Overview of SCHOTT’s protein micro-array slide surfaces

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Choosing the best slide coating chemistry for your protein micro-array application. Poster presented at the Advances in Microarray Technology 2008 conference in Barcelona, Spain.

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Overview of SCHOTT’s protein micro-array slide surfaces

  1. 1. Substrate formats <ul><li>SCHOTT Nexterion ® provides a broad range of high quality substrates suited for protein microarrays </li></ul><ul><li>For almost all protein array applications SCHOTT Nexterion ® offers the ideal solution </li></ul><ul><li>SCHOTT Nexterion ® is established in the microarray market as the substrate specialist since 8 years and is strongly committed to this area </li></ul><ul><li>Lables, marks, barcodes etc. can be applied for customized slide layout </li></ul>SCHOTT’s protein microarray surfaces: Choosing the best chemistry for your application Dietmar Knoll, Corinna Schwung, Alistair Rees and Rüdiger Dietrich Schott Jenaer Glas GmbH Microarray Solutions Germany, 07745 Jena, Otto-Schott-Straße 13, +49 3641 91969 PepTalk 2008 SCHOTT Nexterion ® - Microarray Solutions MTP 96 well SBS microplate format 16 pad slide Customized 8 well slide (customized) 2 pad slide The lower number of protein species to be printed allows smaller array sizes. This allows lower assay volumes and finally cost/time savings due to multiplexing. Protein microarray substrates 3-D thick film coatings (0,1 – 200µm) 3-D thin film coatings (10 – 100nm) Nitrocellulose, Agarose coatings Polymer and hydrogel type Conventional 2-D substrates 3-D thick film coatings-Nitrocellulose Examples 1. NC (Nexterion ® NC-W and -D) 2. Agarose Remarks 1. Nitrocellulose is known since decades 2. High print buffer tolerance (e.g. lysate arrays) 3. Stabilization of protein structure (long shelf life) 4. High background values 5. NC coating not compatible with underlaying signal enhancing coatings (e.g. evanescent waveguide) 3-D thin film coatings Benchmarktest: Binding capacity and Signal intensities <ul><li>Higher signal intensities for 3-D surfaces especially for Nitrocellu-lose slides </li></ul><ul><li>Thin film 3-D substrates have higher binding capacities than conventional 2-D substrates </li></ul><ul><li>Highest signal intensities for NC-W ( w hite) due to higher autoflou- rescence of the white nitrocellulose </li></ul><ul><li>The black pigment inside the NC-D ( d ark) significantly reduces the autoflourescence </li></ul>Benchmarktest: Background <ul><li>Highest background for NC-W slide </li></ul><ul><li>Lower background for NC-D slide </li></ul><ul><li>Very low unspecific binding for slide P and H </li></ul><ul><li>Low background signals for con- </li></ul><ul><li>ventional 2 D substrates </li></ul>printed probe: anti osteopontin Ab – target: Osteopontin S/B ratio printed protein concentration in mg/ml S/B ratio printed protein concentration in mg/ml Benchmarktest: Signal/Background ratio Score of the benchmark test Summary Data have been normalized to allow data comparison printed probe: rabbit anti goat Ab – target: IgG Goat (3,125µg/ml) signal intensity colours refer to antibody concentration printed signal intensity colours refer to target concentration (IgG goat) printed probe: rabbit anti goat Ab (1,2mg/ml) – target: IgG Goat colours refer to target con- centration (osteopontin) colours refer to target con- centration (osteopontin) <ul><li>Highest S/B ratios for slide P and H </li></ul><ul><li>S/B ratios of Nitrocellulose slides and conventional 2D slides comparable </li></ul><ul><li>Dark NC slide has higher S/B ratios than NC-W due to reduced background values </li></ul><ul><li>Excellent correlation between probe and target concentration and signal intensities for all slides </li></ul>  Next® slide H   Next® slide P   Next® slide NC-W Next® slide NC-D Next® slide E Next® slide AL Bind. capacity and signal int. (0,25) +++ +++ ++++ +++ +++ ++ Signal/Backgr. ratio (0,25) ++++ ++++ ++ +++ +++ ++ Probe target linearity (0,2) +++ +++ ++++ ++++ +++ ++ Sensitivity (0,15) ++++ ++++ ++ +++ +++ ++ Print buffer tolerance (0,15) ++ ++ ++++ ++++ +++ ++ Storage of printed slides -20°C -20°C RT RT - 20°C -20°C Standard 25 x 75 mm slide                        SCHOTT Nexterion conventional 2D surfaces (– 10nm) metallic surfaces 3-D surfaces Epoxy,- Aldehydesilane Gold coatings CH 2 CH O l CH 2 CH O l CH 2 CH O l epoxy groups Immobilization •• O NH 2 OH NH P P <ul><li>Remarks </li></ul><ul><li>Robust and reliable surfaces </li></ul><ul><li>Low support of protein structure </li></ul><ul><li>Storage of printed arrays at –20°C </li></ul><ul><li>Examples </li></ul><ul><li>Epoxysilane (Nexterion ® E) </li></ul><ul><li>Aldehydesilane (Nexterion ® AL) </li></ul>Scanning electrone microscope photograph of SCHOTT Nexterion ® NC slide Schott Nexterion ® NC-W/D slides Schematical view of increased capacity by pore structure cross linking compound reactive group polymer - selected to provide lowest unspecific binding and to stabilize protein structure <ul><li>Examples </li></ul><ul><li>Hydrogel coatings (Nexterion ® H) </li></ul><ul><li>Polymer coatings (Nexterion ® P) </li></ul><ul><li>Remarks </li></ul><ul><li>Very low unspecific binding </li></ul><ul><li>Very high S/N ratios </li></ul><ul><li>Stabilization of protein structure </li></ul><ul><li>Tight print buffer tolerance </li></ul><ul><li>Storage of printed arrays at –20° - 4°C </li></ul>slide H slide P slide E slide AL slide NC-W slide NC-D signal intensity colours refer to antibody concentration printed slide H slide P slide E slide AL slide NC-W slide NC-D signal intensity colours refer to target concentration (IgG goat) slide H slide P slide E slide AL slide NC-W slide NC-D slide H slide P slide E slide AL slide NC-W slide NC-D slide H slide P slide E slide AL slide NC-W slide NC-D slide H slide P slide E slide AL slide NC-W slide NC-D

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