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Membrane Surface-Enhanced Raman Spectroscopy (MSERS) for Sensitive Detection of Molecular Behaviour of Lipid Assemblies Keishi Suga, Tomohiro Yoshida, Haruyuki Ishii, Yukihiro Okamoto, Daisuke Nagao, Mikio Konno, and Hiroshi Umakoshi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5048532 • Publication Date (Web): 03 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Analytical Chemistry

Membrane Surface-Enhanced Raman Spectroscopy (MSERS) for Sensitive Detection of Molecular Behaviour of Lipid Assemblies Keishi Suga,1 Tomohiro Yoshida,1 Haruyuki Ishii,2 Yukihiro Okamoto,1 Daisuke Nagao,2 Mikio Konno,2 and Hiroshi Umakoshi1* 1

Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka 560-8531, Japan 2 Department of Chemical Engineering, Graduate School of Engineering, Tohoku University, 6-6-07 Aoba, Aramaki-aza Aoba-ku, Sendai 980-8579, Japan ABSTRACT: The dynamic properties of phospholipid (PL) membranes (phase state and phase transition) play crucial roles in biological systems. However, highly sensitive, direct analytical methods that shed light on the nature of lipids and their assemblies have not been developed to date. Here, we describe the analysis of PL-modified Au nanoparticles (Au@PL) using membrane surface-enhanced Raman spectroscopy (MSERS), and report the properties of the self-assembled PL membranes on Au nanoparticle. The Raman intensity per PL concentration increased to 50-170 times higher by Au@PL, as compared to large unilamellar vesicles (LUVs) at the same PL concentration. The phase state and phase transition temperature of the PL membrane of Au@PL were investigated by analysing the Raman peak ratio (R=I2882/I2930). The enhancement at 714 cm-1 (EF(714)) varied with the hydrocarbon chain length of the PLs and the assembled degree of Au@PLs. In calculation, the EF(714),assembled was estimated to be 111-142 when the distance between AuNPs was 7.0-7.5 nm, which was correlated to the speculative enhancement factor, suggesting that the assembly of the Au@PLs contributed to the MSERS.

INTRODUCTION The functions of biomembranes have been widely studied, especially in regard to their physicochemical properties and responsiveness to external signals.1 In an effort to understand the fundamental behaviours of lipid membranes, artificial self-assembled lipid membranes, such as large unilamellar vesicles (LUVs), giant vesicles (GVs), Langmuir Blodgett membranes, supported lipid bilayers (SLBs) etc., have been investigated; natural and man-made lipids have been applied in the preparation of such membranes. LUVs provide a biomimetic environment, and can be utilized as platforms to regulate the localization, conformation, and function of various biomolecules.2-5 In order to understand such emergent functions arising from the self-assembly of amphiphilic molecules,6 the dynamic properties of membranes, phase state, phase transition temperature, and so on, have been characterized using spectroscopic and microscopic techniques. It is thus important to understand the molecular behaviours of am-

phiphilic molecules in self-assembled membranes, and to utilize such membranes as platforms for (bio)chemical processes. The behaviour of amphiphilic molecules and their assemblies have been evaluated with direct, label-free techniques, such as Raman spectroscopy, infrared spectroscopy, vibrational sum frequency spectroscopy, dielectric dispersion spectroscopy, and differential scanning calorimetry.712,46,47 These methods usually require high lipid concentrations (>50 mM), although labelling techniques, such as fluorescence spectroscopy, do only sub-millimolar concentrations; therefore, more sensitive and quantitative analytical methods that are capable of shedding light on vesicular membrane properties must be developed. Surface-enhanced Raman scattering (SERS) has garnered significant attention as a highly sensitive method that can be used in the analysis of various chemicals and their physicochemical properties.13-18 Au nanoparticles (AuNPs) or Ag nanoparticles (AgNPs) modified with amphiphilic mole-

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cules have been used to induce SERS signals for the enhanced sensing of Ramanactive molecules.19,21,48 Self-assembled monolayers (SAMs) of alkanethiols are typically utilized to form the lipid layer on metal nanoparticles or solid surfaces, and a lipid-alkanethiol hybridized leaflet (so-called hybrid bilayer (HBL)) has been developed as a SERS analysis platform for lipids (Table 1).20,30,49-51 Halas et al. reported a method that could be used to detect the interactions between ibuprofen and lipids using a HBL-modified Au nanoshell.20 Tip-enhanced Raman spectroscopy (TERS) has also been utilized to analyse lipid monolayers prepared by the Langmuir-Blodgett technique.52 Notably, a higher concentration of lipids is required to evaluate membrane properties in conventional Raman spectroscopy (lipid concentration: 100-600 mM).7,10,33,53 To overcome the aforementioned limitations, Raman probes, such as rhodamin 6G, have been utilized for the highly sensitive analysis of molecules within membranes,19,48 because lipophilic molecules are positioned in the interior of amphiphilic assemblies in aqueous media. However, in the aforementioned studies, the hybrid lipid bilayers were not fully characterized, because lipids are not ‘SERS-active’ in most cases.

surfaces.51,57 The rational design of nanoparticles (with a specific diameter etc.) and lipid membranes (with a specific thickness etc.) is needed in order to make a lipid molecule ‘SERS active’. However, the design of ‘SERS active’ lipid membranes has not been established.

