Supported lipid bilayer microarrays created by

non-contact printing

Stefan Kaufmann� Jens Sobek† Marcus Textor‡

Erik Reimhult§

April 10, 2011

Abstract

Arrays of supported lipid bilayers (SLBs) provide great potential for future

drug development and multiplexed biological research, but are difficult to prepare

due to sensitivity of both lipid and protein structural arrangement to air exposure.

A novel way to produce arrays of SLBs is presented based on non-contact dis-

pensing of vesicles to a glass slide through a thin surface confined water film. The

approach presents many degrees of freedom since it is not limited to a specific

substrate, lipid composition, linker or controlled environment. The method allows

adjustment of spot size (180 µm - 360 µm) by repeated dispensing as well as control

over the composition of the spots and subsequent analytes. SLB formation by vesi-

cle adsorption and rupture allows for incorporation of membrane proteins through

pre-formed proteoliposomes. Dispensing through a dip-and-rinse water film avoids

contamination, disruptive drying and need for complex buffer compositions. Fur-

thermore, no humidity control is necessary which simplifies the production step

and prolongs the life-time of the spotting system. We characterize the method with

respect to control over spot size, bilayer mobility and the formation process as well

�Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Switzerland†Functional Genomics Center Zurich, Switzerland‡Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Switzerland§Department of Nanobiotechnology University of Natural Resources and Life Vienna, Vienna, Austria

E-mail: erik.reimhult@boku.ac.at

1

as demonstrate the possibility to fuse bilayer spots with subsequently added vesi-

cles. Since complex lipid compositions and multiple spotting nozzles can be used,

this novel technique is expected to be a promising platform for future applications,

e.g. patterning to monitor peptide/protein-lipid interactions, for glycomics using

glycolipids or lipopolysaccharides, and to study mixing of spatially confined lipid

membranes.

1 Introduction

Patterning techniques are increasingly important for biological research, as for in-

stance for microarrays for genetics,1 glycomics2 and proteomics.3 Different tech-

niques are available to produce patterns in the micro- or even nanometre range, as

e.g. photolithography, soft lithography and spotting.4 Flexibility, low-cost and high-

throughput provided by these methods are desirable aspects of microarray production.

However, patterning techniques and delicate biomolecules are often incompatible since

organic solvents and drying steps are frequently part of the processing. As an exam-

ple, cell membranes, which structurally comprise a lipid bilayer, are stabilized by hy-

drophilic/hydrophobic interactions which are destabilized by drying steps. Mimics of

native cell membranes, such as supported lipid bilayers (SLBs), are particularly suit-

able to study processes of biological relevance as for instance peptide/protein-lipid

interactions in an array format, but are equally susceptible to destruction by solvents

and drying steps.5,6

Techniques compatible with patterning of delicate SLBs are therefore necessary

and several techniques have already been developed to produce micro- and nanometer-

sized SLBs.7–10 However, these methods to produce arrays require prefabricated sub-

strates,7,9 complex or laborious procedures,10,11 accurate climate control (� 98 % hu-

midity),12 specialised printing solutions9 or engineered lipid linkers.13,14

Common ways to produce SLBs are vesicle fusion, Langmuir-Blodgett deposition,

painting or solvent spreading. Advantages of vesicle fusion are the simplicity and ver-

satility of the method and the possibility to integrate functional components into vesi-

cles and therefore to transport and incorporate these components into the SLB while

2

avoiding solvent residues in the membrane. SLB formation by vesicle fusion proceeds

by initial adsorption of vesicles on the substrate until a critical surface coverage is

reached. The adsorbed vesicles fuse and rupture and thereby induce a transition from

vesicles to a SLB15,16 as shown in Figure 1 (a).

Ideally, a patterning method is based on vesicle solutions that form SLBs under

normal conditions on a chosen surface without changing the standard buffer solution;

it can produce micropatterns that are spatially separated and addressable; and it allows

to choose different functionalities at different locations for multiplexed readouts. Fur-

thermore, it should be feasible to produce such platforms in a high-throughput manner.

A common method for molecular patterning by dispensing solutions or suspensions is

the use of robotic array printers, which roughly can be divided into contact and non-

contact printers. Non-contact printers have the advantages of high reproducibility and

absence of undesirable mechanical contact with the clean target surface.

