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Self-partitioned Droplet Array on Laser-patterned Superhydrophilic Glass Surface for Wall-less Cell arrays Kerui Xu, Xiaopu Wang, Roseanne Marie Ford, and James P. Landers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03764 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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Analytical Chemistry

Wall-less cell arrays in self-partitioned droplets 308x262mm (96 x 96 DPI)

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For Submission to


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Self-partitioned Droplet Array on Laser-patterned Superhydrophilic Glass Surface for Wall-less Cell arrays Kerui Xu1, Xiaopu Wang2^, Roseanne M. Ford2 and James P. Landers1,3,4*

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Departments of 1Chemistry, 2Chemical Engineering, 3 Mechanical and Aerospace Engineering, and 4Pathology, University of Virginia, Charlottesville, VA 22904, USA

Keywords: superhydrophilic, glass, CO2 laser, self-patterning, droplet 15

*To whom correspondence is addressed:

^Current address:

Prof. James Landers Department of Chemistry University of Virginia McCormick Road P.O. Box 400319 Charlottesville, VA 22904 Ph 434-243-8658 Fx 434-243-8852 E-mail: [email protected]

School of Petroleum Engineering University of Petroleum Qingdao, 266580, China

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Abstract In this work, we report a novel method for the creation of superhydrophilic patterns on the surface of hydrophobically-coated glass through CO2 laser cleaning. This mask-free approach requires no photolithography for the print of the features and only a single-step surface pre5

treatment is needed. The laser-cleaned glass surface enables self-partitioning of liquid into droplet arrays with controllable, quantitative volumes. We further designed wall-less, cell arrays for the mapping of culturing conditions and demonstrated the potential of this droplet-arraying method.

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1. Introduction One of the most significant advances brought about by the evolution of Micro-Total Analysis Systems (µTAS) is miniaturizing the size of assays to the micro- and nano-liter scale, thus enabling thousands or even millions of chemical/biological assays that are separately controlled but simultaneously interrogatable in a single experiment. This has led to the increased

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analytical throughput and data acquisition in various fields of study, including single-cell phenotyping1–3 and genotyping4–7, drug screening8–11 and cell transfection studies12–15. Microdevices for high-throughput bioassays require arrays of spatially-resolved experimental sites, usually created by inkjet printing16–18, soft lithography19, subdivision in microchamber/wells20,21 or self-patterning22.

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Among these methods, inkjet printing is the fastest, most automated approach for generating microarrays with a range of desired chemical concentrations. However, in order to be printed by the inkjet properly, the solution’s surface tension and viscosity need to be adjusted by 2 ACS Paragon Plus Environment


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adding additive reagent prior to the printing. The surfactant based additive reagents might affect some of the biomacromolecules’ activities23. Besides, another concern is the risk of damage of the biomacromolecules by the shear forces when jet though the nozzle24. On the other hand, soft lithography has been proved capable of making chamber or well arrays25 which allow for high5

throughput bioassays

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or micro-well arraybased single cell analysis26. However, the molding

and curing of stamp structures are rather time-consuming and labor-intensive27. As an emerging arraying technique, surface tension-based self-patterning of liquid solutions has drawn increasing attention, primarily due to its simple, power-free and equipmentindependent nature22,28. 10

In an array where superhydrophilic patches are patterned on a

hydrophobic background, free-moving liquid (usually aqueous solutions) is retained in superhydrophilic regions and repulsed by the surrounding hydrophobic regions, which results the rapid self-partitioning of solution into an array of droplets defined by the array pattern of superhydrophilic patches.

Numerous methods on the fabrication of the superhydrophilic-

hydrophobic patterns have been reported, and these methods can be categorized into two types: 15

(1) differential surface treatment (UV, plasma, chemical deposition etc.) on a mask-mounted or molded substrate where the patterns are laid on the substrate by the features of the physical mask 7,29–33

or (2) direct printing of superhydrophlic substances onto a hydrophobic substrate34–37.

