Inkjet-Printing Patterned Chip on Sticky Superhydrophobic Surface for

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Biological and Medical Applications of Materials and Interfaces

Inkjet-printing Patterned Chip on Sticky Superhydrophobic Surface for High-efficiency Single-cell Array Trapping and Real-time Observation of Cellular Apoptosis Yingnan Sun, Wenhua Song, Xiaohan Sun, and Shusheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10703 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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ACS Applied Materials & Interfaces

Inkjet-printing Patterned Chip on Sticky Superhydrophobic Surface for High-efficiency Single-cell Array Trapping and Real-time Observation of Cellular Apoptosis Yingnan Sun, Wenhua Song, Xiaohan Sun, Shusheng Zhang* Shandong Provincial Key Laboratory of Detection Technology for Tumor Makers, College of Chemistry and Chemical Engineering, Linyi University, Linyi, Shandong 276005, P. R. China. KEYWORDS: Microdroplet array, superhydrophobic surface, inkjet printing, single cell array.

ABSTRACT:

Single-cell assays have broad applications in cellular studies, tissue engineering，fundamental studies of cell-cell interactions

and understanding of cell-to cell variations. Most existing

methods for micron-sized cell patterning are still based on lithography-based microfabrication process. Thus, exploiting new mask-free strategies while maintaining high-precision single cell patterning is still a great challenge. Here, we presented a facile，low-cost and mask-free approach for constructing high-resolution patterning on sticky superhydrophobic (SH) substrates based on inkjet printing with ordinary precision. In this work, the SH surface with both high

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contact angle and relatively high contact angle hysteresis can not only obtain high-resolution spots, but also avoid droplets bouncing behavior. We improved the feature size of printed protein spots as small as 4 μm, which is much smaller than protein spots used for single cell trapping. Moreover, with the assistance of a narrow microchannel, the inkjet-printing patterned chip with fibronectin ink allows for fast and high-efficiency trapping of multiple single-cell arrays. Using this method, single-cell occupancy could reach approximately 81% within 30 min on subcellular sized patterning chip and there was no significant effect on cell viability. As a proof of concept, this chip has been applied to study the real-time apoptosis of single cells and demonstrated the potential in cells heterogeneity analysis.

INTRODUCTION The ability to arrange single-cell array in a predefined patterns could have broad applications in cellular studies, tissue engineering ， fundamental studies of cell-cell interactions

and

understanding of cell-to cell variations.1 Currently, kinds of single-cell approaches have been designed for this purpose, including microfluidic devices,2-5 microcontact printing,6 surface patterning,7-10 and physical constraints.11,12 Until now, most studies employed conventional “lithography-based methods” (such as microfabrication or lithography-based mask) to fabricated microstructures and micropatterns, including the microcontact printing and micro-sized stencils. However, this mask-based method suffers from several drawbacks, including lithography-based microfabrication processing and special equipment requirement, which is limited for restricting their sample-economic fabrication and ease of manipulation in further applications. Therefore, it is highly desirable to propose a facile ， rapid, low-cost and effective mask-free patterning method to fabricate high-precision single-cell trapping chip.