In this paper, we present a detailed investigation of array printing with a non-contact

dispensing system to produce SLB spots in the micrometer range. A straightforward

printing of SLB arrays on dry substrates is not possible since evaporation of the spotted

solution dehydrates SLBs and results in delamination and lipid redistribution. Attempts

to use specialized spotting buffers that reduce these drying effects instead tends to

severely affect the biomolecular self-assembly.14,17 The presented method provides the

advantage that vesicles or SLBs never leave their natural liquid environment and that

neither a high-humidity environment nor specialised printing solutions or substrates are

necessary to perform the printing. The method is applicable to all membrane solutions

that can be tailored to form SLBs on the described timescale on hydrophilic substrates.

2 Results and discussion

2.1 Spotting through a thin buffer film

The problem of evaporation, limited time for SLB formation and edge effects of droplets

usually found when vesicle solution is spotted onto a hydrophilic substrate in an envi-

ronment without humidity control was circumvented by protecting the spotted vesicle

3

solution by pre-coating the target substrate with a thin buffer film which prevents fast

evaporation of the spotted solution.

1) Prewetting 2) Dispensing 4) Rinsing3) Incubation

Formation of a SLB by vesicle fusion

5) Adding buffer and sealing

Figure 1: Steps involved in the formation of SLB microarrays: 1) Pre-wetting of thesubstrate. 2) Dispensing of vesicle solution through the water film resulting in vesicleadsorption, rupture and fusion to a homogeneous and functional SLB. 3) Incubationin a high humidity chamber for a specified time. 4) Buffer rinse to remove residualvesicles. Optional: 5) Adding buffer to maintain the liquid environment of the SLBand if necessary sealing of the liquid chamber for storage.

Microarrays of SLBs were produced on commercially available glass slides, mounted

in a home-built microscopy cell (round, diameter 21 mm) or equipped with a seal-

ing frame (rectangular, 19 mm � 60 mm). The complete process involved five steps:

pre-wetting, dispensing, incubation, rinsing and adding liquid/sealing (Figure 1). In

the pre-wetting step the clean, hydrophilic glass slide was wetted by adding a precise

amount of buffer with a pipette. By tilting the substrate back and forth, a thin, homo-

geneous water film was formed on the glass slide. The added buffer volume defines the

thickness of the water film and hence assures that in the time period of the dispensing

process the substrate is not dehydrated due to evaporation.

After the pre-wetting step, the solution containing vesicles at a concentration of

1 mg/ml was dispensed with a non-contact piezo dispenser onto the glass slide. The

number of droplets was varied from 1 droplet to 20 droplets in 5-droplet steps. Sub-

sequently, the glass slide was placed in a humid environment chamber to prevent the

dehydration of the water film and to let it rest for a specified incubation time. The

rinsing was performed by introducing the slides into a tube with subsequent addition

4

of 50 ml of ultrapure water. This resulted in a fast rinsing with a copious amount of

water, which would not have been be the case using a pipet or a spray bottle. After the

rinsing step buffer was added to the glass slide to maintain the aqueous environment of

the SLB when needed for time-consuming characterization, storage or transportation.

A second glass slide was added to seal the liquid over the sample.

In our approach of spotting through a thin buffer film different parameters com-

pared to conventional spotting onto a hydrophilic substrate play an important role as

for instance the thickness of the buffer film, the incubation time, the diffusion of vesi-

cles in the thin water film and the rinsing procedure. The influence of these parameters

will be described below.

2.1.1 Buffer film thickness

The thickness of the water film was calculated from the area that was hydrated with

buffer solution assuming the formation of a homogeneous water film. A minimal buffer

film thickness of 90 µm was necessary to assure that the substrate was kept wet for the

whole dispensing process of approximately 30 - 60 s. A thickness of � 90 - 180 µm

(corresponding to 100 - 200 µl on substrates of dimensions 19 mm � 60 mm) resulted in

the formation of functional SLBs whereas a thickness of 270 µm only resulted in a low

fraction of adsorbed vesicles. The latter observation was likely due to the dispensed

droplet not having sufficient time to fully penetrate through the too thick water film to

the substrate surface. The resulting lower concentration close to the substrate surface

combined with dilution through diffusion led to insufficient vesicle adsorption. The

low but sufficient thickness of 90 µm was kept constant for further measurements. See

Supplementary Information for images of spots dispensed at buffer film thickness of

90, 180 and 270 µm.