The SERS effect is usually observed when localized surface plasmon resonance (LSPR) appears on rough surfaces (e.g., metal materials with a higher degree of surface roughness).22-26 The assembly of metal nanoparticles can give rise to an enhancement in the Raman signal, where the distance between nanoparticles can be critical.14,26-29 It is therefore assumed that SERS-active regions in self-assembled membranes can facilitate the direct analysis of molecular behaviours of membranes owing to greater Raman intensities. Raman signals of target molecules can be enhanced to single-molecule detection levels on rough surfaces of metal nanomaterials, possibly due to the formation of a plasmonic gap. Interestingly, recent studies have suggested that SERS enhancements originating from molecules outside of the junction gap also generate appreciable SERS signals.54-56 As compared to the ‘pure’ assembly of amphiphilic molecules (such as LUVs), solid-supported lipid assemblies exhibit different properties: for instance, the phase transition temperature of HBL is differ from that of LUV.46,47 Furthermore, the polarization of spectroscopic signals might create issues, especially in the case of solid surfacesupported lipid bilayers, due to the preferential plasmoic polarization at metal

EXPERIMENTAL SECTION

The aim of this study is to design a SERS active platform, constructed by selfassembled PL membranes on AuNPs, which would enable a highly-sensitive, direct analysis of the dynamic behaviours of PL molecules in the membrane. Herein, we describe the membrane surface-enhanced Raman spectroscopy (MSERS) of the assembly of PL-modified Au nanoparticles (Au@PL) in an aqueous solution. The SAM-coated AuNPs20,30,49 were mixed with zwitterionic PLs. Using Au@PLs, the Raman intensities of the PLs were significantly enhanced as compared to those using LUVs. The phase transition behaviours of the membranes on Au@PLs were similar to those on LUVs, indicating that the behaviours of PL molecules in the membrane can be analysed using PL-modified AuNPs. The enhancement factors of the PLs at 714 cm-1 (EF(714)) with different hydrocarbon chain lengths were determined, and suitable conditions for MSERS analysis were discussed.

Materials. 1,2-Dilauroyl-sn-glycero-3phosphocholine (DLPC), 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC), and 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). A solution of 100 nm citrate-stabilized Au nanoparticles (3.8×109 particles per milliliter), 1,6diphenyl-1,3,5-hexatriene (DPH), 1-[4(trimethylamino)-phenyl]-6-phenyl-1,3,5hexatriene (TMA-DPH), and 6-lauroyl-2dimethylaminonaphthalene (Laurdan) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) and used without further purification. Preparation of phospholipid-modified Au nanoparticles (Au@PL). The PL-modified AuNPs (Au@PL) were prepared as described in Figure 1a. SAM of decanethiol was formed on the AuNPs by mixing 2 ml of AuNP solution (3.8×109 particles per milliliter), 3 ml of ethanol, and 10 µl of decanethiol solved in chloroform (volume ratio: chloroform/decanethiol = 300/1). The mixture was stirred

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Analytical Chemistry

Table 1. Summary of Raman spectroscopy technique for assembly of amphiphilic molecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Method SERS

Template Au nanoshell Au nanoshell Ag substrate

SAM dodecanethiol dodecanethiol hexanethiol, decanethiol, octadecanethiol

Amphiphilic molecules DMPC DMPC, ibuprofen

Lipid conc. 0.1 mM 0.1 mM

ref. 49 20

DMPC, DPPC

-

50

octadecanethiol

POPC/DOTAP(80/20)

-

51

-

DOPC/DPPC(50/50) POPC, PAPC, PDPC C(18) : C(10)PC DPPC/PAMAM DMPC

250 300 600 100

52 53 33 10 7

Au substrate

TERS Conventional Raman

Au nanoparticles Au substrate -

mM mM mM mM

Figure 1. Characterization of Au@DMPC. (a) Preparation protocol of Au@PL. (b) TEM image of Au@DMPC. The total concentration of PL was 0.5 mM. (c) Hydrodynamic diameters and LSPR peaks of Au@DMPC. 1, Au@DMPC without ultrasonication treatment; 2, Au@DMPC with ultrasonication treatment for 60 min; 3, extruded Au@DMPC (200 nm pore); and 4, extruded Au@DMPC incubated for 7 h at 37 oC. The DLS results were shown in Supplementary Figure S1c. (d) UV-vis spectra of Au@DMPC with different treatment time for ultrasonication. Ultrasonication treatments were performed by bath type sonicator at 37 oC for 10 min, 30 min, 60 min, and 80 min, respectively.