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As an attractive alternative, inkjet printing has been proved to be a versatile micropatterning technique because of its direct-writing and mask-free properties.13 Various trapping materials have been printed to generate cell-trapping arrays, including collagen, fibronectin (FN), (aminopropyl)triethoxysilane and Poly-L-Lysine (PLL) etc.14,15 However, most of these trapping arrays did not offer single-cell patterning precision and control1. Since the trapping patterns (including FN and PLL et al.) with subcellular feature size has a potential application as means of controlling cellular behavior and activity,15 it is necessary to improve the printing precision of trapping materials. In order to improve the printing resolution of patterns, currently much attention has been drawn toward the improvement of nozzle aperture and instrument performance, thus requiring time-consuming and expensive fabrication procedures.15, 17-19 For example, Gennari et.al.15 reported a novel pyroelectric-jet approach for multiscale printing of APTES and FN used for achieving single cells. Although this nozzle-free p-jet process could print spots with size from 200 μm to 0.5 μm by means of the pyroelectric field, the p-jet method had limited throughput in fabricating single-cell array and poor repeatability in controlling droplet volume. Wittstock and coworkers20 immersed the traditional piezoelectric inkjet nozzle into oil layer to print femtoliter droplets patterns, the volume of which were far smaller than droplets printed in air. However, since the bioink arrays were cover with immiscible oil layer, the patterned chip was not suitable for cell trapping. Song et al.21 printed nanoparticle aqueous inks with a Fujifilm printer with 10 pL cartridges, generating a 10 pL single ink droplet. The average diameter of the printed spots on OTS-treated hydrophobic substrate (contact angle = 110.1°) could be as small as 5 µm. But the nozzle with an inner diameter of less than 10 μm is often difficult to operate in practical applications since easy to blocked and difficult to clean.


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Therefore, it is not the best choice to improve the accuracy of pattern printing by improving the hardware facilities. It is noteworthy that Song’s method provides a new idea to improve the precision of printing based on the properties of substrate hydrophobic surface. Lowering the surface energy of the substrate and endowing the substrate with patterned chemical structure are efficient methods to control the dewetting behavior of printed droplets on the surface and further improve the printing resolution.22-26 Generally speaking, for droplets with same volume, the larger the surface contact angle (CA), the smaller the FN-spot size after dewetting process. However, when the surface becomes superhydrophobic (CA >150°), and since the droplet is ejected from the nozzle at a higher velocity, the droplet will bounce on the interface, resulting in severely scattering droplets and the limited printing accuracy. Therefore, it is indispensable to utilize the SH substrate to obtain high precision patterns with feature size smaller than a single cell based on the printer with ordinary resolution. Furthermore, the practical application of the existing surface-patterned single-cell trapping assay in general laboratories are also limited due to another two challenges: time-consuming process for gravity-induced sedimentary cells trapping, and low occupancy of single cells due to the intractable issues of cluster formation.27 Therefore, construction of an inkjet-printing patterned chip with high resolution and variable widths for high-throughput single-cell trapping, concurrently possessing high throughput, short time consuming, and high single-cell occupancy still remains a challenge. For all we know, only a few publications have dealt with alternative methods for facile，rapid, low-cost and effective construction of surfacepatterned single-cell trapping assay.28 In this work, we combined the methyltrichlorosilane(MTS)-modified sticky SH surface with inkjet printing technology to prepare high-resolution protein patterns for single-cell trapping


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of a large throughput. We prepared the SH surfaces with both high contact angle and relatively high contact angle hysteresis, by one-step silanization process with MTS, which can not only obtain high-resolution spots, but also avoid droplets bouncing and scattering behaviors. To our knowledge, it's the first time that we applied a transparent sticky surface to anti-bouncing inkjet printing for fabricating high-resolution patterns. Based on an inkjet-printer with ordinary precision, the minimum feature size we could obtain with this method was as small as 4 μm. We adopted the 9 μm feature size for further experiments of single-cell trapping. Moreover, different from previous trapping devices based on gravity-induced cell sedimentation, in this paper, with the assistance of a narrow microchannel specially designed for cells loading and culture, large single-cell assay trapping on inkjet-printing patterned chip could be achieved only in half an hour with a high single-cell occupancy. As a proof of concept, we applied our approach for studying the real-time apoptosis of single HeLa cells caused by apoptosis inducer staurosporine (STS). This assay is suitable for multiple cellular types, fast and efficient single-cell trapping and realtime study of single-cell behaviors. This simple, low-cost, and efficient approach has a great potential in cells function and heterogeneity analysis. EXPERIMENTAL SECTION Preparation of Superhydrophobic Substrates. Prior to the surface treated by methyltrichlorosilane (MTS), the silica glasses were sonicated in acetone, ethanol, and water subsequently. Then they were dried by nitrogen flow and placed in a plasma cleaner for 2min. The glasses with specified size was totally immersed in a Teflon bottle with 30 mL of toluene, and then 50μL of MTS and 34μL of concentrated hydrochloric acid were added to the bottle, and finally the bottle was sealed and stored in the refrigerator at 4 °C for 10-30min. After the required reaction time, the glasses were taken out to terminate the reaction, and immediately