The mask or mold based patterning procedure requires additional cost and operations in the fabrication and alignment of the mask or mold and the fast change of the desired pattern in 20

prototyping stage becomes rather inconvenient. On the other hand, most of the direct patterning approaches require multiple layers of deposition of chemicals and their corresponding treatments. Both types of methods are associated with challenges in the promotion of the microarray-based applications due to insufficient flexibility and considerable complexity. 3 ACS Paragon Plus Environment

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In this work, we present a simple, mask-less approach for the fabrication of superhydrophilic-hydrophobic patterns using CO2 laser. CO2 laser patterned hydrophilic spots array against a hydrophobic background has been previously reported but on the surface of wax paper where the surface of paper substrate is exposed after removal of the wax coating38. 5

However in that work an additional step of surface treatment of the exposed paper after laser cleaning is required, in which the silica particles are deposited on the exposed paper surfaces to increase their hydrophilicity so that the exposed spots array can trap liquid droplets. Alternatively, by exploiting the ability of CO2 laser to clean the glass surfaces and facilitate the removal of organic contaminants

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, we demonstrate their effectiveness in directly

removing the hydrophobic coatings on a pre-dip-coated glass surface. Since the surface of glass, or silica, is already exposed by laser treatment, no post-laser surface modification is needed anymore and the cleaned surface of the glass already becomes superhydrophilic. If the glass is cleaned in a pattern of array, the sharp contrast of the wettability between the superhydrophilic laser-cleaned glass surface and untreated hydrophobic areas will enable the self-partitioning of

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aqueous liquid into an array of droplets.

Compared with paper substrate, another distinct

advantage of the glass substrate is that it is transparent, which allows fluorescent detection and measurement even if the droplet arrays are sandwiched between two surfaces. This advantage opened the door for long-term cell culturing in droplet arrays since the evaporation of the droplet can be suppressed by this sandwiched structure. We illustrate the utility of this rapid self20

patterning system with an array-based mapping of the culturing conditions for GFP expression in bacteria cells.



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2. Materials and methods Unless otherwise indicated, all chemicals are purchased from Sigma-Aldrich (Oakville, Ontario, Canada) 2.1 Preparation of hydrophobic glass surface 5

Commercial borosilicate cover glass (Fisher Scientific, Pittsburgh, PA) was treated by piranha solution [30% H2O2 (Avanter Performance Materials, Center Valley, PA) and 98% sulfuric acid (Fisher Scientific, Pittsburgh, PA)], freshly mixed upon use in the volume ratio of 3:7 for 30 min to remove any possible coating or contaminations. After thorough wash by diH2O the glass covers were air dried in the hood and stored in a desiccator. The cleaned glass

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surfaces were dip-coated in Sigmacote® and air dried in the hood. The coated glass was stored in a desiccator for future use. 2.2 Laser cleaning of glass The pattern to be cleaned out by laser was designed in a commercial graphic designing software, CorelDRAW 10.0 (Corel Corporation, Ottawa, Canada), and then printed by

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VersaLaser VLS 3.50 with 50W CO2 laser source (Universal Laser System Inc.) on the coated glass surface. The laser-cleaned glass surface was briefly purged by nitrogen flow and ready to be used. 2.3 Contact angle measurement The contact angles of the Sigmacote®-treated glass surfaces before and after the laser

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cleaning are taken by a Canon EOS Rebel T1i camera (Canon Inc., Tokyo, Japan). For the comparison of the recovery of the surface wettability, the piranha-cleaned, plasma-cleaned and


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laser-cleaned glass surfaces are stored in atmosphere and the contact angle on each surface is measured at the time point of the 0, 1, 2, 4, and 7th days after cleaning. For the study of the preservation of the superhydrophilicity, the laser-cleaned glass surface was stored in a sealed petri-dish (Fisher Scientific, Pittsburgh, PA) with soaked cotton to 5

create a saturated humidity under room temperature. The glass was taken out at the 0, 1, 2, 4, 7, 14 and 30th day after the laser cleaning for the measurement of the contact angles. 2.4 Examination of the surface texture of the glass The Sigmacote®-treated glass and laser-cleaned glass were examined by the Environmental Scanning Electron Microscope (ESEM) to reveal their surface textures. Images

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were obtained by FEI Quanta 650 Scanning Electron Microscope (FEI Company, Hillsboro, OR) 2.5 Determination of the volume of the droplets The volumes of the droplets were determined by the self-partitioning of a solution of 6 µm yellow-green fluorescent beads (Spherotech, Lake Forest, IL) with a known concentration. The beads in each self-patterned droplet is counted under Axio Scope.A1 fluorescence

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microscope (ZEISS, Jena, Germany) and the volume of the droplet was back-calculated using the beads concentration and the counted number in each droplet (detail of calculations is in Supporting Information, Section 1). 2.6 Design of the cell arrays and cell culture E. coli HCB1 (wildtype)40 were genetically tagged with green fluorescent protein (pGLO