for 3 hours at room temperature. The solution was separated to two liquid phases by incubation for 30 min at room temperature, and the bottom phase was carefully transferred to a round-bottom flask. The solvent was removed by evaporation under vacuum condition. The obtained decanethiolmodified AuNPs were kept under a high vacuum for overnight, and the solvent was

completely removed. A solution of PLs in methanol/chloroform (volume ratio: methanol/chloroform = 1/2) was added to the dried AuNPs, and the solvent was removed by evaporation. The thin films including PL and SAM-AuNPs were kept under a high vacuum for at least 3 hours, and were then hydrated with distilled water at room temperature. After the 5 times freeze-thaw

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treatment (-80 °C /50 °C), the aqueous suspension of Au@PL were obtained. Ultrasonication treatment was performed by a bath type sonicator at above phase transition temperature.58

ed. The peak intensities at 2882 cm-1and 2930 cm-1 were evaluated according to previous reports,33,34,35

To isolate the Au@PL from the mixture of Au@PLs and PL vesicles, the sample solutions were centrifuged at 5000 rpm for 10 min. The precipitates were dispersed with adding distilled water. The concentration of the PL was measured with the assay kit (Phospholipid C-Test; Wako Pure Chemical).31 UV-vis spectra of AuNPs and Au@PLs were measured by UV-vis spectroscopy (UV-1800, Shimadzu, Kyoto, Japan). The absorption spectra were measured from 700 nm to 500 nm at room temperature (light path length 10 nm). The hydrodynamic diameters and size distributions of the Au@DMPC suspensions at 25 oC were determined with Marquadt method from measurements of dynamic light scattering (DLS) (ELS-8000, Otsuka Electronics, Osaka, Japan). Images of the samples were taken with scanning transmittance electron microscope (STEM) (HD-2700B, Hitachi HighTechnologies Corporation, Tokyo, Japan) at an accelerating voltage of 80 kV.

where R represents the packing density of lipid membranes. From the obtained R values, the phase transition temperature, Tm(R), was calculated for Au@PL and LUVs. Here, the sigmoidal fitting was applied to each T v.s. R plot, as follows:

Preparation of large unilamellar vesicles (LUVs). A chloroform solution with PLs was dried in a round-bottom flask by evaporation under a vacuum.32 The obtained lipid thin film was dissolved in chloroform again, and the solvent was evaporated. The lipid thin film was kept under a high vacuum for at least 3 hours, and was then hydrated with distilled water at room temperature. The vesicle suspension was frozen at -80 °C and thawed at 50 °C to enhance the transformation of small vesicles into larger multi-lamellar vesicles (MLVs). This freeze-thaw cycles were performed five times. MLVs were used to prepare the LUVs by extruding the MLV suspension 11 times through two layers of polycarbonate membranes with mean pore diameters of 100 nm using an extruding device (Liposofast; Avestin Inc., Ottawa, Canada). Raman Spectroscopic Analysis. Raman spectra of LUVs and Au@PL were measured using a confocal Raman microscopy (LabRAM HR-800, Horiba, Ltd., Kyoto, Japan). The 532nm YAG laser of a 100mW was used for excitation and a 20×objectlens was used to focus the laser beam. All the spectra reported here were measured with an accumulation time of 20 sec, and each spectrum data was accumulated with 5 times. The background signal of the solution was removed, and then the baseline was correct-

R = I2882 / I2930 ,

S(T)=Rmin+(Rmax-Rmin)/[1+exp{(T-Tm)/τ}] where τ represents a constant, and Rmin and Rmax are the minimum and maximum R values, respectively, in each experiment. Evaluation of membrane fluidities and membrane polarities. The membrane fluidities, (1/P), and membrane polarities, GP340, were evaluated based on the previous reports (see supplementary methods).7,36-39

