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Thin Solid Films 516 (2

Patterning parameters for biomolecules microarrays constructed withnanosecond and femtosecond UV lasers

V. Dinca a,b,⁎, M. Farsari a, D. Kafetzopoulos c, A. Popescu d, M. Dinescu b, C. Fotakis a,e

a Institute of Electronic Structure and Laser, Foundation for Research and Technology, Vassilica Vouton, P.O. Box 1527, 71110, Heraklion, Greeceb National Institute for Laser, Plasma and Radiation Physics, Atomistilor Str. 409, P.O. Box 16, 077125, Magurele, Bucharest, Romania Bucharest, Romania

c Institute of Molecular Biology and Biotechnology, FORTH, Vassilika Vouton, P.O. Box 1385, 711 10, Heraklion, Crete, Greeced Department of Biophysics, Faculty of Physics, University of Bucharest, Fizicienilor Str. 1, P.O. Box 11, Magurele, Bucuresti, Romania

e Department of Physics, University of Crete, P.O Box 2208, GR-71003,Greece

Received 8 May 2007; received in revised form 15 January 2008; accepted 26 February 2008Available online 5 March 2008

Abstract

The development of laser techniques for transferring biomolecules on solid surfaces in a controlledmanner has attracted much attention during thelast few years. Laser-induced forward transfer (LIFT) is a direct-write laser technique employed in printingmicron-sized patterns or features of a widerange of materials in solid or liquid state, in ambient atmosphere and temperature or in vacuum. In this work, we use LIFT with nanosecond andfemtosecond UV lasers for patterning the proteins Biotin, Avidin and Titin. In addition to the laser parameters, we also investigate the influence of thesolution viscosity and the receiving surface wettability on the formed patterns. To confirm pattern functionality after transfer, we employ the strategyof patterning a coupling molecule with LIFT, immobilizing an active complementary protein on it and performing a fluorescence microscopy assayafterwards. Our results show that both nanosecond and femtosecond lasers are suitable tools for obtaining micron size protein patterns, withfemtosecond laser pulses requiring a lower energy for transfer. We also show that the smallest patterns are formed just above the threshold energy forthe thinnest target and that the pattern size increases with increasing surface wettability and decreases with solution viscosity.© 2008 Elsevier B.V. All rights reserved.

Keywords: Nanosecond and femtosecond pulsed laser; LIFT; Protein; Micron size patterning

1. Introduction

Immobilizing biomolecules such as oligonucleotides, DNAand proteins in specific predetermined positions on solid surfaceshas attracted intense attention over the last years due to the broadfield of their applications in cell biology [1,2], biosensortechnology [3], drug discovery [4] and tissue engineering [5,6].However, there are still major issues concerning proteinmalfunction after patterning, as proteins easily lose their activitydue to denaturising, dehydration or oxidation [7]. To this end, avariety of methods have been developed for biomoleculespatterning: from simple dispensing or soaking techniques tolocalized contact and non-contact patterning methods.

⁎ Corresponding author. Institute of Electronic Structure and Laser, Founda-tion for Research and Technology, Vassilica Vouton, P.O. Box 1527, 71110,Heraklion, Greece. Tel.: +40 21 4574414; fax: +40 21 4574467.

E-mail address: dinca@iesl.forth.gr (V. Dinca).

0040-6090/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.tsf.2008.02.043

When using dispensing or soaking techniques, the biologicalmolecules are applied in solution onto their binding or adsorbingsubstrates [8]. In contact methods, for generating high densitiesof biomolecules spots on either porous materials or solids, pinsor capillary dispensers are necessary [9]. However, because ofthe liquid nature of the samples and the differences between thepins, the spot size obtained with these methods is variable andthe substrate may be damaged due to the pin impact [10]. Amongthe contact methods, dot-blotting is unsuitable for deposition onnon-porous substrates and cannot produce dots smaller than1 mm [11]. In addition, robotic micro-needle array fabrication[12] is a costly and time-consuming technique that impliesadditional danger of cross-contamination during tip cleaning[13].

