DELIVERABLE REPORT

NFFA-Europe has received funding from the EU's H2020 framework programme for research and innovation under grant agreement n. 654360

WP7 JRA2 – Research on High Precision Manufacturing

D7.7 Test of 3D hierarchical scaffold with and without liposome membranes

31/08/2017

M24

2

PROJECT DETAILS PROJECT ACRONYM PROJECT TITLE

NFFA-Europe NANOSCIENCE FOUNDRIES AND FINE ANALYSIS - EUROPE GRANT AGREEMENT NO: FUNDING SCHEME

654360 RIA - Research and Innovation Action START DATE

01/09/2015

WP DETAILS WORK PACKAGE ID WORK PACKAGE TITLE

WP7 JRA2 – Research on High Precision Manufacturing

WORK PACKAGE LEADER

Christian David (PSI)

DELIVERABLE DETAILS DELIVERABLE ID DELIVERABLE TITLE

D7.7 Test of 3D hierarchical scaffold with and without liposome membranes DELIVERABLE DESCRIPTION

Fabrication and test of 3D hierarchical scaffold with and without lipid membranes EXPECTED DATE ESTIMATED INDICATIVE PERSONMONTHS

M24 31/08/2017 3.5 AUTHOR(S)

Benedetta Marmiroli, Barbara Sartori, Heinz Amenitsch (TUG), Imma Ratera (PRUAB), Adriana Kyvik (PRUAB) PERSON RESPONSIBLE FOR THE DELIVERABLE

Christian David (PSI) NATURE

D - Demonstrator DISSEMINATION LEVEL

☒ P - Public ☐ PP - Restricted to other programme participants & EC: (Specify) ☐ RE - Restricted to a group (Specify) ☐ CO - Confidential, only for members of the consortium

3

REPORT DETAILS ACTUAL SUBMISSION DATE

31/08/2017 09.41 AM NUMBER OF PAGES

17 FOR MORE INFO PLEASE CONTACT

Benedetta Marmiroli (TUG) Tel. +39-040-3758708 Email: benedetta.marmiroli@elettra.eu

Version Date Author(s) Description / Reason for modification Status

0 dd/mm/aaa Name Surname Template 1 18/08/2017 Benedetta Marmiroli Draft 2 21/08/2017 Barbara Sartori Revision 3 28/08/2017 Imma Ratera Revision 4 29/08/2017 Heinz Amenitsch Revision 5 30/08/2017 Christian David,

Francesc Perez Murano

Revision

6 31/08/2017 Benedetta Marmiroli Final

Contents Executive Summary 4

1. Concept 5

2. Design specification 6

2.1 Mesoporous films preparation with and without lipid membrane 6

2.1.1 Mesoporous films with Pluronic P 123 6

2.1.2 Mesoporous films with Brij58 7

2.1.3 Deposition of POPC lipid membranes on the mesoporous substrates 7

2.2 GISAXS experimental setup 7

3. Results 9

3.1 Preliminary GISAXS measurements of the sample components 9

3.2 In situ GiSAXS measurement of humidity ramp 11

3.3 GISAXS measurement of the fluid delivery through the mesopores 13

4. Conclusions and Perspectives 15

References 16

4

Executive Summary Microfluidic systems for handling/sensing chemical and biological samples could improve their performance through the employment of materials with selected functionalities in specific regions of the device.

Among these, mesoporous materials feature ordered tailored structures with uniform pore sizes, highly accessible surface areas and large pore volumes, making them an attractive support for functionalization and catalysis. The ordered mesopores are an ideal host for functional organic molecules or nanoparticles and patterning them would allow the design of devices for different types of advanced applications, like DNA nanoarrays. Patterning of such films to obtain circuits or dot arrays can be reached by coupling the bottom-up route with top-down processing such as lithography. In this way, hierarchically structured materials can be obtained in which organization resides on multiple length scales: porosity (typically 2–10 nm), film thickness (200–500 nm), and pattern size (1 - 500 µm).

The current activity, described in detail in the following chapters, was focused on using the mesoporous films as active support for lipid membranes. In the specific case, the pores were filled with water, and it was examined if and how lipid membranes are kept hydrated by the underlying mesoporous film. This would open the use of such mesoporous films for other biological studies of increasing complexity by selectively functionalizing the mesopores.

