Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

1. Preparation of TiO2-coated Glass Coverslip
<ol>
	<li>Number the coverslips using a diamond scriber. This helps not only to keep track of each coverslips but also to ensure that the correct side of the sample is facing up. Clean the coverslips, first under running ddH2O, then by immersing them in piranha solution (H2SO4:H2O2 = 4:1). After 10 min, rinse the coverslips thoroughly, 8 times in ddH2O. Dry the coverslips under N2 flow.</li>
	<li>Set TiO2 target in the radio-frequency (RF) sputtering system. Attach the coverslips onto a sample holder of the sputtering equipment using a polyimide tape. Place the sample holder in the sputtering chamber. Evacuate the chamber until the pressure reaches 2.0&#215;10-4 Pa.</li>
	<li>Introduce Ar gas into the chamber and set the deposition pressure to 4.0 mTorr. While keeping the shutter closed, gradually increase the RF power to 70 W.</li>
	<li>Open the shutter and sputter for 15 min to obtain a film with a thickness of 120-150 nm (Figure 1: Step A). Growth rate of the film needs to be derived for each machine.</li>
</ol>
2. Surface Coating with Cell-repellent Film
<ol>
	<li>Hydrophilize the TiO2 surface by treating the sample with O2 plasma, following instructions provided by the manufacturer of the plasma reactor. We treat the sample for 5 min at 200 W with O2 flow of 100 sccm. Immerse the sample in ddH2O and confirm that the surface is superhydrophilic. Dry the surface thoroughly under N2 flow.</li>
	<li>Prepare 1 mM octadecyltrichlorosilane (OTS) solution by adding 39.6 &#956;l OTS to 100 ml toluene. Immerse the sample in the solution for 1 hr at RT. Conduct this step inside an N2-filled glove bag (Figure 1: Step B).</li>
	<li>To remove physisorbed molecules, sonicate the sample in toluene, acetone, ethanol, and ddH2O for 5 min each, in that order. Rinse the sample four times in fresh ddH2O and dry the surface under N2 flow. The surface should be hydrophobic with a contact angle of 100-110&#176;.</li>
</ol>
3. Ex-Situ Surface Patterning
<ol>
	<li>Working in a laminar flow hood, draw several scratch marks with a diamond scriber on the surface. The marks help in keeping track of the processed regions and also bringing microscopes into focus. Sterilize the OTS-coated TiO2 by immersing the sample in 70% ethanol for 5 min. Then rinse the sample twice in sterilized ddH2O.</li>
	<li>Place the sample in a 35-mm dish, and add 2 ml of PC12 growth medium (see Step 4.2). Incubate for over 3 hr in a CO2 incubator (37 &#176;C). This procedure is intended to let serum albumins absorb onto the surface. Adsorbed albumins inhibit subsequent adsorption of other proteins and cells (Figure 1: Step C).</li>
	<li>While waiting, set up the inverted fluorescence microscope.
	<ol>
		<li>Turn on the arc lamp, insert the UV filter cube, and set the objective lens to 20X.</li>
		<li>Measure light intensity I (W cm-2) using an UV-intensity meter, and calculate irradiation time t (in seconds) for a dose of d (in J cm-2) as: t = d/I. 
		For instance, to irradiate at a dose of 200 J cm-2 using a light source of 600 mW cm-2, 333 sec of irradiation is required.</li>
		<li>Use a stage micrometer and close the field diaphragm to set the size of the region to be irradiated, e.g., 200 &#956;m.</li>
	</ol>
	</li>
	<li>After the 3 hr incubation, supplement the medium with 200 &#956;l of 3.0 mg ml-1 type-IV collagen (Col-IV; final concentration 300 &#956;g ml-1).</li>
	<li>Transfer the 35-mm dish to the microscope stage. Find the scratch mark, focus the microscope onto the sample surface, and irradiate UV-light at a dose of 200 J cm-2 (Figure 1: Step D). The area of UV irradiation can be altered either by adjusting the opening of the field diaphragm or by replacing the field diaphragm with a metal mask of arbitral geometry.</li>