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bacterial transformation kit, Bio-Rad, Hercules, CA). Culturing medium was prepared by adding 0.50 g tryptone (BactoTM, BD, Franklin Lakes, New Jersey), 0.25 g NaCl (Fisher Scientific,



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Pittsburgh, PA) into 50 mL diH2O followed by autoclaving. The frozen stock of GFP-encoded E. coli HCB1 was thawed at room temperature and a 100 µL aliquot of the stock added into the 50 mL of medium and suspended by vortexing. 10 µL of 100 mg/mL ampicillin and 1 mL of 100 mg/mL L-(+)-arabinose were added into HCB1’s growth medium to provide selective pressure 5

for plasmid-containing, fluorescing bacteria.

Figure 1. Assembly of dip-chip cell droplet array. (A) The conceptual illustration of the creation of cell array under various reagent concentrations. (B) The stepwise assembling of cell array facilitated with the manifold and aligner. (1) A glass manifold with PDMS spacer is inserted onto the PMMA aligner. The PDMS spacer on the manifold can reversibly bond to the glass covers and hold them in desired position and distance; (2) insert the dried reagent array onto the aligner facing up; (3) insert the cell droplet array onto the aligner facing the reagent array; (4) the glass covers are reversible bonded with the PDMS and the two-array assembly can be lift off from the aligner and ready for cell culture.

In the construction of the cell array an in-house constructed glass-PDMS manifold and PMMA aligner was used to facilitate the alignment of the arrays (Figure 1). Briefly, one surface


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of glass was prepared by CO2 laser to form an array with different spot sizes of 0.24, 0.48, 0.72, 0.96 and 1.2 mm in diameter, and total of 96 (12 by 8) spots were arrayed in a 12 by 18 mm area. 1mL of the solution of L-(+)-arabinose was added over the array, and then the excessive solution was absorbed away by a folded KimwipeTM tissue wiper (Kimberly-Clark, Dallas, TX). The L5

(+)-arabinose sampled array (reagent array) was dried and attached to the manifold by the reversible bonding between the back of the cover glass and the PDMS spacer. The bacterial suspension was patterned into an array of droplets on another surface of glass using the same procedure described above, except that the sizes of the spots are uniform with a diameter of 1.2mm and the cell-suspension droplet array was immediately combined face-

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to-face with the reagent array before the cell suspension got dried. The double-layered manifoldcover glass assembly was put in a sealed petri-dish with soaked cotton and cultured overnight in 30 °C. The array was then examined under Axio Scope.A1 fluorescence microscope (ZEISS, Jena, Germany) and the images were taken by a CCD camera. The fluorescent intensity of the droplet array was measured in RFU using ImageJ.

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3. Results and Discussion 3.1 Superhydrophilic-hydrophobic Patterning of Glass Converting the hydrophilic nature of a glass surface to one that is hydrophobic by ‘coating’ the exposed silanols is not a new concept. The functionalization of silanol groups with 20

some form of organosilane was seminal to the creation of reverse phase chromatography in the 1970’s41. It also was exploited to hydrophobically-coat the glass plates used for electrophoretic gel sequencing, a must to assure that, during the post-separation examination the thin sequencing 8 ACS Paragon Plus Environment


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gels could be removed intact for processing. Sigmacote® was a common glass ‘silanizing’ reagent used for this purpose, and by simply ‘dipping’ the glass plate in this reagent, the hydrophilic glass surface was converted to one that was hydrophobic. Figure 2 shows images of the Sigmacote® -treated surfaces before and after laser cleaning, showing the changes in both the 5

contact angles and the textures on the surface of glass. The uncleaned hydrophobic surface yields a contact angle >90° (Fig. 2A), while the contact angle of the laser-treated surface has been reduced to ~13° (Fig. 2B). The increased wettability of the surface might be attributed to either the laser (i) simply cleaning the surface via evaporation and decomposition of hydrophobic polymer39 or, alternatively, (ii) texturizing the glass surface in a manner that exposes more