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washed with toluene, ethanol and deionized water in sequence and dried at 110 °C for at least 5 min29,30. Preparation of Bioink. Bioinks for inkjet-printing here were prepared by mixture human fibronectin (FN, Sigma-Aldrich) with Fibrinogen-Alexa Fluor 488 conjugate (Invitrogen) in PBS (pH 7.2-7.4, RT) at a concentration rate of 3:1 (final concentration of 30 mg/mL and 10 mg/mL, respectively). The ink was filtrated through a PVDF mesh before use. When the single-cell assay was used for subsequent cell staining, the bioink would use fibrinogen instead of fibrinogenAlexa Fluor 488 as ligand recognition conjugate, in order to avoid the green fluorescence emitted from Alexa Fluor 488. Printing Conditions. The method of droplet-array generation on an inkjet platform has been reported in detail in our previous work31. Briefly, (1) adjust the pulse waveform for the purpose of obtaining reproducible and stable droplets without satellites. (2) Analyze the accurate volume of droplet with stroboscope and software. (3) Obtain a specific volume of droplet by fine-tuning the parameters. (4) Print designed patterns of bioink at specific coordinates on substrates. In this work, the ink droplets containing FN were automatically printed onto SH substrates with a 30 μm nozzle in a rectangular array as a software setting and then allowed to rapid and spontaneous evaporate at room temperature. Characterization of substrate. The SH substrates were characterized using SEM (Nova, NanoSEM, FEI, USA) and AFM (Bruker Multimode V, Germany). All bright and fluorescence images were acquired by an inverted fluorescence microscope (TE2000, Olympus, Japan) with a 10×, 20× objectives and a CCD camera (DP73, Olympus). Static contact angle (SCA) and contact angle hysteresis (CAH) were measured on a DSA100 instrument (Krüss, Germany). The average CA was obtained by measuring more than three different positions of the same sample.


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Assembly of the Device. A mixture of PDMS prepolymer (10:1) was poured into a disposable Petri dish and then heated at 80 °C for at least 60 min. After peeled off, a flat and clean PDMS was cut into a rectangular area (2 cm×2 cm) and punched with a 1mm hole. Afterwards, the PDMS cube was attached on the PDMS spacer, which was a thinner PDMS layer with a through-hole and a specific thickness. The fresh PDMS layers and clean glass plate could contact tightly under presssure to prevent floating and leakage during cell loading and culture, the assembled device was then incubated at 37 °C for a certain time to allow cells to adhere to the patterned substrate. Cell Culture. HeLa, L-02, and MCF-7 cells was utilized in our study. Cells were cultured in DMEM (supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum) and incubated at 37 °C in a humidified atmosphere with 5% CO2, and cell passages were performed every two days32. HeLa cells were adopted as the model cell for studying cellular apoptosis based on the Caspase 3/7 kit. Cell Viability Test. According to previous reports33, for cells staining, Hoechst 33342 and FDA solutions were added to the cell culture medium at a final concentration of 10 μg/mL and 2 μg/mL, respectively). After incubation for 10 min, the cells were rinsed twice with PBS and refilled with PBS for fluorescence observation. Cell viability was evaluated with the proportion of living cells (blue color, stained by FDA) to the total cells (green color, stained by Hoechst). Fluorescence images were processed with the use of ImageJ software to calculate cell numbers. Observation of Single-Cell Apoptosis. HeLa cells were loaded with TMRM (ImageiT™ TMRM Reagent, Invitrogen, USA) followed by Caspase-3/7 detection reagent (Invitrogen, USA). A 20× objective and the appropriate instrument filter sets was used to capture the images