2.1.2 Spot quality

Spots were judged to be of good quality if (a) the fluorescence showed homogeneity

over the whole spot; (b) the spot was circular and showed a well-defined edge region

(i.e. no blurred transitions from high homogeneous fluorescence to background); (c)

5

the lipids were laterally mobile as expected for a SLB.18

Apparent factors that influence the spot quality are the cleanliness and quality

(roughness, scratches, composition) of the substrate, the rinsing step and the trajectory

of the droplets when leaving the nozzle. We found that dispensing multiple droplets per

spot could result in deviations from a circular spot shape, especially for higher number

of repetitions, e.g. 20 droplets, because a slight deviation in the trajectory could result

in a non-circular spot shape. The lipid mobility was confirmed by FRAP measure-

ments demonstrating a level of lipid mobility (1.5�0.2 µm2~s and a recovered fraction

of 97�2 %) comparable to that found for homogeneous SLBs prepared by incubating

the whole substrate with the vesicle solution (1.2�0.2 µm2~s and a recovered fraction

of 93� 3 %). For sufficiently high lipid concentration and incubation time (see sec-

tion 2.1.3) recovery was observed for the entire spot, demonstrating that lipids were

mobile also at the edge of the well-defined spots.

The rinsing step has to be handled with care to assure that no tails develop due

to convectively transported and adsorbed vesicles. Insufficiently fast rinsing even

with sufficient volume can result in artifacts in the rinsing direction on the free glass

substrate (see Supplementary Information). Fast and cautious rinsing with a copious

amount of buffer ensures removal of unbound vesicles.

After spotting the SLB array can be sealed with a second glass slide which allows

to store functional SLB arrays at 4 XC for several days without observing any change

in either spot size or morphology. A detailed investigation on the long-term stability of

the spots was however beyond the scope of this report.

2.1.3 Time evolution and formation process of SLB spots

In the spotting process a high concentration of vesicles is locally deposited and there-

fore it is important to determine the influence of vesicle diffusion in the liquid film

during the formation process.

Figure 2 (a) shows microscopy images of adsorbed fluorescent lipids from one dis-

pensed droplet (1 mg/ml, 400 pl droplet volume) incubated for 10 seconds, 2, 3, 10,

20 and 30 minutes (upper row), and compares the resulting spot sizes to the size that

6

10 s 2 min 3 min 10 min 20 min 30 min

2 800 000 2 300 000 2 000 000 500 000 200 0Nvesicles

(a)

200

150

100

50

0

R (mm

)

30252015105t (min)

(b)

3 min

0.5 mg/ml

20 min

1 mg/ml

2 mg/ml

3 min 20 min

3 min 20 min

(c)

Figure 2: (a): Measured and calculated spot profiles of spots with one dispensed droplet(1 mg/ml, 400 pl droplet volume) after 10 seconds and 2, 3, 10, 20 and 30 minutes ofincubation time. The remaining number of free vesicles after a specified incubationtime is indicated below each column. The last calculated spot profile (after 30 min ofincubation) includes a dashed circle presenting the theoretic spot size based on the totallipid mass. Scale bar: 200 µm. (b): Spot radii after 2, 3, 10, 20 and 30 minutes of incu-bation time with average value and standard deviation of A 10 spots. Symbols describespots with experimentally measured 1� dispensed droplet (blue j) and theoreticallydiffusion predicted 1� dispensed droplet (black X) as in figure 2 (a). Lines betweenthe measuring points are added for clarity. (c): Concentration dependent spot sizes for5� dispensed droplets after 3 and 20 minutes incubation time with 100 µm buffer filmthickness. The concentrations are 0.5, 1 and 2 mg/ml. The microscopy images showspots after FRAP where the arrows indicate the position of the bleached rectangularregion. Scale bar: 200 µm.

7

would be expected due to diffusion of vesicles for spots with one dispensed droplet

calculated with a diffusion and adsorption model described in the Supplementary In-

formation (lower row). The numbers of free vesicles in solution is indicated underneath

each figure and the red dotted circle after 30 minutes incubation describes the expected

spot size accounting for the total lipid mass that was dispensed.

In Figure 2 (b) the time evolution of the average experimental spot radius for one

dispensed droplet with standard deviation calculated from A 10 spots is shown. Ex-

cluding the first measurement after 10 seconds, no significant increase in spot size is

observed from the microscopy images in Figure 2 (a) or from the average spot radii in

Figure 2 (b).

