Microarrays of Phospholipid Bilayers Generated by Inkjet Printing

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Microarrays of phospholipid bilayers generated by inkjet-printing Misato Yamada, Hiromasa Imaishi, and Kenichi Morigaki Langmuir, Just Accepted Manuscript • DOI: 10.1021/la400570h • Publication Date (Web): 29 Apr 2013 Downloaded from http://pubs.acs.org on May 14, 2013

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Microarrays of phospholipid bilayers generated by inkjet-printing

Misato Yamada 1, Hiromasa Imaishi1, 2, Kenichi Morigaki 1, 2 *

1: Graduate School of Agricultural Science, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501 Japan 2: Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501 Japan

*Corresponding author: Kenichi Morigaki: E-mail: [email protected], Fax: +81-78-803-5941

1 ACS Paragon Plus Environment

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Abstract We report an efficient and reproducible method to generate a microarray of model biological membranes on a solid substrate by applying the inkjet printing technology. Although inkjet printing is currently widely used for the industrial fabrication processes including biological materials, printing lipid membranes remained technically challenging due to the hydrophobic nature of droplets and instability of lipid bilayer structure against dehydration. In the present study, we printed lipids onto a glass substrate covered with a micropatterned membrane of polymeric phospholipid bilayer. Polymeric bilayers were formed by the lithographic photo-polymerization of a diacetylene-containing

phospholipid,

1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-

phosphocholine (DiynePC). After removing non-polymerized DiynePC with a detergent solution, natural lipid membranes were incorporated into the polymer-free regions (corrals) by using an electric field-based inkjet printing device that can eject subfemtoliter volume droplets. To avoid rapid dehydration and destabilization, we pre-printed aqueous solution containing agarose and trehalose onto the corrals, and subsequently printed lipid suspensions (“two-step-printing method”). After rinsing, stable lipid bilayer membranes were formed in the corrals. The bilayers were continuous and fluid as confirmed by fluorescence recovery after photobleaching (FRAP). We could introduce multiple bilayer patches having different lipid compositions into the neighboring corrals. The present results demonstrate that the combination of patterned polymeric bilayer and inkjet printing technology enables efficient, reliable, and scalable generation of the model membrane microarrays having varied compositions.


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1. Introduction Substrate-supported phospholipid bilayers (SPBs) are being studied as a versatile model system of the biological membrane at the solid-liquid interface.

1,2

One important

feature of SPBs is the possibility to generate micropatterned membranes, which enable to create designed arrays of biological materials for biomedical applications such as high throughput drug-candidate screening. Various approaches have been applied to the micropatterning, including the use of pre-patterned substrates 7,8

3-6

and soft-lithography.

We have previously developed a methodology to create micropatterned membranes

composed of polymeric and fluid lipid bilayers by the lithgraphic polymerization of a 9-12

diacetylene phospholipid.

The polymeric bilayer acted as a framework for

stabilizing the model membrane, whereas embedded lipid membranes were made of natural phospholipids and retained physicochemical properties of the biological membrane.

Although these micropatterning techniques are useful for generating small and well-defined patches of SPBs on the substrate, most of the studies have deposited a single type of lipid membrane into all spots, except for those that used rather technically elaborate micro-fluidic, spotting, or electrochemical techniques.

13-20

Especially, a

variety of spotting techniques (including inkjet printing) have been applied to deposit different kinds of lipids.

13,14,16,19,20

However, spotting small droplets of lipid

suspensions has posed significant technological challenges due to the hydrophobic nature of droplets and instability of lipid bilayer structures against dehydration. The surface of aqueous droplets containing lipid membranes is generally hydrophobic due to a lipid monolayer formed, making it difficult to print the droplets onto hydrophilic


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substrates (e.g. glass). Rapid dehydration of small aqueous droplets also makes the bilayer membrane susceptible to destabilization. These technological barriers have severely limited the reliability of the membrane deposition. One approach that has been taken to avoid the contact between lipid membranes and air was to spot lipids in the aqueous solution.

13,19,20

However, this approach inevitably complicates the handling

and positioning of droplets and raises the technical bar for miniaturizing microarrays.