RESULTS AND DISCUSSION Characterization of Au@PL. The Au@DMPC was characterized using scanning transmission electron microscopy (STEM), dynamic light scattering (DLS), and UV-vis spectroscopy (Fig. 1). A thin layer with a thickness of 8 nm or less was observed on the Au@DMPC after the hydration of the dried DMPC-AuNPs films with distilled water (Fig. 1b). This layer was assumed to be a lipid membrane, since no layer was observed on the untreated AuNPs (Supplementary Figure S1a). The Au@DMPC assembled together, that is consistent with the hydrodynamic diameter of Au@DMPC suspension (315.6 ±73.3 nm, Fig. 1C). The lipid layer formation on AuNPs was also confirmed by LSPR.19,25,27,28 LSPR peaks of the untreated AuNPs and Au@DMPC were observed at 571 nm and 580 nm, respectively. The red-shift of the LSPR peaks of Au@DMPC indicated the increase of their diameter. After ultrasonication for 60 min, the hydrodynamic diameter and the LSPR peak wavelength of Au@DMPC suspension decreased to 141.4 nm and 573 nm, respectively, which were still larger than those of the untreated AuNPS (121.7 nm, Fig. S1a). A thin layer was also observed on the surface of single Au@DMPC in the ultrasonicated suspension (Supplementary Figure S1b). These results revealed that the ultrasonication treatment resulted in disaggregation of the Au@DMPC assembly without removal of the PL membrane from the AuNP surface. To eliminate the multilamellar formation of PL membranes on the AuNP, the extrusion treatment through a 200 nm-pore was performed after ultrasonication. The hydrody-

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namic diameter of the extruded Au@DMPC slightly decreased from 141.4 nm to 119 nm (Fig. 1c), and the LSPR peak was not shifted. After incubation for 7 h at 35 o C, the hydrodynamic diameter of Au@DMPC increased to 133.2 nm, and the LSPR peak shifted from 573 nm to 575 nm. It is assumed that a larger hydrodynamic diameter tends to correlate with a longer wavelength of the extinction maximum in LSPR. To elucidate the effects of ultrasonication, LSPR spectra were measured as a function of time for Au@DMPC (Fig. 1d). Upon ultrasonication of Au@DMPC, the LSPR peaks were blue-shifted from 580 nm to 573 nm after ultrasonication for 60 min, indicating the disaggregation of the Au@DMPC assemblies. In the case of disaggregated Au@DMPC, the presence of the red-shifted LSPR peak implied that the surface of the AuNPs was coated with the PL membrane, whereas in the case of the untreated AuNP, the LSPR peak was observed at 571 nm. Ip et al. reported that lipid coating of AuNPs resulted in a ca. 3 nm red shift of the LSPR peaks, owing to the change in the dielectric constant around the surface of the AuNPs.19 It is expected that Au@DMPC with LSPR peaks at wavelengths longer than 573 nm can exist in an assembled form, and the degree of assembly of Au@DMPC can be controlled by ultrasonication treatment. As shown in Fig. 1b, the assembled Au@DMPC formed a space where the surfaces of the AuNPs were enclosed. Because the gap formation is a key factor in the enhancement of Raman signals of molecules on the surface of metal nanoparticles,16,28,51,59 the enhanced Raman signals of PL membranes are expected by Au@PL. MSERS analysis of PL membranes using Au@PL. The Raman intensities of Au@DMPC and DMPC LUVs were measured (Fig. 2). An enhancement of the Raman intensities was observed in Au@DMPC with a total PL concentration of 0.5 mM (Fig. 2a). On the other hand, no enhancement was observed in a mixture of AuNP (not hydrophobized). The Raman intensities of DMPC LUVs increased as a function of PL concentration (Fig. 2b), and the Raman signal at 0.5 mM PL was hardly detected. The Raman intensities of Au@DMPC decreased with ultrasonication, but were still greater than those of DMPC LUVs at a PL concentration of 0.5 mM. Because ultrasonication induced the disaggregation of Au@DMPCs (Figs. 1 and S1), the assembled form of Au@DMPC was assumed to play a crucial role in the enhancement of the Raman signals. With other Au@PLs, the enhancements in the Raman intensities were observed at lower PL concentrations

(Supplementary Figure S2). In this study, no significant differences were found between the Raman spectra of Au@PLs and those of LUVs. In addition, the enhanced Raman signals were isotropic in optical polarization studies (Supplementary Figure S3). To evaluate the enhancement efficiency, the enhancement factor, EF, was calculated using the following equation:16,28,29,41,42,61 EF = (IMSERS / CMSERS) / (ILUV / CLUV),

(1)

where CMSERS and IMSERS represent the concentration and Raman peak intensity of Au@PL, and CLUV and ILUV are the concentration and Raman peak intensity of the LUVs, respectively. The EF values for DMPC are summarized in Table 2. The Raman spectra of Au@DMPC and DMPC LUVs were almost same from 500 to 3200 cm-1, and the EF values were 80 to 210. These data indicate the possibility for the sensitive analysis of PL molecules and their membrane properties. To evaluate the contribution of the degree of assembly of Au@DMPC in MSERS, the EF values, derived from the headgroup (choline group: 714 cm-1) and acyl chains (methylene group: 2850 cm-1), were measured as a function of ultrasonication time (Fig. 2c). As treatment time increased, both EF(714) and EF(2850) values drastically decreased, indicating that the degree of assembly of Au@PL could be the key factor for the enhanced MSERS intensity. The ultrasonicated Au@DMPC (60 min), and extruded Au@DMPC (through a 200 nm-pore) showed quite similar Raman intensities (Fig. 2d). After incubation for 7 h at 35 oC, an increase in the EF values was observed. Considering the extinction maximum in LSPR of each state of Au@DMPC (Fig. 1c), these results suggested that the degree of Au@DMPC assembly could be essential in MSERS analysis. Subsequently, the dynamic behaviours of the Au@PL membranes were investigated by MSERS, with a focus on the phase state and phase transition properties of the membranes. Characterization of the membrane properties by MSERS. It has been reported that the Raman spectra of PL vesicles, in the hydrocarbon C-H regions (2800-3100 cm-1), exhibit well-defined peaks. The peak intensity ratios are good indicators of the membrane properties:33,34,42 the ratio of the intensity at 2882 cm-1 to that at 2930 cm-1 can indicate the packing density (R) of lipid membranes.35 The temperature dependencies of the Raman spectra were determined for Au@DMPC, DMPC powder,