The theoretically calculated spots grow in size due to diffusion and reach the final

size after approximately 20 minutes when all vesicles have adsorbed. In contrast, the

experimentally measured spot size shows an initial growth but then it reaches a constant

maximum size after 2 - 3 minutes. The first measured spot after 10 seconds represents

an accumulation of mostly intact adsorbed vesicles where the SLB formation was inter-

rupted by the early rinsing step, as was demonstrated by FRAP measurements yielding

only partial fluorescence recovery in the center of the spot (see Supplementary Infor-

mation). On the macroscopic scale the formation kinetics of POPC vesicles on SiO2

has been shown to be on the order of 1 min for 1 mg/ml vesicle concentration, increas-

ing to several minutes at 0.1 mg/ml.19 An incubation time of 10 seconds therefore is

hardly enough time to form a complete SLB and the data are therefore not included

in the plot in Figure 2 (b) which shows that within the error of the measurements no

increase in the spot size is observed after the initial growth with incubation time.

Another parameter which is important in relation to the SLB formation kinetics is

the vesicle concentration. The SLB formation therefore was explored with different

vesicle concentrations of 0.5 mg/ml, 1 mg/ml and 2 mg/ml. Figure 2 (c) shows spots

after 3 and 20 min incubation for spots with 5� dispensed droplets.

The concentration dependence of the spot build-up shown in Figure 2 (c) indicates

that the lower the concentration is, the slower the SLB formation proceeds, but also

concentration has almost no influence on spot size. After 3 minutes incubation of the

8

5� dispensed 0.5 mg/ml droplet only a small fluorescent spot is visible with practically

no SLB formed, as demonstrated by the lack of fluorescence recovery in the bleached

rectangular area. Incubation for 20 min leads to saturation of the spot size, but only the

center of the spot shows the fluorescence recovery indicative of a SLB. The surround-

ing ring without recovery likely is a boundary region of adsorbed intact vesicles which

never reached sufficient local surface coverage to initiate SLB formation.19 These re-

sults also demonstrate that within the impact zone of the droplet the vesicles rupture

and SLB formation starts in the center and proceeds radially outwards. The 1 mg/ml

and 2 mg/ml 5� dispensed droplets show similar size, but do not have diffuse sur-

rounding rings. This indicates that concentrations of 1 - 2 mg/ml form a SLB over the

entire spot and for the 0.5 mg/ml droplets the vesicle concentration is diluted and leads

to a corresponding slow-down of the SLB formation process. Also at the sufficient

concentration for SLB formation across the entire spot a mostly very thin boundary

region containing lipid material which does not recover after bleaching is visible. This

boundary region is likely a mixture of planar membrane islands and vesicles,19 which

is further discussed in Section 2.1.6 and quantified in the Supplimentary Information.

2.1.4 Spot size dependence on dispensing of multiple droplets

The total amount of lipid material deposited to the surface can be increased by either

increasing the vesicle concentration or by repeated dispensing of droplets to the same

spot. Increasing the vesicle concentration led to no significant increase in spot size as

described in Figure 2 (c). In contrast to increasing the vesicle concentration, spotting

of multiple droplets to the same spot increased the spot radius as shown in Figure 3.

The data in Figure 3 further emphasize the finding described above that the spot size is

close to independent on the incubation time and that this holds regardless of number of

dispensed droplets per spot. The absolute sizes of the experimental spots are, however,

also much smaller than predicted based on the total amount of lipid material1. In fact,

the spot size does not increase far beyond the expected impact zone if the convection

1The predicted radius of the spot was calculated based on the total amount of lipid: r �½

VdropletcsolutionρSLBπ

with Vdroplet � 400 pl, csolution=1 mg/ml, ρSLB=4.2 ng/mm2 20.

9

7 min7 min

0 min 0 min

1x 5x

7 min

0 min

10x

8 min

0 min

15x

10 min

0 min

20x(a)

800

600

400

200

0

R (mm

)

1 5 10 15 20# of Drops

(b)

Figure 3: (a) FRAP series of spots with 1, 5, 10, 15, 20 dispensed droplets per spot areshown. (Dispensing: 3 minutes incubation.) Scale bar: 100 µm. (b) Radius of spotsR versus number of dispensed droplets for expected radius R based on total amount oflipid material (�), dehydrated spots (j), 3 min incubation (R) and 20 min incubation(n).

10

(pushing liquid volume in the impact zone outwards) induced by multiple droplet dis-

pensing is taken into account. This extended size of the dispensed liquid in the impact

zone can be estimated by the size of a spot treated by multiple dispensed droplets on

a dry glass slide (see Figure 3 (b) j and Supplementary Information Figure S7). The

FRAP images in Figure 3 confirm that for 5�, 10�, 15� and 20� repetition the entire

fluorescently labeled spot is identified as a SLB.