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silanol groups

42,43

. The SEM images of the Sigmacote®-treated glass surface before and after

the laser cleaning are shown in Figure 2C and 2D, respectively. It is clear that the surface of the glass is well cleaned by the laser treatment, and most of the features attributed to the coating have been removed, which implies that the increased wettability is due to the simple cleaning of the glass surface. The power of the laser is carefully adjusted so that there is no detectable 15

damage to the glass, e.g., cracking or peeling, effects that have been reported when applying a CO2 laser to glass44. Takeda et al. demonstrated that the cleaned surface of glass will lose its hydrophilicity in open air as a result of the re-adsorption of atmospheric contaminants45. We compared the lasercleaned glass surface with those cleaned by the other two popular glass-cleaning methods: (i)

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plasma oxidation and (ii) treatment with ‘piranha’ solution. The effect on the contact angle over time was plotted in Figure 3A. In comparison with these two methods, cleaning by the CO2 laser is slightly less effective than plasma oxidation (implied by the higher initial contact angle), which may be explained by incomplete dissociation of coating residue from the ablated surface. 9 ACS Paragon Plus Environment

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The rate of the loss of the hydrophilicity on the laser-cleaned glass surface is similar to the other two methods’

Figure 2. The wettability of coated glass surfaces before and after laser cleaning and their SEM images. The contact angle of a droplet of water on the surface of Sigmacote®-treated glass (A) before and (B) after laser ablation, together with the SEM images of their surface textures, (C) and

Patel et al. reported that the wettability of cleaned glass could be preserved in a high 5

humidity environment 46. Figure 3B shows that storage of the laser-cleaned surface under high humidity conditions allows for the superhydrophilicity to be maintained with a corresponding contact angle 0.5mm) the number of beads in each droplet is large enough so the Poisson distribution

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can be approximated by normal distribution. But for the smallest spot (0.3mm), because the number of beads in each droplet is very small the counting of the beads still shows typical Possion distribution, with large number of sample size (767 droplets were examined) (detail of calculation is in Supporting Information, Section 1). The volumes of the droplets are, therefore, determined and plotted versus the diameter of laser-cleaned spots (Fig. 5B). The volume (V, in

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nL) of the droplet is dependent on the diameter of the laser-cleaned spot (d, in mm) (Fig. 5B), with a relationship of V=32.9d2.44. However, if all droplets are assumed to have the same 12 ACS Paragon Plus Environment


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geometry (spherical crown), the volume should be proportional to d3. The difference between the ideal and experimentally-determined parameter stems from a non-proportional increase in the droplet height as the base diameter increases. The effect of gravity on the liquid-air interface of the droplet ‘pulls down’ as it becomes larger, so less liquid is retained.

A

B

Figure 5. The volume determination of the self-partitioned droplets. (A) The distribution of bead counts per droplet in an array of 767 droplets partitioned into 300 μm superhydrophilic spots. (B) The relationship between the volumes of the droplets and the sizes of the laser-cleaned spots (n>10, the error bars denote the standard deviation of the mean). 5

3.3 Precise Alignment in Coupling Array Plates The volume-spot size relationship described earlier (Fig. 5B) suggests a simple and convenient method for sampling nanoliter-scale volumes of liquid reagents and, where reagent concentrations or other variables need to be evaluated, can be exploited for array-based high


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throughput bioassays. The basic procedure illustrated in Figure 1 has two laser-cleaned arrays dipped into different reagents/solutions for droplet loading with volumes specified by the hydrophilic spot sizes. One of the arrays is allowed to dry (e.g., the reagent array) prior to sandwiching, in a mirror image fashion, with an array plate containing droplets (e.g., cell 5

suspension); this fuses the dried reagents to the droplets in a spatially-controlled manner. The reason for the reagent array being dried is that it will not affect the total volume of droplet after combining with the cell suspension droplet array. In order for this to be achieved accurately without droplet-to-droplet contamination or crosstalk, the inter-droplet spacing and glass-glass distance needed to be optimized.

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As illustrated in Figure 6A, the glass-to-glass ‘contact’ distance is critical, as this determines when the droplets are in close enough proximity to have the top of a single droplet (cell suspension in this case) reach the surface loaded with reagent. Following droplet fusion, the glass-glass distance is narrowed to the point that the boundary of the sandwiched droplet is in intimate contact with the hydrophobic part of the surface. This ensures that the liquid-solid

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interfaces experience an inward surface tension – from a top view, this results in a regular roundshaped droplet. This distance, defined as the ‘full-coverage’ distance, is the point at which the liquid disk between the two surfaces of the glass fully covers the superhydrophilic area. Based on the volume and base area of the droplet (described in section 3.2), we calculated the minimum contact distance (the borderline between the red and the orange areas in

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Fig. 6B) and maximum full-coverage distance (the borderline between the orange and the yellow area in Fig.6B) at a given diameter of the base area. The desired glass-glass distance and the diameter of spot size were then determined using Figure 6B and adjusted by the PDMS film spacer. (detail of this process is in Supporting Information Section 2). 14 ACS Paragon Plus Environment


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A

B

Figure 6. Precise alignment for spatial coupling of droplet/reagents between two laser-patterned surfaces. (A) The illustrative definition of non-contact distances, contact distance and full-coverage distance. (B) The relationship between glass-glass distance and spot diameters.