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Figure 2 Raman spectroscopic analysis for Au@DMPC. (a) Raman spectra of Au@DMPC (red line), Au@DMPC with ultrasonication treatment for 30 min (black line) and DMPC LUV with AuNP (gray dot line). The excitation wavelength was 532 nm, the Raman measurements were carried out at 25 oC. The total PL concentrations for Au@DMPC and that in (AuNP + DMPC LUVs) were 0.5 mM. Ultrasonication treatments were performed by bath type sonicator at 35 oC (b) Raman spectra of DMPC LUVs with different PL concentrations: black-bold line, 50 mM; black line, 10 mM; gray line, 5 mM; gray-dashed line, 1 mM; and gray-dotted line, 0.5 mM. The excitation wavelength was 532 nm, the Raman measurements were carried out at 30 oC. (c) Relationship of the ultrasonication treatment and EFAu@DMPC at 714 cm-1 and 2850 cm-1 values. (d) Comparison of EF values at 714 cm-1 and 2850 cm-1: 1, Au@DMPC without ultrasonication treatment; 2, Au@DMPC with ultrasonication treatment for 60 min at 37 oC; 3, extruded Au@DMPC (200 nm pore); and 4, extruded Au@DMPC incubated for 7 h at 37 oC. The total PL concentrations were 0.5 mM.

Table 2. Enhance factors of Raman intensities of DMPC Raman shift

DMPC LUVs,

Au@DMPC,

Assignmenta -1

[cm ] 714 873 1062 1087 1126 1298 1442 1738 2850 2882 2930 2960 3040

νs(N-CH3) νa(N-CH3) ν(C-C)trans ν(C-C)gauche ν(C-C)trans τ(CH2) σ(CH2) ν(C=O) νs(CH2) νa(CH2) νs(CH3) νa(CH3) ν(CH3)choline

EF [-] I0/C0 0.08 0.03 0.05 0.05 0.03 0.07 0.14 0.01 0.79 0.75 0.56 0.27 0.05

b

ISERS/CSERS 10.9 5.02 6.35 4.13 3.91 10.8 16.8 1.35 99.2 95.2 71.1 37.5 7.15

a

d

c

143.1 149.7 134.1 89.1 111.9 155.6 123.0 204.7 125.1 126.2 126.8 138.0 152.0

Peak assignment was referred on the basis of previous reports.33,34 determined with 50 mM of phospholipid at 25 ºC. The data was accumulated with fivetime reproduced measurements. c determined with 0.5 mM of phospholipid at 25 ºC. The data were accumulated with fivetime reproduced measurements. d calculated according to equation (1).16,28,29,41,2 b