Films were prepared following different recipes and using different irradiation doses at the Deep X-ray Lithography beamline (DXRL) at Elettra-Sincrotrone Trieste, therefore presenting different pore size and arrangement. The proper lipid membrane was then selected: multilayer 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane, which has a structure that is sensitive to the presence of water. POPC was dissolved in isopropanol, dip coated on half of the mesoporous substrates and left drying under vacuum overnight. Then, static Grazing Incidence Small Angle X-Ray (GISAXS) experiments were done keeping the sample at 4% relative humidity (RH) with a custom made humidity chamber, directly mounted at the Austrian SAXS beamline at Elettra-Sincrotrone Trieste. Subsequently, for each sample in situ GISAXS measurements were performed increasing the humidity from 15% to 90% (at 0.01 % RH/s). The results showed a change of the lattice dimension of the mesoporous material and of the structure of the lipid membrane.

In the last experiment, the sample was equilibrated at 40% RH and a water droplet was added to the area uncovered by the lipids. The experiment was designed to allow sample hydration only by water diffusion through the mesopores.

Time resolved GISAXS patterns were taken on the lipid membrane after adding the drop of water to determine the change in hydration. The first results evidenced the structural changes of POPC due to partial hydration through the mesopores, demonstrating the possibility to use the mesoporous film to convey liquids. It was also proved that by changing the mesoporous material recipe and the irradiation doses, the delivery of fluids can be tuned.

The mesoporous films, with and without the lipid membrane, are now available on request for the users of TA4. One of them is currently used as a reference sample to align the GISAXS stage at the Austrian SAXS beamline at Elettra-Sincrotrone Trieste.

Such preliminary results will be presented at the MNE 2017, Micro-Nano Engineering Conference, in Braga (Portugal) in September 2017.

5

1. Concept Today, microfluidics is highly applied to life sciences, as a result of its versatility and its unique properties such as the ability to precisely handle small volumes of liquid, to control fast transport, to achieve high surface to volume ratio, and to precisely tune concentration gradient [1,2]. Recently, microfluidics has been successfully applied to several biological studies including monitoring cellular responses to chemical gradients [3], drug efficacy [4] and cytotoxicity screening [5]. The further reduction of the channel dimensions down to the nanodomain, also called nanofluidics, is an emerging area that is of high interest for analytical and sensing applications. When the channels size is at the nanometers length scale, surface and interfacial forces become predominant, and open new opportunities for sample manipulation in fields like single molecule enzymology or DNA separation [6, 7].

Micro/nanofluidic systems for handling and sensing chemical and biological samples will improve their performance through the employment of materials with selected functionalities in specific regions of the device such as active monolayers or binding proteins on sensor interfaces. To obtain this goal, top-down techniques can be employed to pattern bottom-up synthesized functional materials. To guarantee the tailoring of the structure for the specific application, suitable investigation techniques must be employed [8].

Self-assembled mesoporous thin films are an important example of bottom-up synthesized materials for which integration in devices, however, requires top-down processing. Specific patterning of mesoporous films aimed at obtaining circuits or dot arrays is a task that cannot be fulfilled by merely dip-coating or spin-coating the precursor solution onto a substrate without further processing. In particular, the bottom-up route needs coupling with top-down processing such as substrate film lithography. In this way, hierarchically structured materials can be obtained in which organization resides on multiple length scales: porosity (typically 2–10 nm), film thickness (50 nm – 500 nm), and pattern size (50 nm - 500 µm). Mesoporous based materials feature ordered tailored structures with uniform pore sizes, highly accessible surface areas and large pore volumes, making them an attractive support for functionalization and catalysis. The ordered mesopores are an ideal host for functional organic molecules or nanoparticles and the patterns, from nano- to micrometer scale, allow the design of devices for different types of advanced applications, for example DNA nanoarrays or lab-on-a-chip devices [9].