Herein, we report on the fabrication of microarrays of model biological membranes by inkjet printing. Inkjet printing technology is currently widely used as a versatile tool for the industrial fabrication processes such as liquid crystal displays and organic transistors. 21,22

Inkjet printing technology is also being applied to biological materials including

DNA, antibodies, proteins, and cells. 23-26 In spite of this rapid expansion of the use in a wide range of settings, inkjet printing technology of lipid membranes is not established, primarily due to the above mentioned technical difficulties. In the present study, we used micropatterned membranes of polymeric bilayer as a scaffold for printing natural lipids. Preformed polymeric bilayers provided a well-defined and stable framework for the incorporation of lipid materials. To avoid dehydration and destabilization of lipid bilayers, we pre-printed aqueous solution containing agarose and trehalose onto the polymer-free regions surrounded by polymeric bilayers (corrals) before printing lipid suspensions (“two-step-printing method”). The combination of patterned polymeric bilayer and two-step-printing method enabled efficient, reliable, and scalable printing of the model biological membranes.


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2. Materials and methods 2.1 Materials 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

(POPC),

1,2-bis(10,12-

tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), 1,2-hexanoyl-sn-glycero-3phosphocholine (DHPC), GM1 Ganglioside (brain, ovine) (GM1), and 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotin-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). N-(6-tetramethylrhodaminethiocarbamoyl)1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TRITC-PE) Texas Red 1, 2-dihexadecanoyl-sn-glycero-phosphoethanolamine

(TR-PE),

N-(7-nitrobenz-2-oxa-

1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine

(NBD-PE),

Cholera toxin subunit B-Alexa Fluor 488 conjugate (CTB488), and Streptavidin-Alexa Fluor 594 conjugate (SAF594) were purchased from Molecular Probes (Eugene, OR). Agarose (Type VII) was purchased from Sigma-Aldrich. Trehalose and glycerine were purchased from Nacalai Tesque (Kyoto, Japan). Deionized water used in the experiments was ultrapure Milli-Q water (Millipore) with a resistance of 18.2 MΩcm. It was used for cleaning substrates, preparing buffer solutions (0.01 M sodium phosphate buffer with 0.15 M NaCl, pH 6.6 (PBS)) and all other experiments.

2.2 Substrate cleaning Microscopy cover slips (Matsunami, Osaka, Japan) were used as substrates for bilayer deposition. The substrates were cleaned with a commercial detergent solution, 0.5% Hellmanex/ water (Hellma, Mühlheim, Germany), for 20 min under sonication, rinsed with deionized water, treated in a solution of NH4OH (28%)/ H2O2 (30%)/ H2O (0.05:1:5) for 10 min at 65°C, rinsed extensively with deionized water, and then dried in


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a vacuum oven for 30 min at 80°C. Before use, these substrates were further cleaned by the UV/ ozone treatment for 20 min (PL16-110, Sen Lights, Toyonaka, Japan).

2.3 Preparation of patterned polymeric bilayers A detailed description of the fabrication method of patterned polymeric bilayers is given in previous papers.11,12,27 Bilayers of monomeric DiynePC were deposited onto glass substrates by the spontaneous spreading of vesicles. Polymerization of DiynePC bilayers was conducted by UV irradiation using a mercury lamp (UVE-502SD, Ushio, Tokyo, Japan) as the light source. The applied UV dose was typically 4 J/cm2 at 254 nm. After the UV irradiation, non-polymerized DiynePC molecules were removed from the substrate surface by immersing in 0.1 M sodium dodecylsulfate (SDS) solution at 30°C for 30 min and rinsing with deionized water extensively. The polymerized bilayers were stored in deionized water in the dark at 4°C and dried before inkjet printing.

2.4 Preparation of vesicle suspensions Lipids dissolved in chloroform were mixed in a round-bottom flask, dried with nitrogen, and subsequently evaporated at least for 4 h in a vacuum desiccator. The dried lipid films were hydrated in PBS (the lipid concentration was 10 mM) overnight. Lipid membranes were dispersed by five freeze/thaw cycles, and the suspension was extruded by using a Liposofast extruder (Avestin, Ottawa, Canada) with 100 nm polycarbonate membrane filter (10 times) and 50 nm polycarbonate filter (15 times).

2.5 Inkjet printing Inkjet printing was conducted by using an electric field-based printing device


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(SLTS0505-KBD: SIJ, Tsukuba, Japan). Solutions were ejected from a nozzle (tip diameter: 2-2.5 µm) by applying a direct and alternating voltage. Two kinds of printing solutions were used. Pre-printed aqueous solution typically contained 10% glycerine, 0.06% (w/v) agarose, and 0.1% (w/v) trehalose. Lipid suspensions typically contained 10mM POPC, 5mM DHPC, a fluorophore (0.1 mM), and 5% (v/v) glycerine. The presence of DHPC stabilized the liquid ejection from the nozzle and promoted vesicle fusion at the same time. 28 The printing solution was loaded into a nozzle and deposited onto the patterned membrane. Aqueous solution including trehalose and agarose was printed into the corrals, and then lipid solution was printed into the same corrals. The printing process was performed at room temperature.