Alternative methods require relatively expensive photolitho-graphic and/or micro-fluidic processing equipment. In photo-lithography, there are biocompatibility and simultaneity issues[14,15].Micro-contact printing is a promising technology for the

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parallel and rapid imprinting of hundreds of spots of bio-reagents. However there are the disadvantages of contaminationand non-controllability of the patterning process [16]. Non-contact printing methods such as the “ink-jet” and “drop-on-demand” approaches have also been used for spotting biologicalliquid samples on different substrates [17,18]. The ink-jet printertechnique [17], however, needs precise pressure control for eachnozzle to achieve uniform deposition during the high-speedprinting. In addition, the printing apparatus may be verycomplex for accommodating many different types of proteins.Moreover, during printing each of the protein samples needsto be ejected out from its own chamber to prevent cross-contamination. In general, these non-contacts methods areinefficient since they effectively deposit only a small fraction ofthe total sample loaded in the dispensing device and are limitedby the viscoelastic properties of the samples [18].

More recently, laser-based non-contact transfer methods suchas Matrix-Assisted Pulsed Laser Evaporation Direct Write(MAPLE DW) [19–22], Laser Induced Forward TransferArrangement (AFA-LIFT) [23–25] and Laser Induced ForwardTransfer (LIFT) [26–29] have been used for specific depositionof different types of biomolecules. The transfer principle issimilar for all these methods: the laser beam is focused onto thethin film containing the biomolecules embedded or not in amatrix to be transferred through its transparent support. Witheach laser pulse, a small quantity of the thin film material isexpelled onto a substrate which is usually placed parallel andfacing the thin film at a short distance. A pattern of thetransferred biomolecules can be ‘written’ on different substrateswith single or multiple shots.

For example, mesoscopic patterns of viable Escherichiacoli and other type of cells have been transferred byMAPLE DW with nanosecond (ns) laser pulses [21,22].Controlled transfer of Trichoderma longibrachiatum conidiaand different types of living cells [23–25] have also beenobtained by modified AFA-LIFT using a ns KrF excimerlaser.

A study of femtosecond (fs) laser-induced forward transferof biomolecules was presented by Zergioti et al. [26] wherethe biomaterials were deposited on a substrate without theassistance of any transferring matrix material or absorbingfilm and from a dried target. Based on the same techniquebut using water as an intermediate layer between targets andreceiving surface, micro-patterning of proteins by femtose-cond laser-induced shockwave was studied recently [27].

Further studies aimed at understanding the LIFT transferprocesses by means of nanosecond laser pulses were carried outby Serra et al. using a liquid target and the assistance of anabsorbing film [28–30]. However, the studies regardingbiomolecules transfer using a liquid target were carried outusing mostly ns laser pulses and there are a lack of studiesinvestigating the transfer parameters by both fs and ns lasers. Inthis work we used both fs and ns excimer lasers to pattern Biotin,Avidin and Titin to predetermined specific positions on solidsurfaces and we compare the parameters involved for the twolaser pulse lengths. Because of the liquid form of the target, wehave constructed an inverse LIFT set-up, where the target sits at

the bottom and the receiving substrate at the top; this helps avoidcontamination of the receiving surface. The aim of this studywasto investigate the controlled positioning of proteins on solidsubstrates by understanding how the UV ns and fs laserparameters affect the transfer process; also to examine theinfluence of the target composition, target-substrate distance andreceiving surface topography on the pattern size and shape.

2. Experimental details

2.1. Materials sample preparation

The proteins used were Biotin (1 mg/ml, Sigma Aldrich),Avidin (100 μg/ml, Sigma Aldrich) and Titin I-band fragmentHuman Recombinant protein (100 μg/ml, Sigma Aldrich),dissolved in a mixture of PBS (Phosphate Buffer Saline) andglycerol (0%–60%). The solutions were spread on a 10 nmthick gold film covered quartz plates previously sterilized for1 h in UV (Fluoarc, UV lamp). For the preparation of films withthickness between 1 and 20 μm, volumes of 2 μl up to 60 μlsolutions were used.

Ordinary glass microscope slides (Waldemar Knittel) andnitrocellulose-coated glass slides (commercial FAST slides,Schleicher & Schuell) were used as substrates. The microscopeslides were cleaned using sonication and immersion in a Piranhasolution as following:

- Sonication: acetone for 15 min, rinsing with double distilledwater, sonication in 70% ethanol solution for 15 min, rinsingwith double distilled water. The surfaces were dried justbefore use to avoid any contamination.