Deep X-ray lithography (DXRL) is a technique which induces chemical and structural changes in matter due to the impact of high energy (3 – 20 keV) photons thus providing high aspect ratios, smooth lateral surfaces and sub-100 nm lateral resolution [10]. Previously, we have patterned mesoporous silica based materials using DXRL where the X-rays simultaneously increase the polycondensation of the exposed silica while partially removing the templating agent. Investigation of the chemical changes upon irradiation was also performed via IR microspectroscopy [11]. To obtain micro-nano devices with tailored properties using mesoporous materials, morphology has to be carefully tuned. For this reason in previous experiments we determined the effects of X-ray irradiation on the films. As demonstrated by our GISAXS measurements, we were able to select the best irradiation dose in terms of pattern quality while maintaining the pore connectivity and structure. After characterizing and fabricating the mesoporous substrate, now we are interested in using the mesoporous films as active support for lipid membranes, in particular phospholipids.

Phospholipids bilayers present similar properties to those of the cell membranes, therefore they could be used as hosts for transmembrane proteins that can serve as sensing elements in biosensing

6

devices. A promising idea is to use mesoporous materials as lipid membranes support, where pore walls provide stability to the membrane and pores would result in a desired lipid environment [12,13]

As a first step, we first considered if it is possible to deliver fluids to hydrate lipid membranes, which is fundamental to apply them in sensor arrays used for example as electronic nose [14]. Such structural changes, driven by humidity, have already been examined by using variuos techniques, among which GISAXS, which is a versatile tool for the study of structure on nanometer length scales, has an important role [15 -18].

In the specific case, we filled the pores with water and examined if and how lipid membranes are kept hydrated by the underlying mesoporous film. This would open the use of such films as supports for other biological studies of increasing complexity, like protein/peptide incorporation for their specific functionalization, such as signaling etc.

In the following we will report our preliminary results on using mesoporous silica films prepared with different recipes and different irradiation doses, to convey water to POPC lipid membranes.

2. Design specification In the following chapter we will describe first the preparation steps in order to obtain a mesoporous material with and without lipid membrane. Then we will give an account of the experimental set-up that has been used to perform GISAXS experiments at the Austrian SAXS beamline at Elettra-Sincrotrone Trieste.

2.1 Mesoporous films preparation with and without lipid membrane Mesoporous silica thin films were synthesized using either Brij58 or Pluronic P123 as templating agents. In both cases, the precursor solution was deposited on clean silicon wafer via spin-coating, at 800 RPM for 1 minute.

2.1.1 Mesoporous films with Pluronic P 123 Samples templated by Pluronic P123 were prepared according to the recipe proposed by Yan et al. [19], as follows: 0.584 g of P123 were dissolved in 26.8 g of ethanol (EtOH). The silica precursor solution was obtained mixing 1.75 g of Tetraethoxysilane (TEOS), 1 g of ethanol and 1.25 g of MilliQ water acidified to pH 1.25 with 2.5mM HCl. The two precursor solutions were stirred for 1 hour, then the P123 sol was added dropwise to the silica sol, and the mixture was stirred at room temperature for 2 hours. Additional 1.925 g of acidified water was added to the mixture, and the solution was stirred for 1.5 hours. Before spin coating, the pH was adjusted to 2.5 with concentrated HCl. Immediately after spin coating, the samples were exposed to high humidity for 10-20 s.

After spin coating, the films were undergoing two different treatments:

1) Thermal treatment as follows: 1hour ramp to reach 450˚C, half an hour at 450˚C

2) X-ray irradiation at the DXRL beamline with a dose of 269 J/cm2 ; development in a solvent composed of 7ml of ethylene glycole, 4 ml of acetone, 2 ml of EtOH for 20 minutes; thermal treatment as in 1). The irradiation dose has been selected following the results of previous experiments focused on the selection of the minimum dose to obtain an ordered structure.

7

2.1.2 Mesoporous films with Brij58 Samples synthesized with Brij58 were prepared according to a modified procedure from Fuertes et al. [20]. Silica precursor solution was prepared stirring 2.1 g of TEOS, 6 g of EtOH, 0.76 g of MilliQ water and 0.0233 ml of concentrated HCl for 1 hour. 0.56 g of Brij58 dissolved in 5 g of EtOH were added dropwise to this mixture, and the final solution was stirred for 1 hour at room temperature. The final molar ratio was 1 TEOS: 0.05 Brij58: 5.2 EtOH: 24 H2O: 0.28 HCl. The pH of the final solution was checked to be below 2 before spin-coating.

