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FAQ - General questions
What is the difference between SCHOTT Nexterion® Glass B and D?
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Nexterion® Glass B is a borosilicate glass that is produced by the microfloat process, resulting in a top fire-polished surface; and a lower surface that has been in contact with the molten tin. On customer request SCHOTT indicates which is the top (fire polished) side of the Nexterion® Glass B slides, and all the slides are kept in the same orientation throughout processing and packing.
The refractive index of Glass B is 1.47 (nd λ = 546nm and ne λ = 588nm).
Nexterion® Glass D is also a borosilicate glass but it is produced by a special down-draw production process that results in two fire-polished surfaces that can be used without any additional processing.
The refractive index of Glass B is 1.52 (nd λ = 546nm and ne λ = 588nm).
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Can SCHOTT offer any advice on the best method of bonding Glass D (D263) to Polydimethylsiloxane (PDMS)?
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The three most common systems / methods used by SCHOTT customers to bond D263 to PDMS are:
- AutoGlow Plasma System
http://www.glowresearch.org/applications.html
- March Plasma treatment system
http://www.marchplasma.com/products.htm
Example of a protocol:
http://engineering.tufts.edu/microfab/index_files/SOP/PDMS_GlassBond_SOP.pdf
- Jelight UV-ozone cleaner
"PDMS elastomer valves are formed by activating both sides of the PDMS membrane (254-ím-thick HT-6240, Bisco Silicones, Elk Grove, IL) with an UV ozone cleaner (Jelight Co. Inc., Irvine, CA)
for 1.5 min to improve PDMS-glass bonding and then sandwiching the membrane between the manifold and the bonded channel wafers."
http://www.cchem.berkeley.edu/ramgrp/alpha/people/Alumni/nick/PCR%20Anal%20Chem%202006.pdf
http://www.jelight.com/uvo-ozone-cleaning.php
"The valves were assembled after bonding in a manner similar to that outlined by Grover et al.27 Briefly, a PDMS membrane (254-ím-thick HT-6240, Bisco Silicones, Elk Grove, IL) was applied to
the bonded channel/RTD wafer stack.glass manifold and spacer were separately cleaned in a UV-ozone cleaner (Jelight Co. Inc., Irvine, CA) and then irreversibly bonded at room temperature for 2 h."
http://www.cchem.berkeley.edu/ramgrp/alpha/people/nick/Pathogen%20Anal%20Chem%202004.pdf
http://www.ocf.berkeley.edu/~wgrover/monolithic_membrane_valves_and_diaphragm_pumps.pdf
http://www.escholarship.org/uc/item/4cj37381
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What is the difference between the three levels of cleanliness of the SCHOTT Nexterion® uncoated slides?
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Please click here for an overview on the levels of cleanliness.
Uncleaned:
These slides are cleaned using deionised water and a conventional washing machine with brushes.
SCHOTT recommends uncleaned slides if the user intends to subject the slides to a thorough cleaning procedure prior the further processing.
Ultrasonically cleaned:
SCHOTT offers as standard uncoated slides that are ultrasonically cleaned. These slides are cleaned ultrasonically under alkaline conditions to remove all particles, debris, and surface contaminants. The slides are also subjected to a 100% QC process (the same standard process used for Nexterion® coated slides) to validate dimensional tolerances and ensure a particle-free surface on every slide produced.
SCHOTT recommends ultrasonically cleaned slides if a basic cleaning procedure is used prior the further processing.
Cleanroom cleaned:
SCHOTT’s highest grade of uncoated slide is cleaned ultrasonically and quality controlled, as detailed in the Ultrasonically cleaned section above. In addition, the slide storage boxes used to transport the slides are sealed in protective foil pouches under inert atmosphere in a class 100 cleanroom environment. The slides can be used immediately from the sealed boxes without subjecting them to a cleaning process.
Cleanroom cleaned slides are recommended if users intend to coat the slides without carrying out any cleaning steps.
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What barcode options can SCHOTT offer?
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The standard barcode is a special laser bonded foil barcode, but also a label barcode is available. The barcodes are fully compatible with commercial automated hybridization stations, and are robust enough to withstand standard hybridization and washing procedures. The barcodes conform to Code 128, and are readable with all commonly available microarray scanners and hand-held barcode readers.
In addition SCHOTT can offer customers the opportunity to customise their slides and glass substrates with graphics, logos, company names, barcodes, reference marks, or 2-D matrix codes. These markings may be added at any location on, or within the glass surface, and may feature a combination of items, for example a company logo with a sequential barcode. Please contact us to discuss your requirements.
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Different barcodes and logos at Nexterion® glass substrates
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Can SCHOTT Microarray Solutions offer custom logos?
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Yes, SCHOTT can offer customers the opportunity to customise their slides and glass substrates with graphics, logos, company names, barcodes, reference marks, or 2-D matrix codes.
Please look at the section above.
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How are the Nexterion® microarray subtrates packaged?
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The Nexterion® substrates are packed in 5, 25 or 30-slide containers. The boxes are made of a specially developed plastic material to minimise out-gassing, and maintain the slide properties.