	<li>Replace the medium with a fresh growth medium (without Col-IV), and place the sample back in the incubator.</li>
</ol>
4. Cell Culture
<ol>
	<li>Routine culture of PC12 cells is carried out on a collagen-coated plastic dish.
	<ol>
		<li>To coat a 60-mm tissue-culture dish with Col-IV, first wet the surface with ddH2O and aspirate all ddH2O.</li>
		<li>Prepare 300 &#956;g ml-1 Col-IV by diluting the original solution (3 mg ml-1) 10x with ddH2O.</li>
		<li>Add 200 &#956;l of 300 &#956;g ml-1 Col-IV per 60-mm dish. Let the solution spread through the surface.</li>
		<li>Dry the dish in a laminar flow hood for approximately 1 hr.</li>
		<li>Rinse the surface twice with 4 ml of Dulbecco&#39;s phosphate-buffered saline (D-PBS). When not using the coated dish immediately, rinse the dish once with 4 ml of ddH2O. After aspirating ddH2O, store the dish in the incubator. Try not to keep it stored for more than two weeks.</li>
	</ol>
	</li>
	<li>PC12 cells are grown in a growth medium that consists of: Dulbecco&#39;s modified Eagle&#39;s medium (low glucose) + 10% fetal bovine serum + 5% horse serum + 1% penicillin-streptomycin solution. Incubate the cells in a CO2 incubator (37 &#176;C, 5% CO2), and replace half of the medium every other day. Passage cells before they reach confluence.
	<ol>
		<li>Aspirate the growth medium, and add 2 ml of PBS+ (D-PBS + 10 mg ml-1 bovine serum albumin (BSA) + 10 mM EDTA) prewarmed to 37 &#176;C. Incubate for 5 min at 37 &#176;C. Gently tap the dish to detach all cells.</li>
		<li>Collect cells in a 15 ml conical tube. Rinse the dish with 3 ml of fresh D-PBS, prewarmed to 37 &#176;C.</li>
		<li>Centrifuge the tube at 150 &#215; g for 4 min.</li>
		<li>Aspirate the supernatant, and add 1 ml of the growth medium.</li>
		<li>For routine culture, split the cells 1:3 to 1:5.</li>
	</ol>
	</li>
	<li>To plate cells on the UV-modified OTS/TiO2 sample, count cell density and add 3.0&#215;105 cells in a 35-mm dish (Figure 1: Step E). Incubate the dish in the humidified incubator (37 &#176;C, 5% CO2) for 1-2 days. Both naive and nerve growth factor (NGF)-differentiated PC12 cells can be used for the patterning experiments. For NGF differentiation, 100 ng ml-1 of 7S-NGF is added to the growth medium several days before plating the cells onto the sample. NGF-differentiation seems to increase the adhesibility of the PC12 cells and makes handling easier, especially in the in-situ patterning experiments.</li>
</ol>
5. In-Situ Surface Patterning
<ol>
	<li>After 1-2 days of culture on the ex-situ modified surface, confirm that the cells are attaching and growing only on the UV-irradiated region. Set up the microscope, as described in Step 3.4.</li>
	<li>Transfer the sample to a new 35-mm tissue culture dish containing the growth medium, 100 ng ml-1 NGF (in the case of using NGF-differentiated cells), and 100 &#956;g ml-1 Col-IV.</li>
	<li>Place the dish on the microscope stage. Find the appropriate position, and irradiate UV light at a dose of 200 J cm-2 (Figure 1: Step F, G). The newly created permissive region is indicated with an asterisk in Figure 1: Step G.</li>
	<li>Try to complete processing of a single sample within 30 min. After irradiation, transfer the sample back into the medium without Col-IV.</li>
	<li>Be extremely careful when carrying the dish with cultured cells and transferring the sample from dish to dish to prevent detaching of the patterned cells.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> 
Figure 1. Schematic illustration of the overall process. See text for details. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