The initial vesicle distribution, more or less defined by the impact zone and spread-

ing of the droplets, contributes to the SLB spot size regardless of the total amount of

spotted lipid material. Therefore, it has to be concluded that lipids are lost during the

process and not available for surface adsorption and SLB formation. The smaller than

expected spot sizes cannot be explained by a lack of incubation time or diffusion of

lipid material. The probable process for the observed loss of lipids is presented below.

2.1.5 Loss of lipids in the dispensing process

As described, the spot size only weakly depends on the amount of deposited lipid

material, which can only be explained by a large and rapid loss of lipids which never

adsorb to the substrate. The most likely process for this loss is adsorption of lipids at

the air-buffer interface, from which they can no longer adsorb to the solid substrate.

Since the air-buffer interface above the deposited lipids provides a large reservoir for

lipid adsorption where they experience zero surface pressure, the sticking coefficient

of lipids at this interface can be regarded as 1. The buffer film thickness is on the

order of the spot radius or smaller and hence a vesicle statistically reaches the air-

buffer interface before reaching the spot edge to adsorb and expand the SLB spot. This

indicates that lipid loss at the air-buffer interface could significantly influence the SLB

formation process. As a result of this loss a concentration depletion zone develops

from the interface which reduces the time for vesicles adsorption to the glass substrate

to the time scale for growth of the depletion zone through the buffer film. A depletion

zone also evolves at the substrate surface since adsorbing vesicles also deplete the

solution in proximity to this interface. Adsorbed vesicles on the glass substrate however

remain immobile on the surface and block the area to further adsorption. For a detailed

11

discussion on the depletion of vesicles including a model for and calculation of the

relevant time scales see the Supplementary Information.

We emphasize that diffusion will put an upper limit to the time needed to form a

depletion zone extending from the air-buffer interface to the substrate since convective

transport due to evaporation of water at the air-buffer interface is likely to significantly

increase the speed with which it forms. Lipid loss at the air-buffer interface is thus

suggested to be the main mechanism for why the observed spot sizes are determined

by the initial distribution of vesicles defined by the impact of the droplets and not by

the total amount of lipid material.

2.1.6 Membrane fusion

The border region surrounding the spots defines if further fusion processes can take

place or not. The possibility to fuse spots with additional deposited vesicles on the free

substrate surface, would allow to perform 2-D reactions at controlled diffusion rates,

e.g. to perform hybridization reactions for DNA tethered to the SLBs21. Furthermore,

backfilling allows to investigate the edge-area of the SLB spot to determine if it is well-

defined, i.e. consisting of SLBs bounded by a thin region of intact vesicles or undefined

lipid agglomerates. The important question is whether the edge allows fusion of a SLB

or whether lipid agglomerates exist that prevents further fusion. Mager and Melosh 11

showed that for SLB deposition via air bubble collapse a gap region is formed con-

sisting of disordered lipid assemblies on the substrate. This region prevented intermix-

ing with subsequently formed surrounding SLBs. Other patterning approaches, based

on pre-fabricated substrates7,9,12 or with specialised lipid compositions22–24 make in-

termixing of SLB spots impossible because of physical or chemical barriers that are

necessary for the patterning process. Methods that allow to study mixing are PDMS

stamping25, photolithographically structured polymers that can be peeled off26 and

dip-pen nanolithography27. Compared to these methods, spotting has advantages, such

as no risk of contamination, reproducibility, low cost, a single processing step and that

multiple lipid mixtures that can be used.

Vesicles with a different fluorescent label, Rhod-PE, were added to a SLB microar-

12

0 min 8.5 min 16.5 min 60.5 min

(a)

160

140

120

100

80

60

40

20

I (a.

u.)

6040200t (min)

8.5

min

16.5

min

60.5

min

(b)

Figure 4: A NBD-PC labeled, deposited SLB spot fuses with an additionally formedSLB which is labeled with Rhod-PE lipids. (a) Microscopy images of the spot withNBD-PC labels (green, upper row) and the lipids with Rhod-PE adsorbing throughvesicles added to the bulk (red, lower row). Scale bar: 100 µm. (b) Intensity at theoriginal spot area versus the mixing time with green X: NBD-PC label and red j: Rhod-PE label.

