Tethered Lipid Bilayers within Porous Si Nanostructures: A Platform for

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Tethered lipid bilayers within porous Si nanostructures: A platform for real-time optical monitoring of membrane-associated processes Elena Tenenbaum, Nadav Ben Dov, and Ester Segal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00935 • Publication Date (Web): 22 Apr 2015 Downloaded from http://pubs.acs.org on April 28, 2015

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Tethered lipid bilayers within porous Si nanostructures: a platform for (optical) real-time monitoring of membrane-associated processes Elena Tenenbaum1, Nadav Ben-Dov1 and Ester Segal1,2* 1

Department of Biotechnology and Food Engineering, Technion – Israel Institute of Technology,

Haifa 32000, Israel 2

The Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa

32000, Israel KEYWORDS: Supported lipid bilayers, Tethered lipid bilayers, Porous Si, Nanostructure, Optical reflectance.

ABSTRACT

The importance of cell membranes in biological systems has prompted the development of artificial lipid bilayers, which can mimic the cellular membrane structure. Supported lipid bilayers (SLBs) have emerged as a promising avenue for studying basic membrane processes and for possible biotechnological applications. Conventional methods for SLB formation involve the spreading of lipid vesicles on hydrophilic solid supports. Herein, a facile approach for the construction of tethered SLB within an oxidized porous Si (pSiO2) nanostructure, avoiding

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liposome preparation, is presented. We employ a two-step lipid self-assembly process, in which a first lipid layer is tethered to the pore walls resulting in a highly stable monolayer. A subsequent solvent exchange step induces the self-assembly of the unbound lipids into a robust SLB. Formation of pSiO2-SLB is confirmed by fluorescence resonance energy transfer (FRET) and the properties of the confined SLB are characterized by environment-sensitive fluorophores. The unique optical properties of the pSiO2 support are employed to monitor in real time the partitioning of a model amphiphilic molecule within the SLB via reflective interferometric Fourier transform spectroscopy (RIFTS) method. These self-reporting SLB platforms provide a highly generic approach for bottom-up construction of complex lipid architectures for performing biological assays at the micro- and nano-scale.

INTRODUCTION

Biological membranes play a key role in cell function, defining the boundaries of living cells and controlling the transport of different ions and molecules in and out of the cell.1, 2, 3, 4, 5 The membrane basic structure is a lipid double layer, mainly held together by non-covalent interactions, embedding a large variety of proteins with different features, such as catalyzing membrane-associated reactions and transferring information across the membrane.5,

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The

importance of understanding the physical properties of lipid bilayers and the different membranal processes created a vast interest in developing model systems. Early artificial models were based on freestanding planar phospholipid membranes7, 8. However, as these systems lack sufficient mechanical stability, a growing number of model membranes on solid supports are developed. Supported lipid bilayers (SLBs) retain the lipid membrane fluidity and exhibit higher stability because of the underlying auxiliary surface, as long as the membrane is hydrated.3, 4, 5 SLBs are


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also a more comfortable platform for membrane characterization by surface specific techniques. Different techniques, such as surface plasmon resonance (SPR)9, impedance spectroscopy (EIS)9,

10, 13, 14

10, 11, 12, 13

and electrical

are employed to monitor SLB formation as well as

interactions of these membranes with molecules of interest. Common SLB preparation methods involve adsorption of vesicles on the surface, their rupture and subsequent fusion to form a supported membrane. 1, 2, 4 In the present work we report for the first time on a simple method for the formation of a supported and tethered lipid bilayer within nanostructured porous silicon (pSi). pSi layers are conventionally fabricated by electrochemical etching of crystalline silicon under a constant current density. The nanostructure features e.g., pore diameter and depth, are easily tuned during the etching process and its surface can be functionalized via a wide repertoire of straightforward chemistries.15, 16 The reflectivity spectrum of these pSi layers is comprised of a series of FabryPérot interference fringes resulting from reflections at the top and bottom interfaces of the porous thin film. By applying a fast Fourier transformation (FFT) on the raw reflectivity data, the effective optical thickness (EOT), which equals 2nL (where n is the effective refractive index and L is the physical thickness of the porous layer), can be extracted. This technique is often referred to as reflective interferometric Fourier transform spectroscopy (RIFTS).17, 18, 19, 20 The resulting pSi-based interferometers are widely employed for biosensing, as their optical properties are highly sensitive to the presence of chemical and biological species inside the pores.15, 21, 22, 23 The formation of supported bilayers onto oxidized porous Si (pSiO2) thin films has been recently studied by several research groups.24,