After spin coating, the films were undergoing two different treatments:

1) Thermal treatment as follows: half an hour ramp to reach 350˚C, 2 hours at 350˚C

2) X-ray irradiation at the DXRL beamline with a dose of 68 J/cm2 ; development in a solvent composed of 4 ml of ethylene glycole, 4 ml of acetone, 4 ml of EtOH, 4 ml of DI water; thermal treatment as in 1). Also in this case, the irradiation dose has been selected following the results of previous experiments to determine the minimum dose to get an ordered structure.

2.1.3 Deposition of POPC lipid membranes on the mesoporous substrates 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was dissolved in isopropyl alcohol at a concentration of 10mg/ml and was deposited via dipcoating on half of the mesoporous films samples. The dip-coated samples were dried overnight at room temperature in a vacuum oven with a pressure lower than 10−2 mbar.

2.2 GISAXS experimental setup GISAXS experiments were performed at the Austrian SAXS beamline at Elettra-Sincrotrone Trieste synchrotron source (TUG partner in NFFA-Europe Consortium) [21].

The wavelength was λ = 0.154 nm and 2D GISAXS patterns were collected with a Pilatus 1M detector (Dectris, Baden Switzerland). The covered q range was 0.32 nm-1 - 5 nm-1. The angular scale of the diffraction pattern has been calibrated with silver behenate [22].

The experiments at controlled humidity were performed in a custom built setup for controlling RH, defined as the ratio of the water vapour pressure to the saturation vapour pressure at the given temperature. The chamber is described in Sharifi et al. [23] and shown in figure 1.

The humidity cell is composed of a metal case equipped with two Kapton windows (0.013mm thickness) to let the incident and the scattered beam pass through. Humidity is controlled by mixing dry (<3% RH) and humid air produced by a supersonic humidifier (>95% RH) outside the chamber, then flowing the mixture in the cell. A humidity sensor inside the chamber in connection with a proportional-integral-differential (PID) controller is used to set the requested RH. The temperature in the whole experimental hutch is constant at (25 ± 1) °C. The samples were directly transferred from the vacuum oven into the sample chamber. GISAXS measurements started after that the sensor had reached the desired RH value.

8

Figure 1: In situ set-up for humidity controlled GISAXS experiments. On the left, the system mounted at the Austrian SAXS beamline; on the right, a detail of the sample chamber is evidenced.

GISAXS experiments of the mesoporous materials with and without lipid membrane, were conducted first at a constant RH of 20%.

Then, for each sample with lipid membrane, a humidity ramp was performed from 15% to 90% and back to 15% RH, at a rate of 0.66 RH%/min. GISAXS measurements were made in situ during the hydration-dehydration cycle for 3s every minute.

Finally, to study the effect of hydration through the mesopores, the samples with lipid membranes were prepared as in figure 2, using a similar system as described in reference [24].

While the silicon substrate is completely covered by the mesoporous film, the lipid membrane is deposited only on one half of the sample. A Polydimethylsiloxane (PDMS) piece around 1 mm wide, was fixed to the sample using kapton adhesive, to act as a barrier between the part covered by the lipid membrane and the other half of the sample. PDMS was chosen as it adapts to surfaces in a conformal way and guarantees the sealing. The sample was inserted in the humidity chamber at a constant RH of 40%. A water droplet was then put on the other side of PDMS with respect to the lipid membrane. Therefore, water could reach the lipid membrane only through the mesopores under the PDMS. Time resolved in situ GISAXS measurements were conducted, taking an image exposed for 3s every minute, to avoid radiation damage on the lipid membrane.

9

Figure 2: Scheme of the sample configuration allowing the diffusion of water through the mesoporous film up to the lipid membrane and the interaction with external humidity. The lipid membrane is then investigated by GISAXS.

The one dimensional cuts were extracted from the two-dimensional patterns using the program Fit2D [25].

3. Results GISAXS measurements of four different mesoporous substrates were conducted, in order to determine their structure:

(a) mesoporous silica prepared with templating agent Pluronic 123, calcined

(b) mesoporous silica prepared with templating agent Pluronic 123, undergone x-ray irradiation with a dose of 269 J/cm2, developed and calcined

(c) mesoporous silica prepared with templating agent Brij58, calcined

(d) mesoporous silica prepared with templating agent Brij58, undergone x-ray irradiation with a dose of 68 J/cm2, developed and calcined

We decided to focus on calcined samples to be sure that the surfactant inside the pores is completely removed and therefore pores are entirely open. In the future, the exposed samples after development and without calcination will also be examined.

