Microcontact Printing
Figure 1 : Octadecanethiol monolayer on silver. (a) Diamminesilver (Ag(NH 3 ) 2 ) is reduced to Ag metal, evidenced by the mirror-like slide bottom, (b) this silver layer is then coated with octadecanethiol, which makes the surface hydrophobic, and (c) water hydrophobicity is demonstrated on a newly formed silver coat. Images (a), (b), and (c) show these results on different prepared surfaces. (d) Distinct behavior of water droplets on side-by-side surfaces: thiol-coated on the left, glass on the right. Images are from a movie posted online by the MRSEC group ( https://
www.youtube.com/watch?v=SvgEd71nHUg .). Notice that the process is the same for regular slides or coverslips.
materials science and nanotechnology research. The group is supported by the UW Interdisciplinary Education Group (UW MRSEC IEG), funded by NSF (National Science Foundation). The missions of the group are dedicated to making complex knowledge available to the public and, at the same time, to stimulate the potential for the resolution of modern and old problems, all under an interdisciplinary umbrella. The UW MRSEC Education Group (IEG) creates and distributes a broad range of educational products that are widely used for K–12 and college-level instruction on topics that build on the center’s materials science discoveries and expertise.
Bacterial plant pathogen . Auburn University’s Department of Entomology and Plant Pathology is the home of a plant pathology lab researching the ionomics of the plant pathogen Xylella fastidiosa and the role of trace elements and nutrients in the infection and progression of disease. T e lab is especially interested in infection processes, phases in host colonization, biofi lm formation, and molecular character- ization of bacterial plant pathogens. T ere is a particular aspect of the interaction of X. fastidiosa that poses a major concern for these investigators. T e question is related to the fact that cell attachment and biofi lm formation have been shown to be aff ected by the xylem chemistry. T e group is investigating the infl uence of calcium, calcium chelators, and ions [ 6 , 7 ]. It is this work where artifi cially prepared surfaces could have a major impact.
Materials and Methods T iol surfaces . For this investigation, we printed an octadecanethiol pattern on gold to assess a pathogen’s prefer- ential binding to this highly hydrophobic surface. Glass slides
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were washed with water, ethanol, and acetone. T e dry glass slides were coated with a thin layer of gold aſt er sputtering for 1 minute. Polydimethylsiloxane (PDMS) stamps were prepared by pouring Sylgard 184 silicone elastomer onto a microfl uidic channel mold and curing at 100°C for 1 hour. Aſt er curing, the pattern was cut away from excess silicone. A few drops of octadecanethiol in ethanol (~1 mg/mL) were placed on the pattern and allowed to dry. T e thiol- coated PDMS was then placed on the gold-coated slide, and light pressure was applied to ensure thiol transfer between the PDMS and substrate. T e PDMS stamp was carefully removed from the substrate, leaving behind a layer of hydrophobic octadecane molecules. T e method promotes localized hydrophobicity by bonding the thiol group to gold and/or silver surfaces, leaving hydrophobic tail exposed ( Figure 1 ). In order to evaluate bacterial
biofi lm formation, we compared X. fastidiosa cultures aſt er growth on a rich growth medium (PD2) by inoculating PD2 medium (control), PD2 medium supplemented with calcium (CaCl 2 ), and PD2 medium plus EGTA (a calcium chelator that binds to calcium). Self-assembled monolayers . T e self-assembled mono- layers of thiolates on metals is becoming an exceptional form of nanotechnology, as anticipated by a group from University of Illinois – Champaign, Urbana [ 8 ]. Self-assembled monolay- ers (SAMs) are convenient, fl exible, and easy to replicate. Assembled monolayers of thiol on top of silver, using a US penny coin pattern ( Figure 2 ), as well as a designed pattern coated with gold ( Figure 3 ), were analyzed in preparation for quantitative biofi lm studies. T e penny experiment was a proof of concept experiment; additional data were generated with the experiment employing the micro fl uidity chamber pattern.
Bacterial growth . Microscope cover slips (1 cm 2 ) previously sputter-coated with gold were printed with octadecanethiol. T ese coated and printed cover slips were sterilized twice with ethanol inside the laminar fl ow hood. Subsequently, slides were placed inside Falcon tubes containing PD2 [ 6 ] growth media and incubated for 5 days at 28°C. Slides were removed, dried inside the laminar fl ow hood, and fi xed with osmium tetroxide. Treatments were: (1) control PD2 medium + Xylella fastidiosa (inoculated media used as positive control); (2) PD2 + 2mM CaCl 2 + Xylella fastidiosa (complex media PD2 supplemented with calcium); and (3) PD2 + 1.5 mM EGTA + Xylella fastidiosa (complex media PD2 supplemented with the calcium chelator EGTA). T e inoculation and growth were carried out at Auburn University laboratories.
www.microscopy-today.com • 2015 September
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