2.6 Fluorescence microscopy observation Fluorescence microscopy observations were performed using an inverted microscope (IX-70, Olympus, Tokyo, Japan) equipped with a xenon lamp (UXL-75XB, Olympus), a 10x objective (NA 0.40) or a 20x objective (NA 0.75), and a CCD camera (Clara, Andor). Three types of filter sets were used: 1) excitation 470-490 nm/ emission 510-550 nm (green fluorescence), 2) excitation 540-550 nm/ emission 575-625 nm (yellow fluorescence), 3) excitation 545-580 nm/ emission > 610 nm (red fluorescence). For the observation of fluorescence recovery after photobleaching (FRAP), an upright microscope (Olympus BX51WI) equipped with a 60x water-immersion objective (NA 0.90) and a CCD camera (DP30BW, Olympus) was used.


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3. Results and discussion SPBs were formed in the corrals between polymeric bilayer matrix by the “two-step-printing method”, in which aqueous solution containing agarose and trehalose was printed before printing vesicle suspensions (Figure 1). Direct deposition of vesicle suspensions resulted in only partial coverage of the corrals with heterogeneous lipid membranes (Figure S1). It is presumably due to the small volumes of liquid droplets and their hydrophobic surfaces with a lipid monolayer. Printed vesicle suspensions formed a spherical droplet and shrank within a corral (Figure S1 left) or flowed out of the corral, covering the surrounding polymeric bilayer region (Figure S1 right). In both cases, liquid droplets rapidly dehydrated and resulted in heterogeneous debris of lipid. In the “two-step-printing method”, we first printed aqueous solution selectively onto the corrals. Positioning of the droplets with respect to the corrals was done relatively easily (after the initial trial and error for alignment), because printed aqueous droplets preferentially covered the corrals, even if the original position was slightly shifted from the center of the corral (Figure 2(A)). It is presumably due to slightly higher hydrophilicity of the corrals (glass) compared with polymeric lipid bilayer. As we printed vesicle suspensions (typically 1h after printing the aqueous solution), the liquid droplets slightly extended but retained their locations and shapes (Figure 2(B)). We could observe fluorescence from vesicles in the droplets (Figure 2(B)). The presence of agarose and trehalose in the pre-printed solution was important for preventing dehydration of liquid droplets and stabilizing them (vide infra). We finally removed excess aqueous solution and vesicles by extensively rinsing the substrate surface with MilliQ water. The droplets were removed and homogeneous SPBs were exposed (Figure 2(C)).


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We confirmed the connectivity and fluidity of printed SPB patches qualitatively by partially photobleaching the embedded fluorophore (TR-PE) and observing the recovery of fluorescence (FRAP) (Figure 3). The boundary between bleached and non-bleached regions blurred with time, suggesting the lateral diffusion of lipid molecules. We noted that lipid molecules slightly remained in the polymeric bilayer regions at the boundary, presmably from the region covered with the droplets (indicated with an arrow in Figure 3A). Extensive rinsing could not remove this “lipid ring”. It is likely that these lipid rings were lipid bilayers that permeated into the defects of polymeric bilayers. Our previous investigation showed that the surface coverage by polymerized lipid bilayer could not reach 100% even with an optimized UV polymerization conditions.

12

Therefore, it is plausible that some defects persisted and lipid molecules were incorporated. Lipid molecules in the rim were immobile, as judged from the FRAP experiment, in agreement with our previous observations that embedded lipid molecules are immobile above the threshold coverage of polymeric bilayers due to the percolation by polymeric bilayer domains.

12

Since the amount of lipids that are distributed at the

rim was considerably smaller compared with that in the corral, we suppose that they should not pose serious complications for the technical applications. We optimized the amount of printed vesicle suspensions by changing the ejection time from the nozzle to ensure that a homogeneous SPB is formed in the corral. As we increased the ejection time of lipid, more vesicles were printed and the fluorescence intensities of residual lipid layers after rinsing increased, finally reaching a plateau value (Figure S2). Further increase of printed lipid did not change the amount of incorporated lipid, clearly suggesting that the coverage of corrals with a single lipid bilayer.