13

ray in buffer. Figure 4 (a) shows a time series of microscope frames where the first

row displays NBD-PC labeled lipids from the spot and the lower row Rhodamine la-

beled vesicles from the bulk solution. The first column shows the spot before adding

the Rhodamine vesicles. In the second column, after addition of Rhod-PE containing

vesicles, the NBD-PC labeled spot develops fuzzy edges and in the Rhodamine chan-

nel a ring surrounding the NBD-PC spot is visible. In the third column the width of the

additional lipid ring has increased and the NBD-PC labeled spot has further decreased

in size. In the last column the NBD-PC labeled spot has disappeared and the surface

is homogeneously covered with a fluid Rhodamine labeled SLB. Figure 4 (b) shows

the intensity change in the region of the original NBD-PC SLB spot as a function of

time. The decrease in the NBD-PC fluorescence signal is perfectly correlated with an

increase of the signal of the Rhodamine-labeled lipids. This demonstrates mixing of

the lipids in the array spot with the subsequently formed surrounding SLB. (See Sup-

plementary Information for two movies showing the detailed image sequence of the

NBD-PC and the Rhod-PE channel.)

The mixing of Rhodamine with NBD-PC labeled lipids demonstrates that SLB

spots formed by dispensing through a thin buffer film do not possess a border area that

prevents further fusion of vesicles. Indeed, the FRAP data presented in Figure 3 and

Figure S1 in the Supplementary Information support the view that the spot is indeed

a SLB and fuses completely and homogeneously with subsequently added vesicles.

This does not exclude the likely presence of a thin region of intact vesicles at the spot

boundary (see Supplementary Information for an estimation), but it demonstrates that

with the described method patterns of homogeneous and uniform SLB microspots can

be produced which possess no chemical or topographical barriers. This is in contrast to

most reported patterning approaches7,9 which need a barrier to restrict SLBs to patterns

and arrays on a substrate.

We further highlight that the adsorption of vesicles at the edge of the SLB seems

to be more favorable than on the bare surface as evidenced by the formation of a ring

of higher Rhodamine fluorescence compared to the background further away from the

original SLB spot. A similar observation was published by Weirich et al. 28 . They

14

stated that vesicles have a greater affinity for SLB edges than for the glass substrate

using temperature-controlled time-resolved fluorescence microscopy. Possible reasons

for such an increased affinity were however not discussed. We base a tentative expla-

nation on local differences in vesicle concentrations due to the presence of the SLB

which does not allow adsorption of vesicles. This would result in locally higher vesicle

concentration close to the surface of the spot due to the vesicles which could not adsorb

to the surface as they could on surrounding free areas. Therefore no depletion zone is

built-up over the SLB and vesicles can diffuse laterally to the edge of the spot where

they get the chance to adsorb and thereby increase the number of vesicles at the edge.

It should also be emphasized that vesicles approaching the edge likely fuse to a SLB

directly rather than first adsorb as vesicles19.

2.1.7 Flexibility

The presented method is not limited to a certain lipid species or surface. In principle,

all vesicle compositions that allow dispensing with a non-contact dispenser can be used

for SLB array creation. However, as implemented here, lipid compositions are required

that form SLBs within the time frame of the survival of the buffer layer. This latter

time requirement can be completely relaxed by putting the sample in a high humidity

environment immediately after spotting. We could e.g. demonstrate spotting of SLBs

from notoriously slow-forming PEG-lipid containing vesicles29,30 as detailed in the

Supplementary Information. Dispensing is only limited for very viscous solutions.

The flexibility of non-contact dispensing allows the formation of user-defined spots

with freely chosen lipids. With multiple nozzles several different lipid solutions can be

dispensed in parallel to produce arrays at a high rate. With a frequency of 500 Hz,

dispensing of 20 droplets takes 40 milliseconds and a microscopy slide of 17�5 spots

is patterned in less than 30 seconds with a single nozzle. Furthermore, the accuracy

in positioning provides the potential to add protein solutions to a pre-fabricated SLB

array with specific SLB spots to screen for protein functions. This procedure is also

compatible with most biomolecules, for instance delicate proteins, since no drying

steps are involved.

15

Further, the proposed method is generally applicable for patterning of surface active

molecules. It avoids evaporation and corresponding redistributing flows in the small

droplet volume which aggregate material31,32. The unique way of defining the spot

size and creating a homogeneous molecular layer on the surface within the spot build-

up would therefore be very advantageous for other biomolecules, as non-homogeneous

biomolecule density and functionality in microarray spots is a common problem33.