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of TMRM (red color) and Caspase-3/7 (green color). In this study, Hela cells were treated with the apoptosis inducer by culturing in STS-containing medium and incubated in chamber (37 °C, humidified 5%). Subsequently, images of the selected arrays were acquired every 30 min over 5 h. Fluorescence images of TMRM and Caspase were processed and the values of intensity were normalized using the ImageJ software (NIH, USA). RESULTS AND DISCUSSION Figure 1 illustrated the overall work flow of the fabrication of the high-resolution protein patterning on SH substrate (Figure 1A) and the assembly of microchannel for rapid single-cell array trapping (Figure 1B). The SH glass substrate was modified with MTS,28,29 which would resist cells adhesion (Fig. S1). Briefly, aqueous bioink droplets (FN here for instance) were inkjet-printed onto a flat SH substrate to form high-throughput droplets array. After the rapid evaporation of water, each droplet shrank into a smaller spot due to the specific dewetting behavior of microscale droplets on substrates,21 generating a high-resolution protein patterning substrate for single-cell trapping (Fig.1A). An flat PDMS cover plate (about 3 mm thickness) with inlet and outlet holes was laid on the pre-patterned substrate, which were separated by thin PMDS gasket (about 1mm thickness), to form space-confined assembly system to sustain cell loading and culture. The PDMS cover plate was silanization with PFOS to provide hydrophobic property to avoid liquid residue and entrainment. Finally, the cell suspended medium with high cell density was directly pipetted (Fig.1B) and the cell-laden device was incubated at 37°C in a certain time to allow cells attachment. As the liquid immersed, printed FN spot arrays served as capture sites, as well as wetting defects34,35 when liquid moved. Under the combined action, single cells could be trapped in short amount of time with a large throughput and high occupancy rate. Subsequently, the assembled chip was gently washed with PBS and then replaced with fresh


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medium for another 30 minutes to 1 hour culture to enhance cells adhesion. In the end, the PDMS cover and spacer were carefully striped away without disturbing the trapped cells.

Figure 1. A non-lithography strategy to fabricate single-cell array on a inkjet-printing patterned substrates. a) FN arrays were fabricated by inkjet printing on a surface functionalized SH substrate. b) The device for cells loading and culture was assembled with a flat PDMS spacer and cove. Loading cell suspended medium and PBS sequentially into the chamber utilizing a pipette. The cover and spacer were removed after cell attachment.

Figure 2 showed the surface morphology of the 3D MTS nanostructures, characterized by SEM (Fig. 2A) and AFM (Fig. 2B). The randomly integrated fibrous quasi-networks distributed all over the surface of the silica glass. The formation of a 3D discrete/quasi-network with MTS nanostructures was useful for the “petal” effect that water droplet could partly infiltrate into the microstructures of the 3D surface, while not into the nanostructures, resulting in both the relatively high CAH and high SCA.36 The wettability of the SH substrate was characterized by static contact angle (Fig. 2C) and contact angle hysteresis (Fig. 2D). The SH substrate treated


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with MTS for 30 min exhibited both the high CA (above 155︒) and relatively high CAH (above 20︒). It was essential for eliminating scattering droplets and printing high-resolution patterns because the ejected droplet would be “stuck” on the SH surface without bouncing and splattering (Fig. S2 and Movie S1).

Figure 2. (A, B) SEM and AFM images of the quasi-network with MTS nanostructures. (C, D, E) Bright micrographs of water droplets on MTS-modified SH surface at 0︒, 40︒ and 180︒ tilt angles. (F)


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The contact angles and hysteresis of water on MTS-modified glass substrates varied with the reaction time. Scale bars in A, C, D and E were 500 nm, 0.5 mm, 0.5 mm and 100 μm, respectively.