- Piranha solution: a 3:1 mixture of sulphuric acid andhydrogen peroxide was used to remove any possible organicresidues. The slides were dipped in this solution for 1 h.Subsequently they were rinsed with double distilled waterand dried just before use in order to avoid contamination.

2.2. Laser-induced forward transfer experimental set-up

A schematic diagram of the excimer laser transfer set-upis depicted in Fig. 1. Single pulses from a KrF laser(248 nm, excimer Lambda EMG150 ET Laser System 15 nsand distributed-feedback dye laser-based femtosecond exci-mer laser system, 500 fs respectively, 1 Hz repetition rate)were used to carry out the ns and fs LIFT experiments.

The centre of the laser beam output illuminated a variable andadjustable aperture in order to give an approximately uniformprofile; afterwards it was imaged onto the lower side of the targetinterface using a 25× microscope objective (N. A=0.4),resulting in spot diameters between 5 μm by 5 μm to 50 μmby 50 μm on the film surface, as estimated by the laser-induceddamage area on the gold coating film.

During the deposition process the target area was viewedthrough the CCD camera. The transferred material wascollected on a substrate, which was placed in close proximity(20–1000 μm) to and parallel to the donor surface. xyztranslation stages allowed precise control of the translation of

Fig. 1. Schematic diagram of the optical set-up for the laser transfer.

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the donor–receptor system with respect to the laser beam. Allexperiments were performed in air at room temperature.

An inverted optical microscope Leica (Germany) equippedwith high magnification objectives (10×, 20×, 50× and 100×)and a digital camera (Unibrain Fire-I 1.2104407) were usedto analyze the features of the deposited patterns.

2.3. Contact angle measurements

Contact angle measurements were performed to measure thewettability of the substrates. Using a Material InterfaceAssociates Inc. automated tension meter a collection of digitalimages of the sessile drops using a 2 μl glycerol, protein andPBS mixture was acquired. The wettability of the samples wasstudied before and after cleaning for each type of substrateused.

2.4. Perfusion chamber experiments: fluorescence assay

The functionality of the deposited patterns was confirmed byperforming fluorescence microscopy assays in a perfusionchamber. The perfusion chamber was built from a cleanedCorning cover glass and the patterned cover glass separated by adouble sided tape spacer. The chamber was pre-coated with aBSA (Bovine Serum Albumine) blocking solution (1–2% BSAin PBS) for 1 to 5 min.

After BSA blocking, the samples containing patterns ofBiotin were incubated with Avidin solution (100 μg/ml) for40 min, washed with double distilled water and incubatedwith 633-Atto Biotin solution (100 μg/ml). The samplescontaining patterns of Avidin were incubated with Biotin(100 μg/ml) for 40 min; washed and then labeled with Atto-565 Streptavidine (Fluka) solution (100 μg/ml).

The chambers containing patterns of Titin were subsequentlyincubated with Monoclonal Anti Titin antibody solution (mouseIgG2b, Sigma Aldrich) at room temperature for 45 min. After theincubation, any unbound molecules were washed away with PBS

and the samples were further incubated with the Cy 5-Goat Anti-Mouse IgG Conjugate antibody (Zymed Laboratories, InvitrogenImmunodetection) in the dark, at room temperature for 45 min.

After incubation with the fluorescence labeling solution, allthe samples were rinsed with PBS and distilled water.Fluorescence was detected using a Zeiss fluorescence micro-scope equipped with a Laser Scanning System Radiance 2100(400–700 nm) and with a Carl Zeiss Axio Camera HR.

3. Results and discussions

Below we present our investigation on LIFT transferparameters and their effect on the functionality of the patternedbiomolecules.

3.1. Effect of pulse length

The effect of the laser pulse length on the transferred patternswas studied for both ns and fs laser systems.

As shown in Fig. 2A and B, well-defined patterns of aglycerol-buffer solution on glass were obtained. With one laserpulse, a small quantity from the viscous solution was transferredto the receiving glass substrate. It can be seen that for the samethickness of the target (3 μm), the droplet size obtained with thens laser pulse was larger than that for the fs laser pulse. This canbe explained by the low angular divergence characteristics of thefs pulse transfer [26,31–32]. The value for the incident fluencewas 0.1 J cm− 2.