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Incorporation of agarose and trehalose was important in the two-step-printing method because of their roles as stabilizer of small liquid droplets against mechanical agitation and dehydration. If we printed the first aqueous solution without agarose, liquid droplets drifted horizontally upon printing lipid vesicles (Figure S3). Presumably, the viscosity of droplets on the substrate was not high enough to resist the movement of the stage during the second printing. In the absence of agarose, printed aqueous droplets in the first step were also more heterogeneous with sprayed small droplets surrounding the corral (Figure S3(A)). On the other hand, we could not print aqueous solutions without trehalose because the nozzle tip was quickly clogged. Trehalose is widely used for its high water retention capabilities.

29

Therefore, trehalose should have helped to prevent

drying and clogging of the nozzle tip. At the same time, trehalose is also known for its stabilization effects on protein conformations because of its extensive hydrogen bonding both to protein and water molecules.

30

Therefore, we assume that trehalose should

potentially have a positive effect on printed bio-molecules in terms of preserving their functions. 31

The attempt to deposite aqueous solution containing agarose and trehalose onto the corrals by spin-coating for bypassing the first printing step failed. The aqueous layer covered the whole surface without any selectivity toward corrals. Printing vesicle suspensions resulted in heterogeneous and irregular spreading of the liquid, causing uncontrolled coverage of the corrals with membranes (Figure S4). This result is in contrast with the report by Kaufmann et al., 20 where they could dispense vesicles onto the substrate through a homogeneous water film. The different behaviors observed in


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the present study should stem from the higher viscosity of the agarose-containing water film. Two-step-printing of agarose/ trehalose and vesicle droplets on glass substrate without polymeric bilayer also did not work (Figure S5). Aqueous droplets containing agarose and trehalose spread out on glass substrate due to the hydrophilic surface, preventing the formation of well defined spots. Although we could print vesicle suspensions onto the spread liquid films, the formed lipid patches after rinsing had irregular shapes. The presence of polymeric bilayer matrix is therefore important for confining the droplets in a well defined region.

For demonstrating the capability to generate SPB microarrays, we printed three types of lipid bilayers having different fluorophores (NBD-PE, TRITC-PE, TR-PE) into the neighboring corrals with a size of 117 µm and displacement of 300 µm (center to center). Figure 4 shows the microscopy observation of printed droplets and SPBs. In Figure 4(A), four corrals in the left were filled with aqueous solution and two corrals in the right remained empty. The slightly unclear appearance of the edges in the filled corrals is due to the optical effects of round-shaped aqueous droplets. Subsequently, three types lipid membranes were introduced into the corrals (Figure 4(B) and (C)). The fluorescence observation clearly showed that different types of SPB were formed in the neighbor corrals. The upper right corral remained void of lipid membrane, since we did not introduce vesicles in the second printing step. As a more biologically relevant example of multiple printed membranes, we printed two types of POPC bilayers containing either GM1 or biotin-PE into neighbor corrals (Figure 5). As a mixture of fluorescently labeled proteins, cholera toxin subunit B (CTB488) and streptavidin (SAF594), was introduced, two proteins specifically recognized GM1 and biotin,


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respectively, and bound selectively to the corrals that contained the binding partner. These results clearly demonstrate that we can construct microarrays of model biological membrane by introducing different types of lipid membranes into individual corrals. The membrane microarray enables to detect multiplex binding events of molecules by molecular recognition.


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4. Conclusions We developed a methodology to generate a microarray of multiple lipid membranes on a solid substrate by utilizing pre-patterned polymeric lipid bilayers and the inkjet printing technology. The intrinsic technical difficulties in printing lipids (hydrophobic surface of droplets and rapid dehydration) have been circumvented by the two-step-printing approach, in which aqueous droplets were pre-printed into the corrals before printing lipid membranes. The use of agarose and trehalose was found to be critical because of their roles to stabilize the printed solutions. Micropatterned polymeric bilayer also played an important role for confining the solutions in pre-defined areas. One may ask whether we could replace the polymeric bilayer scaffold with other materials such as pre-patterned proteins or photoresist. We should point out that some unique features of polymeric bilayers were actually playing important roles in the present printing strategy. First, polymeric bilayers are resistant toward nonspecific adsorption of lipid membranes and proteins. It would be difficult to confine the printed membranes without suppression of nonspecific binding in the surrounding regions. Second, polymeric bilayers are stable in air. Currently, the printing is done in an ambient condition and some protein-based coatings may lose its surface properties upon dehydration. Basically, an air stable material with a resistance toward non-specific binding (e.g. hydrophilic polymer brushes 32) may be a possible alternative of polymeric bilayers. However, there is further advantage of polymeric bilayers that they have the same bilayer structure as the introduced natural lipid membranes. We previously observed that incorporation of lipid membranes into the corrals was enhanced by the presence of pre-formed polymeric bilayers, because their hydrophobic edges acted as a catalyst for the vesicle destabilization and rupture.