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3.4 Cell culture As one of the most canonical model microbes in biology, Escherichia coli has been well studied, at least in part, because its genome could be manipulated robustly with current 5

molecular biology and bioengineering techniques.

A common approach for studying gene

expression, is the introduction of the green fluorescent protein (GFP) reporter gene to the bacterial genome, with the expression level of GFP quantitatively regulated by L-(+)-arabinose. To show proof of feasibility for the laser-patterned superhydrophilic arrays, we cultured a GFP reporter gene encoded E. coli strain (HCB1) 10

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that uses L-(+)-arabinose as the inducing

reagent for the expression of the encoded gene. The L-(+)-arabinose concentration-dependent induction profile was reported in the expressing of the encoded gene49. The E. coli cells are cultured in the droplet arrays with varying concentrations of L-(+)-arabinose and the fluorescence intensity of GFP is used as the indicator of the expression level. After overnight culture of the cell array in a sealed petri-dish there is no observable change in either the size or

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the shape of the droplets.

The E. coli array cultured under different L-(+)-arabinose

concentrations shows a gradient of fluorescence intensity (Figure 7) that is consistent with reported data49–51, showing a non-linear, exponential rising curve versus the logarithm of the arabinose concentration. The reason for this non-linear rising curve is the mechanism of the autocatalytic induction, in which the transporter of the inducing reagent is up-regulated by the 20

inducing reagent itself. This feedback causes the accumulation of the inducing reagent in the already-induced cells and an all-or-none induction in each cell52. So the whole population of the E. coli contains two sub-populations, the already induced and un-induced50.

As the

concentration of the arabinose increases, the fraction of the induced population rises, making the 16 ACS Paragon Plus Environment


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fluorescence of the droplet stronger. The agreement between the results shown here and the reported response further validates this method as a potential tool for quantitative array-based bioassay.

Figure 7. Array of E. coli cultured under different concentrations of L-(+)-arabinose. (A) The fluorescent images of a 12 by 8 array of E. coli at 6 different arabinose concentrations. (B) The relationship between the expression level of GFP (in RFU) and the concentrations of L-(+)-arabinose (n=16). (C) The expression level of GFP versus the spot size of arabinose droplet.

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4. Conclusions In summary, we developed a simple method for the fabrication of superhydrophilic-hydrophobic patterns on a pre-treated glass surface using CO2 laser cleaning. The method requires only two 17 ACS Paragon Plus Environment

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simple steps: (1) dip-coating of glass into a silanizing agent (hydrophobic coating), and (2) direct writing of the desired pattern by laser cleaning of the hydrophobic coating, without any masking, lithography or molding procedures. The pre-coated cover glass in (1) can be conveniently prepared in large quantities and stored for future use upon request, so that the creation of the 5

hydrophilic patterns actually only requires one single step of the laser cleaning in several minutes without any required post-treatment/fabrication steps, which is much shorter than all the previously reported hours-taking methods. The superhydrophilicity of the laser-ablated surface slowly reverts within a week, but this can be suppressed by storage in a humidity-saturated environment.

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The volume of the self-patterned droplets can be tuned by the size of the

superhydrophilic pattern and, thus, provides a quantitative sampling technique with volumes ranging from couple of nanolitres to a few hundreds of nanolitres. Long-term stability of the droplet array with negligible evaporation in saturated humidity enables the culturing of cells while simultaneously exposing them to a variety of reagents, reagent concentrations or culturing conditions. The reported method demonstrates a fast, convenient and low-cost approach to

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construct spatially-resolved sub-colonies of cell cultures, which could provide a much more accessible and cost-effective array-based platform for bioassays.

Acknowledgements The authors would like to thank Prof. Roseanne. M. Ford (Department of Chemical Engineering, University of Virginia) for the resources provided by her research group in the cell culture assay. 20



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