In our current protocol, TiO2 film was formed by RF-magnetron sputtering. We favor this method of deposition since it allows us to reproducibly prepare a photocatalytic TiO2 film with a sub-nm roughness. Although sputter deposition processes are familiar to materials scientists and electronic engineers, it may not be quite accessible to biologists. In that case, spin-coated TiO2 film would be an alternative choice23. In this method, TiO2 nanoparticles dissolved in a solvent is spread out on a substrate surface by centrifugal force. Special equipment required is a spin coater, which is much affordable than a sputtering system. We have used for ourselves a spin-coated TiO2 nanoparticle film and have confirmed that the film can also be used to carry out similar experiments (data not shown). The surface of the spin-coated TiO2 film is comparatively rough with an rms roughness of several tens of nm, and it takes some practice to uniformly coat the substrate reproducibly especially when multiple spin coatings are needed to obtain thick films. Nevertheless, the method might be the first candidate for biologists.
Thus far, we have utilized this method in patterning not only the PC12 cells but also a human pancreatic cancer cell line AsPC-1 and primary neurons obtained from embryonic rat hippocampi19. Adhesibility of AsPC-1 cells are much stronger than that of PC12 cells or hippocampal neurons, and supplementation of scaffolding molecules, such as collagen, was not required in patterning them. We yet note that non-specific adhesion of the cells to the unirradiated region was occasionally observed, which is probably due to defects in the OTS SAM.
Patterning primary neurons were much more challenging since their adhesibility is weak. In fact, the critical step of the current process lies in the proper adsorption of scaffolding molecules. A simple supplementation of collagen or laminin was insufficient to increase affinity of the modified region for the neurons. Conventional culture of hippocampal neurons relies on positively-charged peptide poly-lysine to coat the surface of the coverslip24. In this context, inducing selective attachment of poly-lysine to the UV-irradiated area is desired, but was impossible since poly-lysine adsorbed on the intact OTS SAM region as well. We tried other surface coatings, such as polyethylene glycol-silane SAMs and perfluoro-silane SAMs, to find that none of these were anti-fouling to poly-lysine. To solve this issue, we very recently showed that a cross-linking molecule could be used to induce active adsorption of the scaffolding molecules onto the UV-irradiated region19. Such selection of scaffolding molecules and optimization of its concentration should be carried out for each experiment.
UV irradiation dose should also be optimized since it strongly affects the amount of protein adsorption. In our case, fluorescence intensity measurement was used to derive an optimal UV dose of 200 J cm-2 18. The value is likely to vary depending on experimental parameters such as the thickness and the crystallinity of the TiO2 film.
Among various techniques of in-situ surface modification developed so far, the major advantage of the present method is that it does not require organic synthesis of specialized molecules for the surface processing. The method presented here is, in principle, compatible with organic films prepared from the vast library of commercially-available polymers, peptides, and SAM precursors. It is also favorable in a sense that the surface-modification process can be carried out using conventional lab equipment, such as a wide-field fluorescence microscope equipped with a mercury arc lamp, as was shown here. Using an UV laser as the light source, subcellular resolution down to ~3 &#956;m can be achieved (data not shown).
Some limitations of the current method include the irreversibility of the surface modification, need for supplementing scaffolding molecules in the medium, and generation of ROS. The process described here is applicable to non-permissive to permissive conversion of the surface, but the conversion is one-way and cannot be reversed. For experiments that require reversal conversion of cell affinity, other methods15 need to be considered. In addition, our process requires scaffolding molecules such as collagen to be bath-applied to the cell culture medium. Since the population of pre-patterned cells would inevitably be exposed to the molecules, this may limit some application of this process. Photocatalyzed generation of ROS, such as hydroxyl radicals, at the UV-irradiated region may be toxic to nearby cells, but, at least, this effect was not observed in our experiments. Even if this becomes prominent for other applications, this problem should be solved by supplementing oxygen scavengers in growth medium. Since the oxygen scavenging molecules would not be located between the TiO2 and the non-permissive molecules on its surface, they are expected to eliminate excessive ROS without affecting the patterning process kinetics.

