This work was funded in part by grants from the South Texas Technology Management (POCrr 2009.041), the Institute for Integration of Medicine and Science from the National Center for Research Resources (UL 1RR025767), and from the National Institute of Dental &amp; Craniofacial Research (5R21DE017294).

1. Preparation of Functionalized Slides
<ol>
 <li>Place the microscope slides in a removable slide rack, and wash twice by immersing in a staining jar containing 99% ethanol (histological grade). Wipe the slides clean using paper towels (ensuring not to generate paper dust), and dry using a jet of compressed nitrogen gas.</li>
</ol>
NOTE: Do not use Kim-Wipes to wipe the slides as it would generate fine paper dust.
<ol start="2">
 <li>Immerse the slide rack containing the sl...

<ol>
	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

Jose L. Lopez-Ribot and Anand K. Ramasubramanian own equity in MicrobeHTS Technologies, Inc., which is developing antifungal agents. MicrobeHTS Technologies, Inc. provided no financial support for these studies.

We have developed a cell-based high-density microarray, CaBChip, consisting of nanoliter volumes of Candida albicans biofilms. The microarray was printed on modified glass substrates, which allowed for robust attachment of collagen gel spots while providing hydrophobicity necessary for a non-spreading, hemispherical 3D gel. A single CaBChip can replace approximately eight 96-well plates, and several chips can be printed and processed at the same time. The chip utilizes nano-scale cultures...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>3-aminopropyltriethoxysilane (APTES)</td>
 <td>Sigma-Aldrich</td>
 <td>440140 </td>
 </tr>
 <tr>
 <td>Polystyrene-Co-Maleic Anhydride (PS-MA) </td>
 <td>Sigma-Aldrich </td>
 <td>426946</td>
 </tr>
 <tr>
 <td>Glass microscopy slides </td>
 <td>Fisher Scientific </td>
 <td>12-549-3</td>
 </tr>
 <tr>
 <td>Rat Tail collagen type I</td>
 <td>BD Biosciences </td>
 <td>354236</td>
 </tr>
 <tr>
 <td>Robotic Microarrayer</td>
 <td>Omnigrid Micro </td>
 <td>MICROSYS4000/4100A</td>
 </tr>
 <tr>
 <td>Microarray Scanner </td>
 <td>Genepix Personal 4100A </td>
 <td>GENEPIX4100A</td>
 </tr>
 <tr>
 <td>Hybridization Cassette</td>
 <td>ArrayIt Corporation </td>
 <td>AHCXD</td>
 </tr>
 <tr>
 <td>FUN1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo&#x200B;-1,3-thiazol-2-yl)-methylidene)-1-phenyl&#x200B;quinoliniumiodide] </td>
 <td>Invitrogen Corp. </td>
 <td>F-7030</td>
 </tr>
 <tr>
 <td>Fluconazole </td>
 <td>Sicor Pharmaceuticals, Inc. </td>
 <td>J02AC01 </td>
 </tr>
 <tr>
 <td>Amphotericin B</td>
 <td>Sigma</td>
 <td>A2411 </td>
 </tr>
 <tr>
 <td>RPMI-1640</td>
 <td>Mediatech, Inc.</td>
 <td>50-020-PC</td>
 </tr>
 <tr>
 <td>Ceramic Tip 190 &mu;m orifice</td>
 <td>Digilab</td>
 <td>60020441-00</td>
 </tr>
 <tr>
 <td>GraphPad Prism Software </td>
 <td>GraphPad Software, Inc. </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Genepix Pro V4.1</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

candida albicans biofilm chip (cabchip) for high-throughput antifungal drug screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Watch this Scientific Journal Video about Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening at JoVE.com

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

candida-albicans-biofilm-chip-cabchip-for-high-throughput-antifungal

Research

JoVE Journal

Bioengineering

13.6K visualizações.  University of Texas at San Antonio. Nós desenvolvemos uma plataforma de microarray de alta densidade que consiste em 3D nano-biofilmes de C. albicans Chamada Ca BChip. O perfil de susceptibilidade de drogas testadas em um Ca BChip é comparável com o modelo de placa convencional de 96 poços, sugerindo que o chip fúngica é idealmente adequado para o rastreio de alto rendimento real de drogas antifúngicas.