3.1 Preliminary GISAXS measurements of the sample components The structure has been determined for each kind of sample. The GISAXS images showing the crystallinity of the structures are shown in figure 3. The indexing is performed only in the qz direction, as data were first analysed using a vertical cut shown in figure 3(a).

It can be seen that samples (a) and (b) present a hexagonal structure. (b) is less ordered than (a), (c) and (d) are both composed of two cubic structures with different lattice dimension.

10

Figure 3: GISAXS images of mesoporous substrates prepared with different conditions. Indexing along the qz axis . (a) hexagonal mesoporous silica prepared with Pluronic 123 and calcined. The red rectangle evidences the cut performed to analyse the data (b) hexagonal mesoporous silica prepared with Pluronic 123, exposed, developed and calcined (c) cubic mesoporous silica prepared with Brij58 and calcined. Two cubic structures with different lattice dimension are present (d) cubic mesoporous silica prepared prepared with Brij58, irradiated, developed, and calcined. Two cubic structures with different lattice dimension are present

Then, POPC membranes have been deposited on each sample, following the procedure reported above. In figure 4, the corresponding GISAXS images at the detector are shown. The I-q (intensity vs scattering vector) plots along the cut evidenced in figure 3, are shown in figure 4. In both pictures, green arrows indicate the contribution which is unequivocally related to the POPC.

11

Figure 4: GISAXS images of POPC lipid membranes at 20% RH deposited on the mesoporous substrates which have been prepared with different conditions as described in figure 1. The green arrows indicate the contribution of POPC. (a) Mesoporous silica prepared with Pluronic 123 and calcined. (b) Mesoporous silica prepared with Pluronic 123, exposed, developed and calcined (c) Mesoporous silica prepared with Brij58 and calcined. (d) Mesoporous silica prepared with Brij58, irradiated, developed, and calcined.

3.2 In situ GiSAXS measurement of humidity ramp Each sample (mesoporous substrate+POPC) has been inserted in the custom made humidity chamber and measured in-situ while performing a humidity cycle (from 15% to 90% RH and back). The resulting scattering intensity curves are displayed in figure 5. It can be observed that both lipid membranes and mesoporous silica are subject to changes. But while the lipid membrane undergoes structural changes (as already demonstrated for other lipid membranes in reference [18]), mesoporous silica show a pore lattice strain (as described in reference [23]).

12

Figure 5: I-q plots of mesoporous substrates with POPC lipid membranes undergoing a humidity cycle (15-90% and back). While the peaks corresponding to the mesoporous structure only shift, some peaks of the lipid membrane disappear with humidity changes. (a) Mesoporous silica prepared with Pluronic 123 and calcined. (b) Mesoporous silica prepared with Pluronic 123, exposed, developed and calcined (c) Mesoporous silica prepared with Brij58 and calcined. (d) Mesoporous silica prepared with Brij58, irradiated, developed, and calcined.

For the purpose of the present deliverable, the attention will be focused on the lipid membrane peaks, in particular on the ones evidenced by the pink circles in figure 6.

13

Figure 6: Out-of-plane GISAXS cuts of mesoporous substrates prepared with different conditions with and without POPC lipid membranes deposited on them at 20% RH. The curves are obtained by horizontal rebinning the GISAXS images along the rectangle shown in figure 3(a). The green arrows evidence the contribution due solely to the lipid membrane. The pink circles indicate the reflections of the lipid membrane that will be considered in later analysis of the effect of the drop (a) Mesoporous silica prepared with Pluronic 123 and calcined. (b) Mesoporous silica prepared with Pluronic 123, exposed, developed and calcined (c) Mesoporous silica prepared with Brij58 and calcined. (d) Mesoporous silica prepared with Brij58, irradiated, developed, and calcined.

3.3 GISAXS measurement of the fluid delivery through the mesopores After the preliminary characterization to understand the behaviour of the different components of the samples, the specimens were prepared as shown in figure 2 and were put in the humidity chamber at constant 40% RH. A drop of water was added to the part without POPC and time resolved GISAXS measurement were performed on the part with POPC.