Nexterion coated slides and cleanroom cleaned uncoated substrates are additional sealed in tough protective laminated foil pouches under an inert atmosphere.
The specially developed packaging protects the slides from damage due to breakage and external contamination. It also offers protection from the adverse effects of light and humidity during transportation and long-term storage.
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Packaging of Nexterion® Glass Substrates / Plastic Boxes
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Packaging of Nexterion® Glass Substrates / Foil Pouch
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How firmly are the SCHOTT Nexterion® coatings attached to the glass?
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The Nexterion® silane coatings are generally resistant to most aqueous buffer systems, and many organic solvents, (such as DMSO, alcohols like iso-propanol, ethanol, methanol, and ketones like acetone or toluene), but obviously SCHOTT has not been able to test all possible solvents. The Nexterion® silane coatings will remain attached to the glass in solutions with pHs of between 2 and 10. Delamination of the silane coating is only observed after a prolonged treatment under highly alkaline conditions (over pH 11). SCHOTT coatings remain stable under a large range of temperature as well: For the slide E, AL, A+ and AStar coatings - temperatures of -20°C up to 100°C will not affect the coating. However, please note that these statements refer to the silane bonds to the glass surface. The active groups of the coatings may well be adversely affected by some of these treatments.
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| Coating |
Dye Used |
FLOSS Result
molecules/cm2 |
UV-Vis Result
molecules/cm2 |
A+
Aminosilane |
L1908
(Sulfonyl Chloride) |
(1.0 +/- 0.3)
x1012 |
Below detection limit
<8.4 x 1011 |
E
Epoxysilane |
D2371
(-NH2 modified) |
(5.6 +/- 0.3)
x1012 |
3.7 x1012 |
AL
Aldehydesilane |
D2371
(-NH2 modified) |
(4.8 +/- 0.3)
x1012 |
2.7 x1012 |
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What factors should be considered when selecting dyes?
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Dyes can be purchased from Molecular Probes, Inc., (Alexa), Atto Tec, Siegen, Germany (ATTO 550 and ATTO 647) or Amersham (Cy 3 and Cy 5). Dyes of course differentiate in their quantum efficiency, stability against oxidation and bleaching, reactivity and quality. Ideally one will use dyes that have a comparable signal yield for two colour experiments. We personally have good experiences with ATTO dyes in this regard.
Crucial for the quality in labeling experiments is the purity and the activation degree (NHS-ester) of the dyes. Both factors influence the background and signal intensity. Important is to store the dyes in water free solvents. Amino groups are far more reactive with the NHS ester than water (more than 1000fold). That’s why the labeling can be done in buffered solutions.
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How can I increase the cDNA labeling efficiency when using the aminoallyl-method?
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For most applications the optimal labeling level is about one dye molecule per 12 to 18. If the dye/base ratio is higher, hybridization efficiency may drop (due to steric hindrance by the dyes) and dye to dye fluorescence quenching may occur.
The labeling efficiency is influenced by the reaction time/temperature, dye concentration, template purity, template concentration, type of dye etc. Free amines like Tris-buffer, ammonium acetate, hydroxylamine or proteins will interfere with the dye coupling and should be avoided. Make sure the pH (7,5-9) to guarantee optimal reactivity. Additionally, succinimidyl ester dyes hydrolyze spontaneously in the presence of moisture. It is important to keep the dyes anhydrous and dissolve it only in an anhydrous solvent (usually DMSO). Protect dyes from light (cover tubes with aluminum foil) and oxidation.
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What factors affect Cy3/Cy5 relation?
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You should not expect a constant Cy3/Cy5 ratio over a wide signal range. The Cy3 and Cy5 dyes have different molar extinction coefficients and quantum efficiencies. Fluorescence intensities of the concentration of spots from each tend to produce a sigmoidal shaped curve. However, the sigmoidal shape is not identical for each dye and is more importantly influenced by environmental factors as - pH, oxygen content, temperature, buffer strength, quenching, solvatization, etc. If any of these variables change, the intensity of fluorescence changes differently for the two complexes. Therefore it is essential to keep the same conditions for each experiment.
Additionally the reactivity from boths dyes to DNA is differently. The AT content has been shown to affect uptake rate differently between Cy3 and Cy5. Moreover, some scanners (e.g. Tecan LS400) have different laser powers for the red and green channel.
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| Spectral line |
Measurement wavelength (nm) |
Refractive index |
| C line |
643.8 |
1.46953 |
| d line |
589.3 |
1.47140 |
| e line |
546.1 |
1.47311 |
| F line |
479.9 |
1.47676 |
| g line |
435.8 |
1.48015 |
| h line |
404.7 |
1.48330 |
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Measured at air pressure: 1013.25 hPa / temperature: 22.0 °C
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| Spectral line |
Measurement wavelength (nm) |
Refractive index |
| g line |
435.8 |
1.5354 |
| n F´ |
479.9 |
1.5305 |
| F line |
486.1 |
1.5300 |
| e line |
546.1 |
1.5255 ± 0.0015 |
| e line |
587.6 |
1.5231 |
| n D |
589.3 |
1.5230 |
| n C´ |
643.9 |
1.5209 |
| C line |
656.3 |
1.5204
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Measured at air pressure: 1013.25 hPa / temperature: 22.0 °C
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Which excitation and emission wavelengths has the Glass B and D been tested with for auto-fluorescence?