As shown in Figure 3A, each printed spot here was generated by a single ink droplet with approximately 30.5 pL. All the FN droplets spontaneously shrunk into FN spots when the microscale printed droplets were evaporating on the SH surface21. Since the concentration of FN was relatively low, the contact angle of DI water was employed to characterize the wetting property of various surfaces in this paper (Fig. S3). With the same droplets volume injected from a nozzle with 30 µm (Fig. 3A）), the printed spots (Fig. 3C) with average diameter of 30 ± 0.5 µm, 12 ± 0.5 µm and 4.5 ± 0.5 µm were substrates with water SCA of 63︒± 2︒,123︒±2︒ and 165︒± 2︒ (Fig. 3B), respectively. In addition, the fluorescence images (Fig. 3D) showed that the fluorescence intensity distribution of FN spot varies as the surface hydrophobicity. As shown in Fig. 3E-1, a large array of FN spots with 9 μm diameter was fabricated on the substrate with CA of 165︒ in volumes of 150 pL approximately. To investigate the reliability of the combined approach for generating high-resolution patterns, the RSD of FN array was calculated to demonstrate the uniformity of spots size after printing and evaporating28. As shown in Fig. 3E2, the RSD of 140 spots adhering to substrate were within 4.3%, which indicated both the reliability and the robustness of this method used for fabricating high-resolution patterns. Besides, the FN array on the MTS surface would not fall off after soaking in the cell culture medium or PBS but stick to the substrate tightly (Fig. S4). What’s more, the patterned substrates can be stored for two weeks at 4℃ while retain the FN protein cell-adhesion activity and blank area cell-repellent property, which would be benificial for practically clinical application.


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Figure 3. Wettability of silanization glass surfaces and their assessment of performance. (A) The droplet was ejected from the inkjet nozzle without satellite droplets. The volume was obtained with software analysis. (B) The contact angle of a droplet of water on the surface of glass non-treated (B-1) and hydrophobic (B-2) and superhydrophobic (B-3). (C) and (D) Bright-field images (C1, C2, C3) and the corresponding fluorescence images (D1, D2, D3) of FN spot printed with same droplets volumes. (E) The


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microscope bright (E-1) and fluorescent images (E-2) of large array of FN spots on SH substrates with spot-to-spot spacing of 100 μm. Scale bars in E-1 and E-2 were 100 μm.

To overcome the issues of cells agglomeration,26 we attempted to optimize the success rate of single-cell capture from the following aspects: the size of FN spot for trapping, the density of cell suspension and the incubation time after cell suspension loading. Specially, it would significantly reduce the amount of incubation time in high cell density condition, which was a significant improvement over the current approaches requiring a longer incubation time of several hours. In addition, the limited microchannel height was beneficial to further reduce intercellular adhesion and stack. In this paper, we finally adopted 9 μm FN-spots array and cultured cells at a density of 3×106 cells/mL for 30min, for further experiments, aided by narrow microchannels at a height of 2 mm. Fig. 4A and 4B showed the corresponding bright and fluorescence images (labelled with Hoechst). The results indicate that the spots of subcellular size were more conductive to the success rate of single-cell capture for high-density cells loading, rather than the ones of cellular size (Fig. S5). Based on the repeated experimental results, the statistic result was shown in Figure 4D: about 81% of the positions defined by the inkjetpatterned array obtained one single cells, while about 16% possessed two or more cells. Because high density cultivation could evidently increase the contact probability between cells and proteins, there's less probability of no cell adsorption. In conclusion, the single-cell trapping efficiency of the inkjet-printing patterned chip depended on the feature size of FN spot, packing density of array, and cell density.


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Figure 4. (A, B) Distribution of HeLa cells. Images were taken under 4× objective lens after washing with PBS. (C) A higher magnification (40 × objective) image of trapped cells after another 30min culture. (D) The average number of cells trapped on single FN spot. Scale bars in A and B were 100 μm. Scale bar in C was 50 μm.