Semiconductor lithography processes and its derivatives &#8212; such as photolithography1,2, electron-beam lithography3-6, and microcontact printing7-10 &#8212; have now become an established tool in cell biology to grow living cells in a defined position and geometry. The patterning method relies on the use of microfabricated substrates, consisting of micro-island of cell permissive coating in a non-permissive background. Such substrate serves as a template to pattern the cells. These technologies have provided us the novel methods to engineer cells and their function at a single- and multi-cellular level, to extract the intrinsic properties of cells, and to increase the throughput of cell-based drug screening11.
The degree-of-freedom in cell patterning would greatly increase if the template pattern geometry could be altered in situ, i.e., while cells are cultured on the surface. The conventional methods for pattern formation cannot be directly applied here, since they process samples in atmosphere or in vacuum. Therefore various new surface modification techniques have been proposed, which are based, e.g., on photoreactive compounds12,13 or laser ablation5,14, just to name a few. The proposed methods have been nicely reviewed by Robertus et al.15, and more recently by Choi et al.16 and by Nakanishi17.
Here in this article, we describe a novel protocol of in-situ surface modification, which takes advantage of photocatalytic decomposition of organic molecules on a titanium dioxide (TiO2) surface18,19. In this method, a TiO2 film is inserted between the glass substrate and the organic film that interfaces the cells, and the organic film is decomposed in situ by locally irradiating ultraviolet (UV) light to a region of interest (&#955; &#60; 388 nm). We show that the new protocol can be used to create micropatterns of extracellular matrix proteins and living cells both ex situ and in situ. TiO2 is biocompatible, chemically stable, and optically transparent, features of which makes it friendly to introduce in cell-culture experiments. This protocol provides a materials science-based alternative for modifying cell-culture scaffolds in cell-culture environment.