2.2 Conclusion

We described and characterized non-contact dispensing of vesicle solution through

a thin water film for the production of SLB microarrays. Fully fluid SLB spots are

formed which have well defined boundaries and without lipid agglomerates preventing

lipid diffusion as proven by FRAP measurements for recommended optimal spotting

conditions of 90 - 180 µm buffer film thickness and 1 - 2 mg/ml vesicle concentra-

tion. With this method the initial vesicle distribution in the thin water layer defined

by the initial impact distribution of the dispensed droplets defines the spot size and the

lipid concentration in the dispensed volume determines the spot formation kinetics. A

mechanism based on interfacial lipid loss was described to explain this observation. It

was shown that repeated dispensing instead can be used to increase the spot size in a

controlled manner. This spotting method is unique in that fluid SLB microarrays could

be made without a major edge density gradient, aggregation, chemical or topographical

barrier, as demonstrated by complete fusion of array spots with a subsequently formed

surrounding SLB. Thus arbitrary micron-sized patterns of continuous SLBs are possi-

ble to create through repeated dispensing, including with multiple lipid mixtures using

multiple nozzles.

The proposed method is applicable to membrane protein assays since the risk of

dehydrating the lipid bilayer or its associated fragile molecules can be prevented as the

membrane never leaves the liquid environment. In such storage membrane spots were

shown to be stable over at least several days. Furthermore the technique provides a

high degree of flexibility since non-contact dispensing does not require pre-fabricated

or patterned substrates (although such can be used in combination) and can be used in

16

principle with all lipid compositions that form SLBs on a desired substrate in a wide

choice of applicable buffer conditions.

3 Experimental

3.1 Materials

POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), NBD-PC (1-palmiotyl-2-

[12[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycerol-3-phosphocholine)

and Rhod-PE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine

B sulfonyl) were purchased from Avanti Polar lipids (Alabaster, AL, USA). Tris buffered

saline (TBS) composed of 10 mM Tris(hydroxymethyl)-aminomethan (Tris) (Sigma-

Aldrich Buchs, Switzerland), 150 mM NaCl (Sigma-Aldrich Buchs, Switzerland) dis-

solved in ultrapure water (Millipore, USA) adjusted to pH 7.4 with HCl was used

for all spotting and SLB formation. Glass substrates (Nexterion microarray substrates

borosilicate glass, Glass D (D263T) and Glass B, 24 mm � 24 mm � 0.17 mm and

25 mm � 75.6 mm � 1 mm) were bought from Schott (Germany) and were rinsed

with ethanol (Scharlau, Spain) and MilliQ water before use and treated in a UV/ozone

cleaner (Bioforce Nanosciences, IA, USA) for 30 min.

3.2 Vesicle preparation

POPC lipids dissolved in chloroform were mixed at the desired mole fraction (3 mol%

NBD-PC and 1 mol% Rhod-PE were added as dyes) and dried under a stream of ni-

trogen for 1 hour. The dried films were resuspended in TBS and extruded 31 times

through two stacked polycarbonate membranes (Avestin, Ottawa, Canada) with a pore

size of 100 nm using a Liposofast extruder (Avestin, Ottawa, Canada) to obtain unil-

amellar vesicles. Vesicle size distribution was determined to be 107�6 nm by dynamic

light scattering (Malvern Nano-ZS, Malvern Instruments, UK).

17

3.3 Microscopy

Microscopy measurements and FRAP measurement were performed on a Zeiss LSM510

confocal laser scanning microscope (Carl Zeiss AG, Germany) equipped with a 25-

mW Argon laser (line used: 488 nm) and a Plan-Neofluar 10x/0.3 objective or a Plan-

Apochromat 63x/1.4 Oil DIC objective (FRAP measurements, bleach circle with 25 µm

diameter on spots with sizes of 180 µm - 360 µm.). The diffusion coefficients were

determined using MATLAB (The Math Works) and the method described by Jönsson

et al. 34 . For high-throughput evaluation of multiple spots a laser scanner LS400 from

Tecan was used (Tecan, Austria) with a resolution of 4 µm per pixel. The brightness of

the microscopy images was slightly adjusted for the figures with Corel Draw X3 (Corel

Coperation, Canada) when needed.

3.4 Spotting

For liquid dispensing, a piezoelectric microarray spotter was used (sciFLEXARRAYER

S11, Scienion, Berlin, Germany). The nozzle ejected droplets of a constant volume of

400 pL with a frequency of 500 Hz. Droplet speed was according to specifications ap-

proximately 2 m/s. The nozzle to surface distance was set to 500 µm. Before spotting,

droplet quality and stability of droplet ejection was checked using a charge-coupled

device (CCD) camera. Spotting conditions were kept constant throughout the experi-

ments.

