of size, number and shape of complexes, which DLS cannot show as compared to LSPR. This highlights the higher sensitivity of LSPR and shows its efficiency to get more information about binding and structural changes. From these, we determined a refractive index of B1.45 at the end of interaction, which corresponds to a Dn of B0.15. It is worthy to note that we determined a mean refractive index because the complexes size becomes much larger (micrometric) than that of silver nanoparticles especially from the beginning of phase separation. To conclude, we showed that the LSPR could be a powerful means to study phase separation in a wide number of macromolecular systems. In addition, it provides results in concordance with those determined by the classical analysis methods as shown in different works.16–24 The idea behind this work is to determine the structures of complexes formed by complementary experiments, other

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1 Do-K. Kim, K. Kerman, Ha M. Hiep, M. Saito, S. Yamamura, Y. Takamura, Y.-S. Kwon and E. Tamiya, Anal. Biochem., 2008, 379, 1. 2 N. Nath and A. Chilkoti, Anal. Chem., 2002, 74, 504. 3 G. Barbillon, J.-L. Bijeon, J.-S. Bouillard, J. Plain, M. Lamy de la chapelle, P.-M. Adam and P. Royer, J. Microsc., 2008, 229, 270. 4 N. J. Anker, W. P. HAll, O. Lyandres, N. C. Shan, J. Zhao and R. P. Van duyne, Nat. Mater., 2008, 7, 442. 5 S. S. Acímovic´, M. P. Kreuzer, M. U. Gonza´lez and R. Quidant, ACS Nano, 2009, 3, 1231. 6 G. Barbillon, J.-L. Bijeon, J. Plain and P. Royer, Thin Solid Films, 2009, 517, 2994. 7 G. Barbillon, J.-L. Bijeon, J. Plain, M. Lamy de la chapelle, P.-M. Adam and P. Royer, Gold Bull., 2007, 40, 240. 8 S. Maier, Plasmonics: Fundamentals and Applications, 2007, vol. XXV, 223, p. 140 illus., Hardcover. 9 G. Barbillon, A. C. Faure, N. El York, P. Moretti, S. Roux, O. Tillment, M. G. Ou, A. Descamps, P. Perriat, A. Alex, J.-L. Bijeon, C. A. Marquette and B. Jacquier, Nanotechnology, 2008, 19, 035705. 10 M.-K. Lee, T. G. Kim, W. Kim and Y.-M. Sung, J. Phys. Chem. C, 2008, 112, 10079. 11 J. M. McMahon, Y. Wang, L. J. Sherry, R. P. Van Duyne, L. D. Marks, S. K. Gray and G. C. Schatz, J. Phys. Chem. C, 2009, 113, 2731. 12 M. J. Jory, P. S. Cann and J. R. Sambles, Appl. Phys. Lett., 2003, 83, 3006. 13 D. A. Stuart, A. J. Haes, C. R. Yonzon, E. M. Hicks and Van R. P. Duyne, Nanobiotechnology, 2005, 152, 13. 14 Angelique M. C. Lokate, J. Bianca Beusink, Geert A. J. Besselink, Ger J. M. Pruijn and Richard B. M. Schasfoort, J. Am. Chem. Soc., 2007, 129, 14013. 15 G. Raschke, S. Kowarik, T. Franzl, C. So¨nnichsen, T. A. Klar, J. Feldmann, A. Nichtl and K. Ku¨rzinger, Nano Lett., 2003, 3, 935. 16 F. Weinbreck, R. de Vries, P. Schrooyen and C. G. de Kruif, Biomacromolecules, 2003, 4, 293. 17 M. Thongngam and D. J. McClements, Langmuir, 2004, 21, 79. 18 V. B. Tolstoguzov, Food Hydrocolloids, 2001, 17, 1. 19 S. L. Turgeon, M. Beaulieu, C. Schmitt and C. Sanchez, Curr. Opin. Colloid Interface Sci., 2003, 8, 401. 20 C. G. de Kruif and R. Tuinier, Food Hydrocolloids, 2001, 15, 555. 21 C. Sanchez, C. Schmitt, E. Kolodziejczyk, A. Lapp, C. Gaillard and D. Renard, Biophys. J., 2008, 94, 629. 22 F. Weinbreck, R. de Vries, P. Schrooyen and C. G. de Kruif, Biomacromolecules, 2003, 4, 293. 23 C. Sanchez, G. Mekhloufi, C. Schmitt, D. Renard, P. Robert, C.-M. Lehr, A. Lamprecht and J. Hardy, Langmuir, 2002, 18, 10323. 24 C. Schmitt, C. Sanchez, S. Desobry-Banon and J. Hardy, Crit. Rev. Food Sci. Nutr., 1998, 38, 689. 25 G. Mekhloufi, C. Sanchez, D. Renard, S. Guillemin and J. Hardy, Langmuir, 2005, 21, 386. 26 C. Schmitt, C. Sanchez, S. Despond, D. Renard, F. Thomas and J. Hardy, Food Hydrocolloids, 2000, 14, 403. 27 C. Schmitt, T. P. da Silva, C. Bovay, S. Rami-Shojaei, P. Frossard, E. Kolodziejczyk and M. E. Leser, Langmuir, 2005, 21, 7786. 28 C. Sanchez, G. Mekhloufi and D. Renard, J. Colloid Interface Sci., 2006, 299, 867. 29 L. S. Jung, C. T. Campbell, T. M. Chinowsky, M. N. Mar and S. S. Yee, Langmuir, 1998, 14, 5636.

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