The effect of water delivery through the mesopores is displayed in figure 7, where the I-q curves are represented for each kind of sample. Only one of the characteristic peaks of the lipid membrane was considered, namely the ones marked by the pink circles in figure 5, which showed most evidently changes. The black curve is the position of the peak at a constant humidity value of 40%. The blue peak corresponds to the one just after drop addition. The red curves represent the peaks corresponding to part of the humidity ramp shown in figure 6. In this way, we can compare the peak

14

correspondent to the addition of the drop to a change in the hydration state of the lipid membrane due to environmental humidity change. Our observations showed that:

i) Adding a drop to sample a + POPC is like rising the humidity to around 65%

ii) Adding a drop to sample b + POPC does not cause any change. The experiment should be repeated to confirm that this is not caused by an experimental fault.

iii) Adding a drop to sample c + POPC is like rising the humidity to around 50%

iv) Adding a drop to sample d + POPC is like rising the humidity to around 78%.

Figure 7: I-q plots of mesoporous substrates with POPC lipid membranes after the addition of a water drop in a configuration like the one described in figure 2. Only one of the characteristic peaks of the lipid membrane is considered, the one most evidently showing changes. The black curve is the position of the peak at a constant humidity value of 40%. The blue peak corresponds to the one just after drop addition. The red curves represent the peaks corresponding to part of the humidity ramp shown in figure 6. (a) Mesoporous silica prepared with Pluronic 123 and calcined. (b) Mesoporous silica prepared with Pluronic 123, exposed, developed and calcined (c) Mesoporous silica prepared with Brij58 and calcined. (d) Mesoporous silica prepared with Brij58, irradiated, developed, and calcined.

15

This experiment demonstrates, in particular sample a, that the mesoporous materials can be used to convey liquids to substances deposited above them (i.e. lipid membranes), and that the quantity of fluid that can be delivered can be tuned by selecting the preparation recipe and the irradiation dose. This opens the possibility to use mesoporous materials as “active” sample holders.

Samples with different treatment and with or without lipid membrane will be available on demand for the TA4 users.

4. Conclusions and Perspectives In this report we successfully demonstrated that mesoporous silica films can be used as “active” sample holders to convey fluids through the mesopores. We also evidenced that the fluid delivery can be up to a certain extent tuned by selecting the synthesis recipe of the mesoporous film, and the irradiation dose through DXRL.

The validation of this statement has been conducted by feeding water to lipid membranes deposited on mesoporous films. Such lipid membranes changed structure upon hydration, which has been shown by GISAXS measurements.

These first results open the use of such mesoporous films for other biological studies of increasing complexity by selectively functionalizing the mesopores.

The first activity that we intend to conduct now, in order to proceed towards this goal, is the study of the functionalization of the pores to improve their hydrophilicity. We will use different molecules, changing the binding sites and modifying the surface properties of the mesoporous materials. Such study will be conducted together with PRUAB partner, which has the functionalization expertise.

Mesoporous silica wafers are already available for users of TNA4 access to align the GISAXS stage.

Samples with different synthesis recipe or irradiation treatment, and with or without lipid membranes, are available on demand.

The present results will be exposed at the MNE 2017, Micro-Nano Engineering Conference, in Braga (Portugal), in September 2017.

16

References [1] H. Craighead; Future lab-on-a-chip technologies for interrogating individual molecules; Nature; 442; 387; 2006

[2] Special issue on fundamental principles and techniques in microfluidics; Lab Chip ; 17; 2009

[3] N. L. Jeon, H. Baskaran, S.K. Dertinger, G.M. Whitesides, L. Van de Water, M. Toner M; Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device; Nature Biotechnology ; 20; 826; 2002

[4] A. Khademhosseini, R. Langer, J. Borenstein, J P. VacanJ; Microscale technologies for tissue engineering and biology; PNAS; 103;8; 2480; 2006

[5] Z. Wang, M.C. Kim, M. Marquez, T. Thorsen; High-density microfluidic arrays for cell cytotoxicity analysis; Lab Chip; 7; 740; 2007

[6] M. Napoli, J. C. T. Eijkel, S. Pennathura; Nanofluidic technology for biomolecule applications: a critical review; Lab Chip; 10; 957; 2010

[7] M. Tagliazucchi, I. Szleifer; Transport mechanisms in nanopores and nanochannels: Can we mimic nature?; Mater. Today; 18; 131; 2015