<table><tbody><tr><td>Glass coverslip</td><td>Warner Instruments</td><td>CS-15R15</td><td>15 mm diameter, #1.5 thickness</td></tr><tr><td>Diamond scriber</td><td>Ogura Jewel Industry</td><td>D-Point Pen</td><td></td></tr><tr><td>RF sputtering system</td><td>ANELVA</td><td>SPC350</td><td></td></tr><tr><td>TiO&lt;sub&gt;2&lt;/sub&gt; sputtering target</td><td>Kojundo Chemical Lab</td><td>Titanium (IV) oxide, target</td><td>Purity, 99.9%</td></tr><tr><td>Plasma reactor</td><td>Yamato</td><td>PR301</td><td></td></tr><tr><td>n-octadecyltrichlorosilane (OTS)</td><td>Aldrich</td><td>104817</td><td></td></tr><tr><td>Toluene</td><td>Wako</td><td>204-01866</td><td></td></tr><tr><td>Tissue-culture dish (35&amp;#160;mm)</td><td>Greiner</td><td>627160</td><td></td></tr><tr><td>Tissue-culture dish (60&amp;#160;mm)</td><td>BD Falcon</td><td>353002</td><td></td></tr><tr><td>Type-IV collagen</td><td>Nitta Gelatin</td><td>Cellmatrix Type IV</td><td></td></tr><tr><td>D-PBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM)</td><td>Gibco</td><td>11885-084</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>12483-020</td><td>Heat-inactivate and pass through a 0.22&amp;#160;&amp;#956;m filter before use</td></tr><tr><td>Horse serum</td><td>Gibco</td><td>26050-088</td><td>Pass through a 0.22&amp;#160;&amp;#956;m filter before use</td></tr><tr><td>Penicillin-streptomycin (100x)</td><td>Nacalai tesque</td><td>26253-84</td><td></td></tr><tr><td>7S nerve growth factor (NGF)</td><td>Alomone Labs</td><td>N-130</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>EDTA</td><td>Dojindo</td><td>N001</td><td>Stock solution in 0.5 M</td></tr><tr><td>TiO&lt;sub&gt;2&lt;/sub&gt; nanoparticle</td><td>Tayca</td><td>TKD-701</td><td></td></tr></tbody></table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Figure 2A shows a cross-sectional scanning electron microscopy (SEM) image of the sputter-deposited TiO2 film. From the observation, thickness of the film was estimated to be approximately 150 nm. Noticeable here is the flatness of the deposited TiO2 film. Further analysis by atomic force microscopy (AFM) revealed that the root-mean-square (rms) roughness of the surface was 0.2 nm (Figure 2B).
When the TiO2 surface is modified with OTS and then immersed in a serum-containing growth medium, serum albumins adsorb onto the OTS surface and inhibit subsequent protein adsorption and cell adhesion18,20. Subsequently, when focused UV light is irradiated to the surface, reactive oxygen species (ROS) are generated at the TiO2 surface21, which decompose the OTS self-assembled monolayer (SAM) on TiO2 and remove the SAM and the adsorbed albumins from the surface. This renders the irradiated region hydrophilic and permissive to protein adsorption. Direct interaction of the ROS with albumins might be occurring simultaneously, but the decomposition of OTS should be the main reaction, since OTS SAM is interfacing albumin and TiO2. In the current protocol, an extracellular matrix protein Col-IV is supplemented in the medium during irradiation, and Col-IV adsorbs preferentially to the UV-irradiated region where albumins are removed (Figure 3A). Other proteins such as fibronectin can be patterned in the same protocol (Figure 3B), indicating the versatility of this approach.
Serum albumins, which are also present in the medium at the time of UV irradiation, should also adsorb onto the irradiated region, but the results suggest that the amount is not high as that on the intact OTS SAM. To confirm this, we investigated the difference in the affinity of BSA to UV-irradiated and intact OTS SAM by fluorescence microscopy of dye-labelled BSA. For this experiment, focused UV light was irradiated to the OTS/TiO2 sample that has not been immersed in the serum-containing medium, and then the sample was immersed in a solution of fluorescein- labeled BSA. Figure 3C shows the adsorption pattern of BSA. Brighter fluorescence in the intact OTS SAM region than the UV-irradiated area indicates that the amount of adsorbed BSA is higher in the intact region. The mechanism of preferential adsorption can be understood by the difference in hydrophobicity of the two regions. The amount of BSA adsorption is known to be higher on hydrophobic surfaces22, and the intact OTS SAM region is more hydrophobic than the UV-irradiated region18.
The pattern of Col-IV thus created serves as a scaffold for cell adhesion. Figure 4A shows NGF-differentiated PC12 cells cultured on an ex-situ patterned region for 1 day. Next, UV light was irradiated to the side of a micropattern, and the cells were cultured for additional 5 days. As shown in Figure 4B-D, cells further migrated to the modified region, indicating that cell affinity of the surface was successfully modified in cell-culture environment.
<img alt="Figure 2" src="/files/ftp_upload/53045/53045fig2highres.jpg" width="700" /> 
Figure 2. Characterization of the TiO2 film.&#160;(A) Cross-sectional SEM image of the sample. (B) AFM image of the TiO2 surface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53045/53045fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53045/53045fig3highres.jpg" width="700" /> 
Figure 3. Site-selective adsorption of proteins onto the UV-irradiated region.&#160;(A) Fluorescein-labelled collagen (200 &#956;m octagonal pattern) and (B) rhodamine-labelled fibronectin (100 &#956;m square pattern). Preferential adsorption of these proteins relies on the absence of albumins that block protein adsorption (C). Note that in (C), OTS SAM was not immersed in serum-containing medium prior to application of fluorescein-labelled BSA. Scale bars, 100 &#956;m (A) and 50 &#956;m (B, C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53045/53045fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53045/53045fig4highres.jpg" width="700" /> 
Figure 4. Ex-situ and in-situ patterning of PC12 cells. (A) Adhesion of PC12 cells onto an ex-situ patterned region. Dotted octagon indicates the site of in-situ UV irradiation. (B-D) Migration of PC12 cells to the in-situ patterned region, at 2 days (B), 3 days (C), and 5 days (D) after irradiation. Scale bar, 200 &#956;m. Modified with permission from Yamamoto et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53045/53045fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K Views.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Video: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition

Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and reduction half reactions, and fast electron (hole) diffusion and charge separation. Nanowires present suitable architectures to meet these requirements. Axially segmented Ag|ZnO and radially segmented (coaxial) TiO2-Ag nanowires with a diameter of 200 nm and a length of 6-20 &#181;m were made by templated electrodeposition within the pores of polycarbonate track-etched (PCTE) or anodized aluminum oxide (AAO) membranes, respectively. In the photocatalytic experiments, the ZnO and TiO2 phases acted as photoanodes, and Ag as cathode. No external circuit is needed to connect both electrodes, which is a key advantage over conventional photo-electrochemical cells. For making segmented Ag|ZnO nanowires, the Ag salt electrolyte was replaced after formation of the Ag segment to form a ZnO segment attached to the Ag segment. For making coaxial TiO2-Ag nanowires, a TiO2 gel was first formed by the electrochemically induced sol-gel method. Drying and thermal annealing of the as-formed TiO2 gel resulted in the formation of crystalline TiO2 nanotubes. A subsequent Ag electrodeposition step inside the TiO2 nanotubes resulted in formation of coaxial TiO2-Ag nanowires. Due to the combination of an n-type semiconductor (ZnO or TiO2) and a metal (Ag) within the same nanowire, a Schottky barrier was created at the interface between the phases. To demonstrate the photocatalytic activity of these nanowires, the Ag|ZnO nanowires were used in a photocatalytic experiment in which H2 gas was detected upon UV illumination of the nanowires dispersed in a methanol/water mixture. After 17 min&#160;of illumination, approximately 0.2 vol% H2 gas was detected from a suspension of ~0.1 g of Ag|ZnO nanowires in a 50 ml 80 vol% aqueous methanol solution.

Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition

Procedures are outlined to prepare segmented and coaxial nanowires via templated electrodeposition in nanopores. As examples, segmented nanowires consisting of Ag and ZnO segments, and coaxial nanowires consisting of a TiO2 shell and a Ag core were made. The nanowires were used in photocatalytic hydrogen formation experiments.

Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and ...

Engineering

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Biology

Ambient Method for the Production of an Ionically Gated Carbon Nanotube Common Cathode in Tandem Organic Solar Cells

A method of fabricating organic photovoltaic (OPV) tandems that requires no vacuum processing is presented. These devices are comprised of two solution-processed polymeric cells connected in parallel by a transparent carbon nanotubes (CNT) interlayer. This structure includes improvements in fabrication techniques for tandem OPV devices. First the need for ambient-processed cathodes is considered. The CNT anode in the tandem device is tuned via ionic gating to become a common cathode. Ionic gating employs electric double layer charging to lower the work function of the CNT electrode. Secondly, the difficulty of sequentially stacking tandem layers by solution-processing is addressed. The devices are fabricated via solution and dry-lamination in ambient conditions with parallel processing steps. The method of fabricating the individual polymeric cells, the steps needed to laminate them together with a common CNT cathode, and then provide some representative results are described. These results demonstrate ionic gating of the CNT electrode to create a common cathode and addition of current and efficiency as a result of the lamination procedure.

A method of fabricating, in ambient conditions, organic photovoltaic tandem devices in a parallel configuration is presented. These devices feature an air-processed, semi-transparent, carbon nanotube common cathode.