In order to systematically investigate the effect of the laserpulse length on the functionality of the transferred pattern, thefollowing strategy was followed: a coupling molecule wasfirstly patterned with LIFT and subsequently an activecomplementary protein immobilized on it. For this purpose,Biotin and Avidin patterns were transferred on nitrocellulose-coated glass slides and the effect of pulse duration on proteinsfunctionality after transfer was studied. Those slides were usedto form flow cell chambers as previously described.

Fig. 2. (A) Optical microscope images of glycerol and PBS patterns on glass surfaces obtained using fs (A) and a ns (B) laser system.

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The flow cells containing patterns of Avidin were incubatedwith a solution containing Biotin and subsequently labeled withfluorescent Avidin. As shown in Fig. 3A (Avidin patternobtained by fs laser pulses, 0.66 J cm− 2), and 3B (Avidin patternobtained with ns laser pulses, 0.37 J cm− 2), the patterns werespecifically labeled, proving protein functionality after transfer.

Fig. 3. Fluorescent microscope images of proteins pattern obtained using fs and nssubsequently labeled Avidin. (C) fs-transferred Biotin incubated with Avidin and lalabeled Biotin. (E) fs- and (F) ns-transferred Titin on nitrocellulose after incubation

Transferred Biotin was incubated with a solution contain-ing Avidin and subsequently labeled with fluorescent Biotin.As shown in Fig. 3C (0.64 J cm− 2), the patterns were visiblylocalized to specific areas proving protein functionality and nosplashing was noticed. In addition, Biotin was patterneddirectly on the nitrocellulose surface and subsequently

laser systems. (A) fs- and (B) ns-transferred Avidin incubated with Biotin andbeled Biotin (D) ns-patterned Avidin on nitrocellulose surface incubated withwith the first and secondary labelled antibody.

Fig. 4. Droplet diameter dependence on the energy for fs and ns lasers. Thetarget contains glycerol and PBS solution (40:60 in volume).

Fig. 5. Droplet diameter dependence on the target-receiving substrates distance.Thickness of the glycerol/PBS target: 3 μm.

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incubated with labeled Biotin (Fig. 3D, patterns were obtainedwith ns laser pulses, 0.27 J cm− 2). As shown by thefluorescence assay, both transferred proteins (Biotin andAvidin) maintain their functionality when thereafter subjectto lock-key ligand-recognition reaction. In control experi-ments, when only a solution of buffer and glycerol wasprinted, there was no fluorescence observed.

Titin was also transferred on nitrocellulose-coated slides andafter incubation with the first and secondary labeled antibody,the fluorescence signal was observed. As shown in Fig. 3E (forpatterns obtained using ns laser pulses 0.27 J cm− 2) and Fig. 3F(for patterns obtained using fs laser pulses, 0.20 J cm− 2), thetransferred patterns remained active after transfer.

The results of the imunoassays presented in Fig. 3(A–E)show that the biomolecules maintain activity for differentvalues of the fluencies used. Moreover, it is possible to controlthat there are no splashes around the transferred patternsconfirming a localized transfer. As mentioned earlier thedifference in size of the transferred pattern can be seen bycomparing the fluorescence spots from the two figures (forexample Fig. 3E–F).

3.2. Effect of energy

In addition to the effect of laser pulse length, the effect ofenergy on the patterns shape and size was also studied. For thisexperiment, a solution consisting in PBS and glycerol (40:60 involume) was used. It was found that there is a threshold for thepulse energy, below which there is no material transferred; thisthreshold is significantly higher for the ns pulse case (1.34 μJ)compared to the fs pulse (0.9 μJ). The graphic in Fig. 4illustrates the effect of the transfer energy and the size of thedroplets deposited on the solid surface. It is noticed in bothcases that the increase of the laser pulse energies correspond toan increase of the droplet diameter.

The corresponding fluences have values between 0.1 and0.8 J cm− 2 for fs laser pulse and 0.1 to 0.5 J cm− 2 for the nslaser pulse regime.

The mechanism for protein transfer with respect to the laser-material interaction can be described as follows.