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and DMPC in chloroform (Fig. 3a). With Au@DMPC, the Raman peak intensity at 2882 cm-1 decreased with increasing temperature, while that at 2930 cm-1 increased, which was consistent with the spectral change of DMPC LUVs.7 On the other hand, with DMPC powder and DMPC dissolved in chloroform, the Raman spectra were not affected by temperature. Based on these spectra, the packing densities of PL membranes (R = I2882/I2930),33,34 were evaluated (Fig. 3b). The R values of Au@DMPC varied depending on temperature; the R values were higher (R >2.0) below 20 ºC, and they significantly decreased above 25 ºC (R 2.0) in the temperature range of 10-35 ºC, while those of DMPC in chloroform (liquid-like) were lower (R < 1.0). Because the packing density of vesicular membranes varies with the phase states of PL membranes,7,33,34,42 the Au@DMPC membrane was found to be dynamic, as was that of DMPC LUVs. The nature of the Au@DMPC membrane phases were estimated to be solid-ordered (solid-like) at T < 20 ºC and liquid-disordered (liquid-like) at T > 25 ºC; the phase transition temperature (Tm) of the DMPC vesicle was reported to be 23.6 ºC.60 From the obtained Raman data, the calculated phase transition temperatures, Tm(R), were evaluated for the Au@PLs and LUVs (Table 3). Although the lipid membrane properties of the supported lipid bilayer might be different from those of vesicles,46,47 our results indicated that the membrane properties of Au@PL (R, Tm(R)) were similar to those of PL LUVs (Supplementary Figure S4). Using fluorescent probes, e.g., DPH, TMA-DPH, and Laurdan,7,36 the membrane properties of Au@PL and PL LUVs were shown to be similar (Supplementary Figure S5), when the compositions of Au@PL and LUV membranes were the same. After ultrasonication, the membrane fluidities, 1/P, were not changed, indicating that the fluorescence of Au@PL was not related to the LSPR (Supplementary Figure S6). These results revealed that the dynamic behaviours of the PL membrane could be analysed by MSERS using Au@PLs. Characterization of MSERS-active regions formed on Au@PLs. The MSERS phenomena was observed upon assembly of Au@DMPC (Fig. 2a); the ultrasonication of Au@DMPCs resulted in their disaggregation (Fig. 1d), and the EF(714) and EF(2850) values of Au@DMPC decreased with ultrasonication (Fig. 2c). In the Raman spectra, the signals attributed to C-H (2800-3000 cm-1) are dominant, and the C-H signal intensities might be affected to the number of hydrocarbon chain length. On the other

hand, the concentration of choline group (-N+(CH3)3, 714 cm-1) is proportional to PC concentration, herein, the EF(714) values are used to find out the key factor of Raman enhancement. In the case of the disaggregated Au@DMPC, the distance between AuNPs might be too large for MSERS to be observed. Thus, the assembly of Au@PLs and the distance between AuNPs are critical to the design the MSERS-active regions on Au@PLs. The Raman intensities of Au@PLs with different lengths of hydrocarbon chains including DLPC (C12:0/12:0), DMPC (C14:0/14:0), DPPC (C16:0/16:0), and DSPC (C18:0/18:0)) were compared to those of LUVs. It is hypothesized that the peak are sensitive to intensities at 714 cm-1 the membrane thickness because the choline headgroups are located at the farthest point from the surface of AuNPs. The EF(714) values of Au@PLs were compared with the number of hydrocarbon groups

Figure 3 Analysis of membrane properties by MSERS. (a) Temperature dependencies of Raman spectra. Top column: Au@DMPC (total PL concentration: 1 mM); Middle column: DMPC powder putted on the glass plate; and Bottom column: DMPC dissolved in chloroform (total PL concentration was 50 mM). (b) R values of DMPC powder (black circle), Au@DMPC (red circle), and DMPC in chloroform (green circle). The exciting wavelength was 532 nm, and the measurements were repeated at least three times.

Table 3. Summary of Tm(R) for Au@PL and LUV DMPC LUVs Au@DMPC DPPC LUVs Au@DPPC a

Tm [oC]a 23.6േ1.5 41.3േ1.8 -

Tm(R) [oC]b 21.9 23.0 42.5 40.8

Tm was based on previous report.60

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b Tm(R) was calculated by the sigmoidal fitting of R values.

in the acyl chains (Fig. 4). The EF(714) values of Au@PLs increased in molecules with shorter hydrocarbon chains (Au@DLPC, Au@DMPC: EF(714)=173.7), and the EF(714) of Au@DLPC was ca. 3 times greater than that of Au@DSPC. In addition, ultrasonication resulted in a decrease of the EF values; the EF(714) values of ultrasonicated Au@DLPC were ca. 5 times lower than that of Au@DLPC. Notably, there were no significant differences in the Raman intensities of PL LUVs (Supplementary Figure S7a). On the contrary, in the case of Au@PLs, there was a remarkable difference in the Raman intensity at 714 cm-1, depending on the type of PL molecules (Supplementary Figure S7b). The MSERS signals disappeared after ultrasonication, while ultrasonication did not affect the Raman intensities of the LUVs. The above results indicated that the assembly of Au@PLs played an important role for MSERS. In the case of Au@DSPC, the assembled Au@DSPC showed an LSPR peak at 580 nm, which was similar to that of the assembled Au@DMPC; however, the EF(714) value was lower than that of Au@DMPC. It was assumed that the distance between AuNPs drastically contributed to MSERS activity. In the case of Au@DOPC, the EF(714) value was ca. 94, although the hydrocarbon chain length of DOPC (C18:1/18:1) is as long as that of DSPC. In general, the membrane thickness of lipid bilayer depends on the phase state: ca. 3.7 nm for liquid-disordered phase, and ca. 4.6 nm for solid-ordered phase. Considering the phase transition temperature (18.3 ºC60) and the phase state (liquiddisordered phase7) of DOPC membrane, the thickness of the membrane formed on Au@PL is the possible enhancement factor for the MSERS intensity. Speculative enhancement model of MSERS between Au@PL. The obtained results suggested that the surface of Au@PL in assembled form contributed to MSERS activity, whereas the surface of Au@PL in nonassembled form did not. To discuss the effect of the gap distance between AuNPs on Raman signal enhancement, the thickness of the Au@PL membrane (dT) and the minimum distance between AuNPs (dG,min), represented in Figure 5a, were calculated, and the speculative enhancement factor, MEM, were estimated. The maximum length of a hydrocarbon chain (-(CH2)n-) was calculated using the following equation:43 d=0.154+n0.1265,