To access the cell viability after entrapment, a cell viability test using Hoechst and FDA was performed. It's worth mentioning that we waited another 30 min to give the cells enough time to further strengthen immobilization on the substrate before cells staining. Figure 5A-1and 5A-2 showed the fluorescent images of HeLa cells array under 10× objective lens after 1h since cells trapping, respectively. In this study, the viability of HeLa cells trapped on the FN-patterned array was about 94 ± 3.5%. This observation clearly suggested that the use of our inkjet-printing patterned chip and the optimization steps posed hardly any effects on cell viability upon cell trapping. To prove the versatility of our chip, we also tested MCF-7 cells and L-02 cells on the


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same way. As shown in Figure 5B, the bright and fluorescence images were taken from the trapped cells (MCF-7 and L-02, respectively) after 1.5 h since cells trapping as well. Statistically, there was also no clear evidence of the significant difference in cell viability between different cell types.

Figure 5. Cell viability test after cells trapping: Blue = cell nuclei (stained by Hoechst) and Green = live cells (stained by FDA). (A-1) and (A-2) fluorescence images of HeLa cells array. (B) The bright and fluorescence images of MCF-7 and L-02 cells array in larger magnification. Scale bars in A-1, A-2 and B were 50 μm.


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As a proof of concept for the inkjet-printing patterned chip in single-cell assay, this platform was applied to study the real-time apoptosis of single HeLa cells induced by an apoptosis inducer STS and demonstrate the heterogeneity of cellular activities. Apoptosis could be evaluated by examining multiple cellular events such as loss of mitochondrial membrane potential, translocation of cytochrome from mitochondria to the cytosol and DNA fragmentation. The Caspase-3/7 Green detection reagent could label cells to report apoptosis and allowed to be used with other fluorescent probes in live or fixed cells. Of particular value is the ability of this reagent to report caspase activity in live cells in real time, while simultaneously detecting other parameters related to apoptosis.37 In this work, we adopted Casepase-3/7 and TMRM to simultaneously detect multiple parameters of apoptosis in single cells, involving DNA fragmentation and loss of mitochondrial membrane potential, respectively. We conducted singlecell apoptosis treated with 30 μM STS and control group without STS. Figure 6A and 6B showed dynamic observations of single-cell apoptosis on the inkjet-printing patterned chip. Over the 5 hrs time course, STS induced a loss of mitochondrial membrane potential followed by activation of caspase 3/7, as indicated by a decrease in TMRM signal (red color) and an increase in caspase 3/7 (green color), respectively. Figure 6C displayed the real-time plots of the normalized fluorescent intensity of individual cells based on 5 randomly chosen cells as a function of culture time. Not unexpectedly, there was a cells heterogeneity during the apoptosis process among the single cells. Specifically, the apoptosis onset time and apoptosis rate were different since the fluorescence intensity reduced or increased soon upon the inducer treatment. In the control group, there was a slight decrease in red fluorescent intensity of TMRM signal, which might due to the fluorescence partial quenching. The agreement between the results shown


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here and the reported response further validated this method as a potential and feasible tool for quantitative single-cell array-based bioassay and analyzing heterogeneity of individual cells.

Figure 6. Detecting multiple parameters of apoptosis. Over the 5 hours time course, STS induced a reduction in TMRM signal (red) (A), followed by an increase in caspase 3/7 signal (green) (B), respectively. (C) Real-time plots of the normalized fluorescent intensity of individual cells based on 5 randomly chosen cells as a function of culture time (Circular marks represent TMRM signal; triangular marks represent caspase 3/7 signal). Scale bars in A and B were both 100 μm.