25, 26, 27, 28

Nevertheless, in all these

studies planar lipid bilayers are formed on top of the porous layer by vesicle rupture. As such, they employ variations in the intensity of the reflected light to monitor the behavior of the


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phospholipids bilayers. Our approach utilizes the large surface area of the porous nanostructure by tethering a lipid monolayer to the pSi inner surface, followed by lipid self-assembly to form a bilayer. As this method allows for the bilayer formation throughout the porous nanostructure, it enables monitoring EOT changes upon processes occurring at the membrane level. Bilayer formation is confirmed by environment-sensitive fluorescent probes as well as by Fluorescence resonance energy transfer (FRET) assay. The partitioning of molecules within the bilayer is monitored by real-time RIFTS, as changes in the relative EOT values. Our preliminary results demonstrate the potential of the developed system as a convenient tool for studying membranelevel processes.

EXPERIMENTAL

Materials Highly-doped p-type Si wafers (0.9 mΩ cm resistivity, - oriented, B-doped) were purchased from Siltronix Corp., France. Aqueous HF (48%) and ethanol absolute were supplied by

Merck.

(3-Aminopropyl)triethoxysilane

(APTES),

Ethyl-3-(3-dimethylaminopropyl)-

carbodiimide (EDC), N-Hydroxysulfosuccinimide sodium salt (NHS), Morpholinoethanesulfonic acid

(MES),

MES

sodium

salt,

succinic

anhydride,

acetonitrile,

octyl

gallate,

Diisopropylethylamine (DIEA), Diphenylhexatriene (DPH), Nile Red and all buffer salts were obtained from Sigma Aldrich Chemicals, Israel. Methanol and acetone were obtained from Gadot, Israel. Chloroform was obtained from Frutarom, Israel. Acetic acid was obtained from BioLab Ltd., Israel. 18:00-18:1 Phosphatidylethanolamine (PE), L-α-PhosphatidylethanolamineN-(lissamine rhodamine B sulfonyl) (Rhodamine-labeled PE), and 1-palmitoyl-2-[12-[(7-nitro-21,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphoethanolamine

(NBD-labeled


4

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PE) were obtained from Avanti Polar Lipids, USA. Phosphate buffered saline (PBS) at pH 7.4 was prepared by dissolving 50 mM Na2HPO4, 17 mM NaH2PO4, and 68 mM NaCl in doubledistilled water (ddH2O, 18 MΩ). MES buffer was prepared by dissolving 6 mM MES and 4 mM MES sodium salt in ddH2O. Fabrication of pSiO2-SLB Preparation of pSiO2: Si wafers are electrochemically etched in a 3:1 (v/v) solution of aqueous HF (48%) and ethanol for 30 s at a constant current of 333 mA/cm2. CAUTION: HF is a highly corrosive liquid, and it has to be handled with extreme care and under secured working conditions. Si with an exposed area of 0.3 cm2 are contacted on the backside with a strip of aluminum foil and mounted in a Teflon etching cell; a platinum ring is used as the counter electrode. After etching, the surface is rinsed three times with ethanol and dried under a stream of nitrogen gas. The resulting freshly-etched pSi is then thermally oxidized at 800°C for 1 h in ambient air to form a porous silica scaffold. High-resolution scanning electron microscopy (HR-SEM): The morphology of the neat pSiO2 is characterized using Carl Zeiss Ultra Plus instrument at an accelerating voltage of 1 keV. The diameter range of the pores is determined from top-view micrographs. Functionalization of pSiO2: pSiO2 samples are amino-silanized by incubation in 2% APTES in 50% v/v methanol/ddH2O and 0.6 % v/v acetic acid for 1 h. Subsequently, the amine-modified surface is immersed in a solution of succinic anhydride (10 mg/ml) in acetonitrile and 5% v/v DIEA for 3 h. After removal of the solution, the surface is washed extensively with acetonitrile two times and with ddH2O. The samples are reacted with EDC (20 mg/ml) and NHS (10 mg/ml) in MES buffer at pH 6. The samples are rinsed with ethanol and dried under a nitrogen gas. Surface modification is verified using attenuated total reflectance Fourier transform infrared