A method of fabricating organic photovoltaic (OPV) tandems that requires no vacuum processing is presented. These devices are comprised of two ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Chemistry

Digital Printing of Titanium Dioxide for Dye Sensitized Solar Cells

Silicon solar cell manufacturing is an expensive and high energy consuming process. In contrast, dye sensitized solar cell production is less environmentally damaging with lower processing temperatures presenting a viable and low cost alternative to conventional production. This paper further enhances these environmental credentials by evaluating the digital printing and therefore additive production route for these cells. This is achieved here by investigating the formation and performance of a metal oxide photoelectrode using nanoparticle sized titanium dioxide. An ink-jettable material was formulated, characterized and printed with a piezoelectric inkjet head to produce a 2.6 &#181;m thick layer. The resultant printed layer was fabricated into a functioning cell with an active area of 0.25 cm2 and a power conversion efficiency of 3.5%. The binder-free formulation resulted in a reduced processing temperature of 250 &#176;C, compatible with flexible polyamide substrates which are stable up to temperatures of 350 &#730;C. The authors are continuing to develop this process route by investigating inkjet printing of other layers within dye sensitized solar cells.

This paper investigates the suitability of inkjet printing for the manufacturing of dye-sensitized solar cells. A binder-free TiO2 nanoparticle ink was formulated and printed onto a FTO glass substrate. The printed layer was fabricated into a cell with an active area of 0.25 cm2 and an efficiency of 3.5%.

Silicon solar cell manufacturing is an expensive and high energy consuming process. In contrast, dye sensitized solar cell production is less ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Morphology Control for Fully Printable Organic&#8211;Inorganic Bulk-heterojunction Solar Cells Based on a Ti-alkoxide and Semiconducting Polymer

The photoactive layer of a typical organic thin-film bulk-heterojunction (BHJ) solar cell commonly uses fullerene derivatives as the electron-accepting material. However, fullerene derivatives are air-sensitive; therefore, air-stable material is needed as an alternative. In the present study, we propose and describe the properties of Ti-alkoxide as an alternative electron-accepting material to fullerene derivatives to create highly air-stable BHJ solar cells. It is well-known that controlling the morphology in the photoactive layer, which is constructed with fullerene derivatives as the electron acceptor, is important for obtaining a high overall efficiency through the solvent method. The conventional solvent method is useful for high-solubility materials, such as fullerene derivatives. However, for Ti-alkoxides, the conventional solvent method is insufficient, because they only dissolve in specific solvents. Here, we demonstrate a new approach to morphology control that uses the molecular bulkiness of Ti-alkoxides without the conventional solvent method. That is, this method is one approach to obtain highly efficient, air-stable, organic-inorganic bulk-heterojunction solar cells.

A method for fully printable, fullerene-free, highly air-stable, bulk-heterojunction solar cells based on Ti alkoxides as the electron acceptor and the electron-donating polymer fabrication is described here. Moreover, a method for controlling the morphology of the photoactive layer through the molecular bulkiness of the Ti-alkoxide units is reported.

The photoactive layer of a typical organic thin-film bulk-heterojunction (BHJ) solar cell commonly uses fullerene derivatives as the electron-accepting ...

Electrospinning of Photocatalytic Electrodes for Dye-sensitized Solar Cells

This work demonstrates a protocol to fabricate a fiber-based photoanode for dye-sensitized solar cells, consisting of a light-scattering layer made of electrospun titanium dioxide nanofibers (TiO2-NFs) on top of a blocking layer made of commercially available titanium dioxide nanoparticles (TiO2-NPs). This is achieved by first electrospinning a solution of titanium (IV) butoxide, polyvinylpyrrolidone (PVP), and glacial acetic acid in ethanol to obtain composite PVP/TiO2 nanofibers. These are then calcined at 500 &#176;C to remove the PVP and to obtain pure anatase-phase titania nanofibers. This material is characterized using scanning electron microscopy (SEM) and powder X-ray diffraction (XRD). The photoanode is prepared by first creating a blocking layer through the deposition of a TiO2-NPs/terpineol slurry on a fluorine-doped tin oxide (FTO) glass slide using doctor blading techniques. A subsequent thermal treatment is performed at 500 &#176;C. Then, the light-scattering layer is formed by depositing a TiO2-NFs/terpineol slurry on the same slide, using the same technique, and calcinating again at 500 &#176;C. The performance of the photoanode is tested by fabricating a dye-sensitized solar cell and measuring its efficiency through J-V curves under a range of incident light densities, from 0.25-1 Sun.