CONCLUSIONS


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In this paper, we presented a simple method for the rapid generation of high resolution protein patterns with controllable and uniform diameter were on the superhydrophobic surface through piezoelectric inkjet printing. Large array of protein spots with diameter as small as 4 μm was available to prepare based on this method. High-efficiency single-cell array trapping was achieved by adjusting the size of FN capture-spots, the density of cell suspension and the incubation time with assistance of a narrow microchannel specially designed for cells suspension loading and culture. By this approach, the real-time and heterogeneous apoptosis behavior of single cells was carried out. The presented method is suitable for different cellular types, fast and efficient single-cell trapping and real-time study of single-cell behaviors. Compared with the microchannels- or microwells-based platforms, our method has several obvious advantages as following: First, this direct-writing approach dispenses with mask preparation for the creation of micropatterns, and different patterns are easily carried out. Second, multiple bioink can be “printed” at subcellular scales only in condition of sufficient hydrophobicity, and only a singlestep surface pretreatment is needed. Third, the assembled chip allows fast and high-efficiency trapping of multiple cell types at the single-cell level with negligible impacts on cell viability. Last but not least, high resolution patterns can be created using general printing equipment with normal resolution on a wide range of substrates, which is still greatly challenging to implement these parameters in a low-cost way. In summary, the reported method offers a facile, efficient and low-cost approach for constructing high-resolution patterns and studying single-cell function and heterogeneity measurement.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Supporting information (PDF). Movie S1. Printing on different surfaces.mp4. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Phone: 86 0539-8766107. Fax: +86-0539-8766867. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We are appreciative for the financial supported from the Postdoctoral Science Foundation of China (2016M572420), National Natural Science Foundation of China (21804065, 21535002, 21775063), and Shandong Provincial Natural Science Foundation (ZR2016QZ001). REFERENCES


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(1) Vermesh, U.; Vermesh, O.; Wang, J.; Kwong G. A.; Ma, C.; Hwang, K.; Heath. J. R. HighDensity, Multiplexed Patterning of Cells at Single-Cell Resolution for Tissue Engineering and Other Applications. Angew. Chem. 2011, 123, 7516-7518. (2) Chung, K.; Rivet, C. A.; Kemp, M. L.; Lu, H. Imaging Single-Cell Signaling Dynamics with a Deterministic High-Density Single-Cell Trap Array. Anal. Chem. 2011, 83, 7044-7052. (3) Zhang, K.; Chou, C. K.; Xia, X.; Hung, M. C.; Qin, L. Block-Cell-Printing for Live SingleCell Printing. Proc. Natl. Acad. Sci. 2014, 111, 2948-2953. (4) Hong, S.; Pan, Q.; Lee, L. P. Single-cell Level Co-culture Platform for Intercellular Communication. Integr. Biol. (Camb)，2012, 4, 374-380. (5) Kim, S. H.; Yamamoto, T.; Fourmy, D.; Fujii, T. Electroactive Microwell Arrays for Highly Efficient Single‐Cell Trapping and Analysis. Small, 2011, 7, 3239-3247. (6) Chen, C.; Mrksich, M.; Huang, S.; Whitesides, G.; Ingber, D. Geometric Control of Cell Life and Death. Science, 1997, 276, 1425. (7) Tseng, P.; Judy, J. W.; Di Carlo, D. Magnetic Nanoparticle-mediated Massively Parallel Mechanical Modulation of Single-cell Behavior. Nat. Methods, 2012, 9, 1113-1119. (8) Giam, L. R.; Massich, M. D.; Hao, L.; Wong, L. S.; Mader, C. C.; Mirkin, C. A. Scanning Probe-enabled Nanocombinatorics Define the Relationship Between FibronectinFeature Size and Stem Cell Fate. Proc. Natl. Acad. Sci. 2012, 109, 4377-4382. (9) Vermesh, U.; Wang, J.; Kwong, G. A.; Ma, C.; Hwang, K.; Heath, J. R. High-Density, Multiplexed Patterning of Cells at Single-Cell Resolution for Tissue Engineering and Other Applications. Angew. Chem. Int. Ed. Engl. 2011, 50, 7378-7380. (10) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Protein Nanoarrays Generated by Dip-pen Nanolithography. Science, 2002, 295, 1702-1705.


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Table of Content 254x140mm (149 x 149 DPI)



Figure 1 177x81mm (300 x 300 DPI)


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154x190mm (149 x 149 DPI)



121x190mm (149 x 149 DPI)


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226x169mm (149 x 149 DPI)



168x190mm (149 x 149 DPI)


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174x147mm (149 x 149 DPI)


Inkjet-Printing Patterned Chip on Sticky Superhydrophobic Surface for

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