(2)

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where d and n represent the length of the hydrocarbon chain and the number of carbon

Figure 4 Evaluation of EF(714) values. The EF(714) compared with the number of hydrocarbon in acyl chains: filled bars and open bars indicate before ultrasonication treatment and after ultrasonication treatment, respectively.

atoms in a molecule, respectively. The length of the polar head group was 0.8 nm.43,44 The minimum distance between AuNPs was assumed to be achieved when dG,min = 2dT, wherein dT is assumed to be a variable of hydrocarbon length. In order to simplify the contribution of the MSERS signal from the surface of Au@PL in assembled form (EF(714),assembled) and those from the surface of Au@PL in non-assembled form (EF(714),non-assembled), the EF(714) values were calculated as follows: EF(714),Au@PL ≃ EF(714),assembled + EF(714),non-assembled, (3) where the EF(714),Au@PL values and EF(714),nonassembled values are shown as closed symbols and open symbols in Fig. 4, respectively. The calculated EF(714),assembled values, shown in Table 4, were 111.1 and 141.9, when the estimated dG,min values were 7.0 nm and 7.5 nm, respectively. The speculative enhancement factor, MEM, was calculated using the following equation:59,62 MEM(x, 0) = A[b/(b-q)]4. (4)

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Figure 5 (a) Schematic illustration of the gap distance dG between the surface of Au@PL. The distance between AuNPs is indicated as dT, distance between AuNP and membrane; and dG, distance between AuNPs. (b) Speculative enhancement factor MEM shown as a function of the minimum gap distance dG,min, calculated by eq. (4).

Table 4 Summary of the number of hydrocarbon, dG, EF(714),non-assembled, EF(714),assembled DLPC DMPC DPPC DSPC a b

n-carbon 12 14 16 18

dTa [nm] 3.5 3.8 4.0 4.3

EF(714),non-asssembled [-] 31.8 23.8 26.6 5.2

dG,min [nm] 7.0 7.5 8.0 8.5

EF(714),assembledb [-] 141.9 111.1 49.7 46.1

calculated according to equation (2). calculated according to equation (3).

Eq. (4) shows the perpendicular decay in the x direction of the electromagnetic enhancement factor, where A is constant relating to the concentration ratio of SERS-active regions. b and q were defined as follows: b = 1 + dG,min/D ,

q = (1-(x/r))1/2,(5)

where D is diameter (=100 nm), and r is radius (= 50 nm). When the contribution of electromagnetic enhancement at the gap centre is considered, x becomes 0. In that case, it is assumed that MEM (0, 0) is consistent with EF(714),assembled because the choline head group is expected to be located in the gap centre. The change of MEM(0, 0) for dG,min showed a strong correlation with the experimental values when A was 0.002 (Fig. 5b). Under our experimental conditions, the bulk concentration of lipid CSERS was used to define the experimental EF values. CSERS does not fully characterize the number of lipid molecules in the vicinity of the gap region, which was expected to contribute to MSERS.61 The experimental EF values and the speculative enhancement factor MEM were in good corre-

lation at A=0.002, indicating that the MSERS active-region (the surface of Au@PL in assembled form) was much less than the surface of Au@PL in non-assembled form. It is assumed that the MSERS could be regulated by the thickness (gap distance) of the membrane, although the actual dG value might be a function of hydrocarbon chain length, phase state, and lamellarity of PL membranes. It was concluded that the MSERS-active regions were formed in the assembled Au@PLs, where the MSERS signals were due to the PL membranes.

CONCLUSION In the present study, PL-modified AuNPs were utilized for MSERS analysis, which can shed light on the molecular behaviours of PL membranes. An enhancement in the Raman intensities of PL membranes was obtained using Au@PL, where the EF values for Au@DMPC were 80 to 210 for each Raman peak in the range of 500-3800 cm-1. The surface of Au@PL in assembled form can contribute to the MSERS. As a possibility, when the gap distance between AuNPs (with unilamellar PL membrane) is assumed to

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7.0-7.5 nm, the Raman signal is enhanced 111- to 142-fold, where the MSERS active region is much less than the total region of PL membranes. Au@PLs may be useful for constructing self-assembled PL membranes, and, therefore, Au@PL membranes can be considered to be a suitable model of curved biomembranes, similar to conventional LUVs. Moreover, MSERS of Au@PLs could be used to elucidate lipid membrane properties. It is expected that Au@PLs can provide insight into the behaviours and functions of lipid membranes, such as a protein-membrane interactions, molecular storage in lipid membranes, and membrane fusion.