[8] B. Marmiroli, H. Amenitsch; X-ray lithography and small angle X-ray scattering: a combination of techniques merging biology and materials science; Eur. Biophys. J; 41; 851; 2012

[9] P. Falcaro, S. Costacurta, L. Malfatti, M. Takahashi , T. Kidchob, M. F. Casula, M. Piccinini, A. Marcelli, B. Marmiroli, H. Amenitsch, P. Schiavuta, P. Innocenzi; Fabrication of Mesoporous Functionalized Arrays by Integrating Deep X-Ray Lithography with Dip-Pen Writing; Adv. Mater; 20; 1864; 2008

[10] M. Tormen, G. Grenci, B. Marmiroli, F. Romanato; X-ray Lithography: fundamentals and applications; in Nanolithography; edited by Stefan Landis, ISTE-Wiley; 2010

[11] P. Falcaro, L. Malfatti, L. Vaccari, H. Amenitsch, B. Marmiroli, G. Grenci, and P. Innocenzi; Fabrication of Advanced Functional Devices Combining Soft Chemistry with X-ray Lithography in One Step; Adv. Mater.; 21; 48; 4932; 2009

[12] M. Claesson, N-J Cho, C. W. Frank, M. Andersson; Vesicle Adsorption on Mesoporous Silica and Titania; Langmuir; 26; 22; 16630; 2010

[13] T-H Yang, C. K. Yee, M. L. Amweg, S. Singh, E. L. Kendall, A. M. Dattelbaum, A. P. Shreve, C. J. Brinker, A. N. Parikh; Optical detection of ion-channel proton transport in supported phospholipid bilayers; Nano Letters; 7; 8; 2446; 2007

[14] A. D. Wilson, M. Baietto; Applications and Advances in Electronic-Nose Technologies; Sensors 9, 5099-; 2009

[15] M. Rappolt, H. Amenitsch, J. Strancar, C. V. Teixeira, M. Kriechbaum, G. Pabst, M. Majerowicz, P. Laggner; Phospholipid mesophases at solid interfaces: in situ X-ray diffraction and spin-label studies; Advances in Colloid and Interface Science; 111; 63; 2004

[16] M. Rappolt; Bilayer thickness estimations with “poor” diffraction data; Journal of applied physics; 107; 08741; 2010

17

[17] J. Katsaras, K. R. Jeffrey, D. S-C. Yang, R. M. Epand; Direct evidence for the partial dehydration of phosphatidylethanolamine bilayers on approaching the hexagonal phase; Biochemistry; 32; 10700; 1993

[18] L. Yang and H. W. Huang; Observation of a membrane fusion intermediate structure; Science; 297; 1877; 2002.

[19] M. Yan, M. J. Anderson, A, Gibaud; Grating induced micelle alignment of mesostructured silica films, Appl Phys. Lett.; 91; 023104; 2007

[20] M. C. Fuertes, F. J. López-Alcaraz, M. C. Marchi, H. E. Troiani, V. Luca, H. Míguez, G. J.de Avila A. Soler-Illia; Photonic Crystals from Ordered Mesoporous Thin-Film Functional Building Blocks; Adv. Funct. Mater.; 17; 1247; 2007

[21] H. Amenitsch, M. Rappolt, M. Kriechbaum, H. Mio, P. Laggner and S. Bernstorff; Journal of Synchrotron Radiation; 5; 506; 1998

[22] T. C. Huang, H. Toraya, T. N. Blanton, Y. J. Wu; X-ray powder diffraction analysis of silver behenate, a possible low-angle diffraction standard; J. Appl. Cryst.; 26; 180; 1993

[23] P.Sharifi, B. Marmiroli, B. Sartori, F. Cacho-Nerin, J. Keckes, H. Amenitsch, O. Paris; Humidity-driven deformation of ordered mesoporous silica films; Bioinspired, Biomimetic and Nanobiomaterials; 3; BBN3; 2014

[24] D. R. Ceratti, M. Faustini, C. Sinturel, M. Vayer, V. Dahirel, M. Jardat, D. Grosso; Critical effect of pore characteristics on capillary infiltration in mesoporous films; Nanoscale; 7; 5371; 2015

[25] P. Hammersley; Fit2D Reference Manual V3.1.; Internal Report ESRF98HA01T; ESRF, Grenoble, France; 1998