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(ATR-FTIR) spectroscopy. Spectra are recorded using a Thermo 6700 FT-IR instrument equipped with a Smart iTR diamond ATR device. Preparation of pSiO2-SLB: NHS-modified pSiO2 samples are immersed in 0.5 mg/ml PE in chloroform overnight at 4ºC. Next, the chloroform is replaced to acetonitrile and incubated for 15 min. The samples are gently rinsed with PBS to remove solvent remnants. For control, pSiO2 samples are treated with PE in chloroform and are thoroughly rinsed with chloroform, ethanol and PBS to remove unbound PE. Characterization of pSiO2 -SLB Properties Nile red and DPH fluorescence assays: pSiO2-SLB samples and appropriate controls are placed in a 24-well plate with PBS. Nile red solution (final concentration of 300 µM) is prepared by dissolving Nile red in ethanol, followed by dilution with PBS (1% v/v ethanol:PBS ). DPH solution (final concentration of 2 µM) is prepared by dissolving DPH in acetone, followed by dilution with PBS (0.1% v/v acetone:PBS). Nile red or DPH solution is added to each well. The fluorescence

is

recorded

using

a

plate

reader

(Thermo

Scientific

Varioskan)

at

excitation/emission of 540/640 nm for Nile red and 365/425 nm for DPH. The fluorescence is measured at multiple points along the lateral axis of pSiO2 sample and results are presented as the average value. Each experiment is performed in triplicates. Conventionally, the behavior of these solvatochromic probes is characterized by transmittance spectrometry.29 However, in the present study, the lipids are confined within the porous nanostructure, characterized by a profound light absorbance, and therefore the probe fluorescence can only be monitored in reflectance mode. Due to this limitation, the Nile red and DPH assays are adapted to rely on fluorescence intensity.


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Confocal laser scanning microscopy (CLSM) imaging: pSi-SLB samples and the controls are labeled with Nile red as described above and thoroughly rinsed with PBS. Images are taken using CLSM (Carl Zeiss LSM 700) equipped with Plan-Apochromat 63x/1.40 Oil objective. pSiO2 photoluminescence is excited at 405 nm and observed at 435 nm. Nile red is excited at 555 nm and its fluorescence is observed at 585 nm. Image acquisition and processing is conducted using ZEN software (Carl Zeiss). Fluorescence resonance energy transfer (FRET) assay: NHS-modified pSiO2 is reacted with NBD-labeled PE (0.05 mg/ml in chloroform) as described earlier. The surfaces are thoroughly rinsed with chloroform to remove unbound lipids. The resulting labeled pSiO2 is incubated in a solution of Rhodamine-labeled PE (0.05 mg/ml) in chloroform for 1 h, to allow proper infiltration into the porous nanostructure. Subsequently, the chloroform is replaced with acetonitrile and incubated for 15 min and the samples are washed with PBS. The samples are studied by the CLSM, at an excitation wavelength of 488 nm (for NBD molecules) and emission intensity is acquired at 518 nm. Image acquisition and processing is conducted with ZEN software (Carl Zeiss). Arithmetic mean of pixel intensity is calculated for each optical crosssection. The values are presented as averaged values of 10 focal cross-sections. Measurement of Interferometric Reflectance Spectra Interferometric reflectance spectra of the samples are collected using an Ocean Optics chargecoupled device (CCD) USB 4000 spectrometer fitted with an objective lens coupled to a bifurcated fiber-optic cable. A tungsten light source is focused onto the center of the sample surface with a spot size of approximately 1 mm2. Reflectivity data are recorded in the wavelength range of 400-1000 nm, with a spectral acquisition time of 100 ms. All optical experiments are conducted in a custom-made Plexiglas cell, which is fixed during the experiment


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in order to ensure that sample's reflectivity is measured at the same spot. Both illumination of the surface and detection of the reflected light are performed along an axis coincident with the surface normal. Data are recorded by Ocean Optics software and analyzed by Igor Pro software as RIFTS, as previously described by Massad-Ivanir et al.

30

Briefly, illumination of white light

on the pSi results in a reflected interference spectrum due to the difference in the refractive indices between the porous layer and the bulk Si. Applying FFT on the collected data results in a single characteristic peak, whose position is monitored. This position corresponds to the value of the EOT of the porous film, which equals to 2nL, where n is the average refractive index of the porous layer and L is its thickness. Alteration of the medium inside the pores changes the average refractive index and accordingly the EOT value. In this work, data are presented as the relative ∆EOT, defined as: ∆EOT/EOT0. Optical monitoring of octyl gallate partitioning in pSiO2-SLB Optical experiments are carried out immediately following pSiO2-SLB preparation. Baseline is acquired by incubation of the samples with PBS for 75 min. Subsequently, the samples are incubated with 1 mg/ml of octyl gallate solution (dissolved in ethanol and diluted with PBS to 2% v/v ethanol) until a constant EOT value is observed; after which the films are rinsed with PBS. The spectra are recorded every 15 s throughout the experiment.