ASSOCIATED CONTENT Supporting Information SEM image and DLS histograms of original AuNPs and Au@DMPC before and after ultrasonication treatment (Figure S1); MSERS signal of Au@PL (Figure S2); Polarized MSERS of Au@DMPC (Figure S3); Packing densities of Au@PLs compared with PL LUVs (Figure S4); Membrane properties of Au@PLs and PL LUVs analyzed by TMA-DPH, Laurdan, and DPH (Figure S5); Membrane property of Au@DMPC before and after ultrasonication treatment analyzed by DPH (Figure S6); and Raman spectra of PL LUVs and Au@PLs before and after ultrasonication treatment (Figure S7). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Corresponding Umakoshi

author:

Prof.

Phone: +81-6-6850-6287; [email protected]

Dr.

Hiroshi

E-mail:

b-

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Funding Program for Next Generation World-Leading Researchers of the Council for Science and Technology Policy (CSTP) (GR066), JSPS Grant-in-Aid for Scientific Research A (26249116), and JSPS Grant-in-Aid for Research Activity Start-up (25889039). One of

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the authors (K.S.) also expresses his gratitude for the Japan Society for the Promotion of Science (JSPS) and GCOE scholarships.

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Figure 1. Characterization of Au@DMPC. (a) Preparation protocol of Au@PL. (b) TEM image of Au@DMPC. The total concentration of PL was 0.5 mM. (c) Hydrodynamic diameters and LSPR peaks of Au@DMPC. 1, Au@DMPC without ultrasonication treatment; 2, Au@DMPC with ultrasonication treat-ment for 60 min; 3, extruded Au@DMPC (200 nm pore); and 4, extruded Au@DMPC incubated for 7 h at 37 oC. The DLS results were shown in Supplementary Figure S1c. (d) UV-vis spectra of Au@DMPC with different treatment time for ultrasonication. Ultrasonication treatments were performed by bath type sonicator at 37 oC for 10 min, 30 min, 60 min, and 80 min, respectively. 338x190mm (96 x 96 DPI)

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Figure 2 Raman spectroscopic analysis for Au@DMPC. (a) Raman spectra of Au@DMPC (red line), Au@DMPC with ultrasonication treatment for 30 min (black line) and DMPC LUV with AuNP (gray dot line). The excitation wavelength was 532 nm, the Raman measurements were carried out at 25 oC. The total PL concentrations for Au@DMPC and that in (AuNP + DMPC LUVs) were 0.5 mM. Ultrasoni-cation treatments were performed by bath type sonicator at 35 oC (b) Raman spectra of DMPC LUVs with different PL concentrations: black-bold line, 50 mM; black line, 10 mM; gray line, 5 mM; gray-dashed line, 1 mM; and gray-dotted line, 0.5 mM. The excitation wavelength was 532 nm, the Raman measurements were carried out at 30 oC. (c) Relationship of the ultrasonication treatment and EFAu@DMPC at 714 cm-1 and 2850 cm-1 values. (d) Comparison of EF values at 714 cm-1 and 2850 cm-1: 1, Au@DMPC without ultrasonication treatment; 2, Au@DMPC with ultrasonication treatment for 60 min at 37 oC; 3, extruded Au@DMPC (200 nm pore); and 4, extruded Au@DMPC incubated for 7 h at 37 oC. The total PL concentrations were 0.5 mM. 338x190mm (96 x 96 DPI)

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Figure 3 Analysis of membrane properties by MSERS. (a) Temperature dependencies of Raman spectra. Top column: Au@DMPC (total PL con-centration: 1 mM); Middle column: DMPC powder putted on the glass plate; and Bottom column: DMPC dissolved in chloroform (total PL con-centration was 50 mM). (b) R values of DMPC powder (black circle), Au@DMPC (red circle), and DMPC in chloroform (green circle). The exciting wavelength was 532 nm, and the meas-urements were repeated at least three times. 264x189mm (96 x 96 DPI)

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Figure 4 Evaluation of EF(714) values. The EF(714) compared with the number of hydrocarbon in acyl chains: filled bars and open bars in-dicate before ultrasonication treatment and after ultrasonication treatment, respectively. 121x189mm (96 x 96 DPI)

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Figure 5 (a) Schematic illustration of the gap distance dG between the surface of Au@PL. The distance between AuNPs is indicated as dT, distance between AuNP and membrane; and dG, distance between AuNPs. (b) Speculative enhancement factor MEM shown as a function of the minimum gap distance dG,min, calculated by eq. (4). 323x143mm (96 x 96 DPI)

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