33

We suppose that the presence of


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polymeric bilayers can stabilize embedded fluid bilaeyrs by the structural compatibility at the junction. This unique feature is especially important if we are constructing much smaller microarrays of membranes. It was observed that fluid lipid membranes can be much more easily incorporated if the distance between polymeric bilayer edges (i.e. the size of corrals) was smaller. 12,33 Although the technical difficulties to align the positions of patterned membrane and printed spots currently prevent us from obtaining smaller patterns, we expect that it should be possible to create smaller microarrays of integrated model membranes with the present printing approach by improving the instrumental precisions of the spot alignment. Such microarrays should provide valuable tools in a wide range of settings spanning from the basic research of membrane protein functions to biomedical applications such as drug-candidate screening.


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Acknowledgements We thank Mr. Takashi Irie (National Institute of Advanced Industrial Science and Technology (AIST)) for producing photomasks. This work was supported by Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).


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Figures:

Nozzle Second step: Vesicle suspension

First step: Aqueous solution

Rinse

Aqueous solution

Substrate

Figure 1: Schematic illustration of the two-step-printing method: In the first step, aqueous solution containing trehalose and agarose is printed into the polymer-free area (corrals). In the second step, vesicle suspensions are printed into the same droplets to induce vesicle fusion in the corrals. After rinsing with MilliQ water, SPBs remained in the corrals.


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Bright field

Fluorescence

(A)

(B)

(C)

100μm

Figure 2: Bright field and fluorescence microscopic observation of SPBs printed by the two-step-printing method: Polymeric and incorporated lipid bilayers were observed with green and red fluorescence, respectively. (A) After printing agarose/ trehalose solution (trehalose0.1%, agarose0.06%, glycerol 10%). (B) After printing vesicle suspenstions (POPC10mM/ DHPC5mM/ TR-PE 1% (mol/mol)) together with glycerol (5%). (C) After rinsing excess vesicles with MilliQ water. The liquid droplets were exposed to air in (A) and (B), whereas the patterned bilayer was immersed in water in (C).


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(A)

(B)

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(C)

Figure3: Lateral mobility of printed lipid membranes assessed by FRAP: Before (A), immediately after (B), and 4 min after (C) photobleaching. The arrow in (A) indicates the “lipid ring” that surrounded the corral. The scale bar is 50µm


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Bright field

Fluorescence

(A)

(B)

(C)

100μm

Figure4: Deposition of multiple types fluorescent vesicles: POPC vesicles having three different fluorophores (NBD-PE, TRITC-PE, and TR-PE) were printed into three adjacent corrals. (A) Printed agarose/ trehalose solution (trehalose0.1%, agarose0.06%, glycerol 10%). Four corrals in the left are filled with the solution, and two corrals in the right remain empty. (B) Three types of vesicle suspensions (POPC10mM/ DHPC5mM/ fluorescent dye 1% (mol/mol)) were printed into different corrals together with glycerol (5%). The vesicles contained either NBD-PE (upper left), TRITC-PE (lower left), TR-PE (lower right). The upper right corral was kept void of lipids. (C) After rinsing excess vesicles with MilliQ water. The left and right images were observed by the bright field and fluorescence microscopy, respectively. The fluorescence observation of printed lipids are shown with false colors, NBD-PE in green; TRITC-PE in blue; and TR-PE in red. (Due to the spectral overlapping of TRITC-PE and TR-PE, the corral with TR-PE looks slightly violet.)


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Green fluorescence Red fluorescence (CTB488) (SAF594)

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Merged image

Figure5: Selective adsorption of CTB488 and SAF594 onto lipid bilayer patches containing either GM1 or biotin-PE. POPC vesicles having GM1 or biotin-PE (2 mol%) were deposited into neighbor corrals (left: GM1, right: biotin-PE). After rinsing excess vesicles with MilliQ water, a solution containing both CTB488 and SAF594 was added. Fluorescence micrographs were taken after rinsing the protein solution. The size of corrals was 117 µm.


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ToC graphic:

Two-step-printing Nozzle Aqueous solution

Vesicle Rinse

Polymeric bilayer

Printed fluid bilayers


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Microarrays of Phospholipid Bilayers Generated by Inkjet Printing

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