First, the incident laser energy interacts with the absorptiongold layer on the target. At low intensities, the processes relateto a simple excitation in the layer, with low thermal effects andonly minor perturbation of the target [29]. For sufficient laserenergies the absorption layer acts as an energy conversionmaterial. In the case of ns pulses, as energy density increases,the absorption depth increases resulting in a larger interactionvolume and increased heat conduction to the surroundingmaterial. Even at low energy densities, radiative coupling ofenergy from the ablation plasma may induce thermal effects,depending on the sensitivity of the material. This explains ourcase, when the liquid that is in contact with the metallic layer isvaporized and expelled onto the substrate.

In the case of fs pulses, the heat-affected zone is minimal, soseveral of the side effects characteristics [33–34] of longerpulses are reduced or minimized and material transfer occurswell after the pulse [34]. The 500 fs laser pulse duration is lessthan the relaxation time of the target material [26,32] and thetransfer at the focal point will be initiated by the mechanicalforce due to the shockwave propagation towards the air and theviscous thin film, the cavitation bubble generation and collapse,and the jet formation. In this case, electronically excited andionized states of the molecules are generated and their energy isquickly distributed to vibrationally excited states of themolecules. Due to the molecular motions in the confinedspace, temperature elevation as well as pressure elevation isinduced. The pressure will propagate as a shockwave, followedby a pressure decrease. This, combined with the temperatureincrease, leads to explosive vaporization and generation ofcavitation bubbles. As these cavitation bubbles collapse, theycause the forming of a jet flow of the liquid [33], which leads to avery fast removal of the proteins thin film and its subsequentdeposition onto the substrate. A possible explanation is that, atfluence values only slightly above the ablation threshold, theablation plume consists of liquid droplets and jets that emergefrom randomly distributed sites within the irradiated surface areawhile at higher fluencies, jets tend to become more numerousand mix [27,33]. This explains our observation: the size and theshape of the transferred pattern depend on the energy; for low

Fig. 6. (A) Threshold energy dependence on the thickness of the film; (B)droplet diameter dependence on the thickness of the film.

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energy, close to the threshold energy, the transfer is uniformwithno splashes. When increasing the energy, the shape becomesirregular, with splashes. When the speed of the expelled materialis smaller, the impact with the substrate is not as strong and

Fig. 7. Droplet-size variations on the bioassay chip surfaces on the nitrocellulose-coaoptical microscope (C)–(D).

therefore the liquid solution can be transferred as uniformdroplets.

Also, for the fs lasers, the negligible loss due to thermaldiffusion enables processing at much lower energies than for thens lasers. Most importantly, the high intensities attained canresult in highly efficient multiphoton processes with wellreduced optical penetration depth [35,36]; such processes enablepattern transfer without using any absorbing gold layer [26].

3.3. Effect of distance

The effect of the distance between the target and the substrateand how this affects the transfer process has been investigatedfor both laser systems. A target consisting of PBS and glycerol(50:50 in volume) with a thickness of 3 μm was used. Thedistance was varied between 20 μm and 1 mm and the diameterof the transferred droplets was measured using opticalmicroscopy. The laser fluence was kept at 0.4 J cm− 2.

The results are shown in Fig. 5. For the fs laser, the distanceup to which uniform transfer was achieved, was 0.5 mm, whilein the ns case, the transfer occurred even for distances higherthan 1 mm and no significant change in the sizes of thetransferred droplets with distance is noticed.

3.4. Effect of target film thickness

The effect of the target film thickness was investigated forboth ns and fs LIFT transfer systems. The target film thicknesswas varied between 1 and 20 μm and the diameters of thetransferred droplets were measured using the optical micro-scope. The graphic in Fig. 6A illustrates the dependence of theenergy required for transfer and the thickness of the film.

In both cases the increase of the laser pulse energycorresponded to an increase of the film thickness and the

ted surface (A) and glass surface (B) with different wettabilities observed from

Table 1Smallest spot sizes transferred for the two types of substrates used in this study:glass slides and nitrocellulose slides

Laser pulseregime

Smallest spot transferredon ordinary glass (μm)

Smallest spot transferred onnitrocellulose-coated glass (μm)

500 fs 10 1215 ns 20 22

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transferred droplet diameter increases linearly with filmthickness (Fig. 6B).

Fluence was kept at a value of 0.1 J cm− 2.Miniaturization of the transferred samples is preferable towork

with thinner films as the droplet diameter is smaller and it requiressmaller values for the transfer energy. The sharper increase of thethreshold energy, observed for the ns pulses than the one observedfor the fs pulses is of considerable importance for further proteintransfer processes as the minimal thermal effects induced by thelower threshold energy required for the transfer will be translatedinto less damage on the protein structure [37–39].