RESULTS AND DISCUSSION

pSiO2 -SLB preparation The pSi films are prepared from a highly doped p-type crystalline Si wafer by an electrochemical etching process at 333 mA/cm2 for 30 s. This technique allows to easily tune the properties of the resulting porous nanostructures, in terms of pore dimensions, morphology and


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porosity by adjustment of the etching parameters (e.g., Si dopant type, dopant level, current density and HF concentration).16 Favorable properties of this nanomaterial are its large surface area23 and its diverse and tunable optical properties31. The current density is adjusted to yield cylindrical pores, with a diameter range of 45-75 nm, as determined by HR-SEM

30, 32, 33

(see

Figure S1). The resulting freshly-etched pSi is thermally oxidized at 800°C to create a hydrophilic pSiO2 scaffold. The pore dimensions are tuned to accommodate lipid bilayers, which are typically 3–5 nm thick in animal cells and 7–9 nm in bacterial cells34, and yet eliminate the accommodation and formation of closed lipid bilayers35,

36

. The synthetic approach for

fabricating the lipid bilayer within the porous nanostructure is outlined in Figure 1. The pSiO2 is first amino-silanized using APTES (Figure 1b). In the following step, the amino groups are reacted with succinic anhydride, forming a carboxylated surface (Figure 1c). Next, NHS and EDC coupling chemistry is used to conjugate the lipid, PE, through its amine terminus, forming a tethered lipid monolayer onto the surface, as illustrated in Figure 1e,f-I. In order to form a lipid bilayer, the chloroform is removed and replaced with acetonitrile, a higher polarity solvent. This induces the unbound lipids to self-assemble, with their head groups facing the acetonitrile and their hydrophobic tails facing the tethered lipids (Figure 1f-II), to minimize the contact between their hydrophobic tails and polar environment.37, 38 Finally, acetonitrile is replaced with PBS.


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Figure 1. Surface immobilization steps and pSiO2-SLB formation. (a) neat pSiO2; (b) APTESmodified surface; (c) carboxylated-surface; (d) surface activated with amine-reactive NHS; (e) PE-modified surface; (f) formation of pSiO2-SLB: after tethering the first lipid layer (I), the unbound excess lipids are allowed to self-assemble in acetonitrile (II).


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The formation of molecular tethers onto the pSiO2 surface is validated using Attenuated Total Reflectance Fourier Transform Infra-Red (ATR-FTIR) spectroscopy (Figure 2). The spectrum of a neat pSiO2 depicts a typical Si-H vibrating mode at 801 cm-1 and a peak at 1056 cm-1 that is attributed to the Si-O-Si stretching mode (data not shown). After the silanization step, a peak at 1631 cm-1 is observed, attributed to the bending of the primary amines.23,

30

Following the

modification with succinic anhydride, the spectrum shows two strong bands at 1558 and 1637 cm-1, attributed to amide II and amide I bonds, respectively.23, 30 In addition, a peak at 1401 cm-1 is detected, assigned to the C-O stretching and O-H deformation vibrations of carboxylic acid groups (Figure 2c).39 Functionalization with amine-reactive NHS results in a typical peak at 1738 cm-1 and at 1786 cm-1, attributed to the asymmetric and symmetric stretching bands of succinimidyl ester, respectively.23, 30 Following lipid immobilization, the spectrum (Figure 2e) shows two new peaks at 2855 and 2923 cm-1, assigned to the symmetric and asymmetric stretching of the CH2 groups on the PE backbone.39 The peak observed at 1733 cm-1 is indicative of C=O acyl chain stretching.40


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Figure 2. ATR-FTIR spectra of the different modification steps: (a) neat pSiO2; (b) amineterminated surface after silanization; (c) carboxylated surface; (d) surface activated with aminereactive NHS; (e) PE-modified surface.

Confirmation of bilayer formation Environment-sensitive fluorescent probes are employed in order to confirm the lipid bilayer formation within the porous nanostructure. Nile Red is an uncharged fluorophore that is sensitive to its local environment polarity and hydration.

29, 41

Specifically, it partitions into lipid

membranes based on its intrinsic hydrophobicity.42 pSiO2-SLB samples are immersed in PBS and Nile red is introduced. The fluorescence intensity of the sample is presented in Figure 3a. For comparison, both carboxylated-pSiO2 and pSiO2-monolayer are studied under the same conditions. The fluorescence intensity of the carboxylated-pSiO2 is observed to be significantly lower in comparison to pSiO2-monolayer and pSiO2-SLB samples (t-test, p

Tethered Lipid Bilayers within Porous Si Nanostructures: A Platform for

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