The glass slides were mounted in a home-built microscopy cell (round, diameter

21 mm) or equipped with a sealing frame (rectangular, 19 mm � 60 mm). The surfaces

were pre-wetted by adding a precise amount of buffer with a pipet to the clean, hy-

drophilic glass slide. A homogeneous water film was obtained by tilting the substrate

back and forth. The solution containing vesicles, if not otherwise stated at a concen-

tration of 1 mg/ml, was dispensed with the non-contact piezo dispenser onto the glass

slide. The glass slide with the dispensed vesicle solution was placed in a humid en-

vironment chamber to prevent the dehydration of the water film and to let it rest for a

specified incubation time. The rinsing was performed by introducing the slides into a

18

tube with subsequent pouring of 50 ml of ultrapure water. The substrates were rinsed

with TBS and in the case of the 25 mm � 75.6 mm � 1 mm glass slides a second, clean

glass slide was used to seal the aqueous chamber.

3.5 Calculation of spot sizes

For the calculation of the spot sizes, including the influence of diffusion, a procedure

in IGOR Pro 6 (WaveMetrics, USA) was written. A vesicle radius of 50 nm, a droplet

volume of 400 pL and a droplet diameter of 90 µm, corresponding to the diameter

of the nozzle and camera images, were assumed throughout the calculations. With

D �kBT

6πη2rvesicleand η � 1006 � 10�6 Pa�s, T � 300 K and kB � 1.3806504 � 10�23 J~K a

diffusion coefficient of 2.18 � 10�12 m2~s was calculated. The assumption of a lipid

bilayer density of 4.2 ng~mm2 as published by Gizeli et al. 20 results in a total amount

of 3.03 � 106 vesicles per droplet.

The solution to the diffusion equation s�r,t� � 1º4πDt

e�r2

4Dt is used to calculate the

vesicle distribution. The range of r is divided into 100 nm sections (approximate size

of the adsorbing vesicles) and due to the rotationally symmetric problem the sections in

the 1D case merge into rings in 2D. The number of vesicles in every ring is calculated

via the normalized area of s�r� for every time point. The starting time t0 is chosen such

that 99.9 % of all vesicles are in the range from r = 0 - 45 µm (the size of the impacting

droplets), i.e.

45µm

R0

s�r,t0�dr

ª

R0

s�r,t�dr� 0.999. For further information on the definition of the

initial conditions for the calculation of the time evolution or for the time evolution of

spot sizes for multiple droplet dispensing see the Supplementary Information.

A ring is supposed to be full of vesicles if the area of the ring segment is completely

covered by the surface area of vesicles, i.e. when 2i+1 vesicles are in the ith ring. Rings

are filled every 1 ms with vesicles that are able to adsorb, i.e. vesicles that are within

100 nm from the surface. The 1 ms adsorption cycle was chosen as the time during

which the mean square displacement of a diffusing vesicle is approximately 100 nm

with D � 2.18 � 1.1 � 10�12 m2~s, i.e. a�100nm�2f � 4� 2.18 � 10�12 m2/s �10�3s. The

adsorbed vesicles reduce the total number of free vesicles left in the simulation. For the

19

adsorption two assumptions are made: (1) the thickness of the buffer film is taken into

account by only allowing a fraction of all vesicles to adsorb. Vesicles are assumed to be

homogeneously distributed within a ring in z-direction up to the buffer film thickness,

which is assumed to stay constant throughout the calculation. Hence the development

of a depletion zone close to the surface is neglected. Therefore a fraction fproximity �h0H

of all vesicles in the ring is able to adsorb during each adsorption cycle, where h0 is the

distance to the surface a vesicle has to approach to be able to adsorb (set as 100 nm)

and H is the height of the buffer film (set as 88 µm). (2) If a ring is almost full, free

sites for adsorption are reduced and a vesicle only has the possibility to adsorb if it

finds an empty adsorption site. To include this slow-down in the adsorption process an

additional factor is added which is fprobability �free vesicle sites in ith ring

total number of vesicle sites in the ith ring .

fprobability could slightly underestimate the calculated spot growth rate since the

presence of SLB spots seems to speed up vesicle adsorption and SLB growth and

fproximity, i.e. the assumption of no depletion zone, could overestimate the spot growth

rate.

4 Acknowledgement

The authors thank Peter Jönsson for the MATLAB code for the FRAP analysis, the

Functional Genomics Center Zurich (FGCZ) and the Light Microscopy Center (LMC)

at ETH Zurich. This work was supported by the Swiss Competence Center for Ma-

terials Research and Technology (CCMX) of the ETH Domain, Switzerland, National

Centres of Competence in Research (NCCR), Switzerland and EU-FP7-NMP-ASMENA.

20

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