3.5. Effect of substrate wettability and solution viscosity ondroplet size

The protein patterning depends not only on the laser transferparameters, but also on substrate wettability as well as on thesubstrate surface topography [38]. We have investigated proteinimmobilization onto two different substrates: nitrocellulose-coated glass slides (FAST slides), which provide a three-dimensional binding area, and normal glass slides which havebeen used as control substrates. The glass slides were sterilizedand cleaned as described earlier.

Our results show that on glass surfaces, which are morehydrophobic than nitrocellulose surfaces, the transferreddroplets are up to 3 μm smaller. This is shown by the contactangle measurements; the contact angle is 18.7° in the case ofnitrocellulose-coated glass and 22.0° for glass slides sterilizedby sonication (Fig. 7A for nitrocellulose; and 7B for sterilizedglass).

Fig. 7C and D reveal the differences in the pattern features;patterns were obtained for the nitrocellulose and for the glasscontrol surface using fs laser pulse; and the same transferconditions (1 Hz, 0.4 J cm− 2). The difference in the droplet sizewas noticed from simple optical micrographs. By changing the

Fig. 8. Droplet-size dependence (filled symbols for the femtosecond regime andhollow symbols for the nanosecond regime) on surface wettability (FAST slides,glass slides after Piranha cleaning, glass slides after sonication cleaning, glassslides) and percentage of glycerol in solution (black square symbol for 10%glycerol, red circle symbol for 20% glycerol, triangle symbol for 40% glyceroland pentagon symbol for 60% glycerol). (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of thisarticle.)

other transfer parameters, the difference between the dropletstransferred on the two surfaces is maintained.

This leads to the conclusion that for obtaining smallerpatterns, hydrophobic surfaces must be used. However, thereare several issues regarding the use of hydrophobic substrates asthere is the need for an intermediate step for biomoleculesimmobilization. The advantage of using nitrocellulose coatingin our case is that it offers much higher binding capacity andbetter spot-to-spot consistency than hydrophobic surfaces [39].Also, no immobilization after transfer is required as the liquid isretained between the nitrocellulose fibers.

In addition to surface tension, solution viscosity alsoinfluences the spot size. To investigate this effect, PBS solutionswere mixed with glycerol in different concentrations (0–60%)to produce solutions with different viscosities. The resultingsolutions were transferred onto FAST and glass slides with bothns and fs laser pulses and the results are shown in Fig. 8.

As shown, smaller droplets are generated when solutions ofhigher viscosity are used. The spot size decreases with theincrease of viscosity and increases with surface wettability.Also, there are differences in the spot size transferred to theglass surface before and after the cleaning procedure. Theminimum droplet sizes obtained are presented in Table 1 for afluence of 0.3 J cm− 2 and for a percentage of glycerol of 60%.

4. Conclusions

Transfer of proteins on biocompatible materials wasperformed using Laser Induced Forward Transfer with both nsand fs laser pulses. The functionality of the immobilizedproteins was confirmed by the immobilization of activecomplementary ligands in a lock-key reaction.

Comparison between the patterns obtained using ns and fslasers reveals that smaller patterns are obtained for fs laser.Although the threshold energy is lower for the fs laser pulsetransfer, both laser pulse regimes can be used for the patterningof the proteins. The main advantage of using the ns laser systemis that the distance for transfer is longer than in the case of the fslaser system. However, the size of the transferred pattern can becontrolled by the dimensions of the laser beam, energy,thickness, glycerol percentage in the composition of the targetand surface wettability. The addition of high percentages ofglycerol prevents the drying of the protein solution, thuspreventing the denaturation of the proteins.

These results combined with the fact that regular transfer ispossible for distances higher than few μm and differentpercentages of glycerol can be added without the risk ofcontamination or clogging demonstrate the flexibility of theLIFT method for biomolecules patterning.

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Acknowledgment

This work forms part of a research program funded by EUMarie Curie Fellowship Program: Advanced Training in LaserSciences (MEST-CT-2004-008048), in IESL-FORTH, Herak-lion, Crete, Greece.

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