The overall goal of this project was to use electrospinning to fabricate a photoanode with improved performance for dye-sensitized solar cells.

This work demonstrates a protocol to fabricate a fiber-based photoanode for dye-sensitized solar cells, consisting of a light-scattering layer made of ...

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical photocatalytic structures. However, the influence of such chemical bonding on the optical and surface properties of the photocatalyst and thus its photocatalytic activity/reaction selectivity behavior has not been systematically studied. In this investigation, TiO2 has been supported on the surface of SiO2 by means of two different methods: (i) by the in situ formation of TiO2 in the presence of sand quartz via a sol-gel method employing tetrabutyl orthotitanium (TBOT); and (ii) by binding the commercial TiO2 powder to quartz on a surface silica gel layer formed from the reaction of quartz with tetraethylorthosilicate (TEOS). For comparison, TiO2 nanoparticles were also deposited on the surfaces of a more reactive SiO2 prepared by a hydrolysis-controlled sol-gel technique as well as through a sol-gel route from TiO2 and SiO2 precursors. The combination of TiO2 and SiO2, through interfacial Ti-O-Si bonds, was confirmed by FTIR spectroscopy and the photocatalytic activities of the obtained composites were tested for photocatalytic degradation of NO according to the ISO standard method (ISO 22197&#8722;1). The electron microscope images of the obtained materials showed that variable photocatalyst coverage of the support surface can successfully be achieved but the photocatalytic activity towards NO removal was found to be affected by the preparation method and the nitrate selectivity is adversely affected by Ti-O-Si bonding.

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported TiO2.

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical ...

Developing Photosensitizer-Cobaloxime Hybrids for Solar-Driven H2 Production in Aqueous Aerobic Conditions

Developing photocatalytic H2 production devices is the one of the key steps for constructing a global H2-based renewable energy infrastructure. A number of photoactive assemblies have emerged where a photosensitizer and cobaloxime-based H2 production catalysts work in tandem to convert light energy into the H-H chemical bonds. However, the long-term instability of these assemblies and the need for hazardous proton sources have limited their usage. Here, in this work, we have integrated a stilbene-based organic dye into the periphery of a cobaloxime core via a distinct axial pyridine linkage. This strategy allowed us to develop a photosensitizer-catalyst hybrid structure with the same molecular framework. In this article, we have explained the detailed procedure of the synthesis of this hybrid molecule in addition to its comprehensive chemical characterization. The structural and optical studies have exhibited an intense electronic interaction between the cobaloxime core and the organic photosensitizer. The cobaloxime was active for H2 production even in the presence of water as the proton source. Here, we have developed a simple airtight system connected with an online H2 detector for the investigation of the photocatalytic activity by this hybrid complex. This photosensitizer-catalyst dyad present in the experimental setup continuously produced H2 once it was exposed in the natural sunlight. This photocatalytic H2 production by the hybrid complex was observed in aqueous/organic mixture media in the presence of a sacrificial electron donor under complete aerobic conditions. Thus, this photocatalysis measurement system along with the photosensitizer-catalyst dyad provide valuable insight for the development of next generation photocatalytic H2 production devices.

Developing Photosensitizer-Cobaloxime Hybrids for Solar-Driven H2 Production in Aqueous Aerobic Conditions

We have directly incorporated a stilbene-based organic dye into a cobaloxime core to generate a photosensitizer-catalyst dyad for photocatalytic H2 production. We have also developed a simple experimental setup for evaluating the light-driven H2 production by photocatalytic assemblies.

Developing photocatalytic H2 production devices is the one of the key steps for constructing a global H2-based renewable energy infrastructure. A number ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO₂ Photocatalysis

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