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The glass slides have been tested by SCHOTT at the Cy3 (excitation 555 nm, emission 570 nm) and Cy5 (excitation 649 nm, emission 670 nm) wavelengths.
The autofluorescence of Glass B (Borofloat 33) has also been tested by the Department of Chemistry, at University of Cincinnati:
Lab Chip, 2005, 5, 1348 - 1354,
DOI: 10.1039/b508288a
The autofluorescence of plastic materials and chips measured under laser irradiation
Aigars Piruska, Irena Nikcevic, Se Hwan Lee, Chong Ahn, William R. Heineman, Patrick A. Limbach and Carl J. Seliskar
http://www.rsc.org/publishing/journals/LC/article.asp?doi=b508288a
http://www.rsc.org/delivery/_ArticleLinking/DisplayArticleForFree.cfm?doi=b508288a&JournalCode=LC
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What is the shelf life of the Nexterion® coated and uncoated slides? What are the recommended storage conditions for the slides?
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Here are the shelf lives of the Nexterion products when stored in the original packaging under the recommended conditions.
Coated substrates
- Nexterion® Slide A and other aminosilane coated substrates 12 months
(Store at 20 to 25°C)
- Nexterion® Slide AStar and other aminosilane coated substrates 12 months
(Store at 20 to 25°C)
- Nexterion® Slide A+ and other aminosilane coated substrates 6 months
(Store at 20 to 25°C)
- Nexterion® Slide AL and other aldehydesilane coated substrates 9 months
(Store at 20 to 25°C)
- Nexterion® Slide E and other epoxysilane coated substrates 12 months
(Store at 20 to 25ºC)
- Nexterion® Slide H and other H-coated substrates 12 months
(Store frozen at -20°C)
- Nexterion® Slide P and other P-coated substrates 12 months
(Store frozen at -20°C)
- Nexterion® Slide NC-C/N and other nitrocellulose coated substrates 12 months
(Store at 20 to 25°C)
Uncoated slides
- Nexterion® Glass B (clean room cleaned) 24 months (Store at 20 to 25°C)
- Nexterion® Glass D (clean room cleaned) 24 months (Store at 20 to 25°C)
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What is the thickness and micro-roughness of the SCHOTT Nexterion®
Slide H coating?
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The coating on Nexterion® Slide H has been measured using atomic force microscopy (AFM) and ellipsometry. As the coating swells in the presence of water it has been measured in both the dehydrated and hydrated state.
The dehydrated Nexterion® Slide H coating is approximately 10 - 20 nm thick with a peak-to-trough roughness of less than 1 nm (rms roughness = 0.29 nm), similar to the surface of the underlying glass.
After 30 minutes hydration, the film thickness stabilizes at 50 - 100 nm, and has a final peak-to-trough roughness of approximately 10 - 20 nm (rms roughness = 3.20 nm).
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How do proteins interact with, and bind to nitrocellulose coated slides?
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Protein binding to nitrocellulose is based on a variety of different forces, including hydrophobic interactions, electrostatic interactions and hydrogen bonding. However, the quantitative contribution of each force to the binding between proteins and nitrocellulose varies under different conditions (such as type of print buffer, protein type etc.), and is still not fully understood. Several models on how proteins are gradually attached and stably bound to the membrane structure are discussed in the literature.
The most accepted model suggests that proteins are initially attracted to a membrane surface by electrostatic interaction. Long-term attachment is achieved by a combination of hydrogen bonding and hydrophobic interactions. Internal R&D tests have shown that a major part of protein fixation on nitrocellulose is mediated by hydrophobic interaction, since the use of 1% non-ionic detergent is able to wash away about 90% of the pre-bound protein. In addition, proteins can also be eluted with high salt (1 M) or by a strong variation of pH.
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How can we optimize the conditions for protein binding to nitrocellulose surfaces?
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Normally the binding to the nitrocellulose membrane is very easy to achieve. However, in cases where the isoelectric point (pI) is only achieved at very low or high pHs, or if the proteins are very small, conditions may need to be fine tuned to improve or optimise binding to nitrocellulose surfaces. As several different forces (hydrophobic interactions, electrostatic interactions and hydrogen bonding) contribute to the binding between protein and nitrocellulose, the optimal conditions for proper binding, as well as maintaining protein activity (e.g. enzymes, antibodies), have to be carefully validated in biochemical assays. For optimal protein binding, the pH of the printing buffer should be close to isoelectric point of the printed protein. In order to receive stable attachment of the proteins, a drying step immediately after printing and before any additional processing steps (e.g. blocking), is strongly recommended.
Different proteins show varying binding affinities to nitrocellulose (see comment about isoelectric point above). Low molecular weight proteins show less affinity (due to fewer potential binding structures) and might need to be “pre-precipitated” (partially denatured) by alcohol (up to 5%), in order to tighten their interaction with the nitrocellulose. This technique is commonly used in the diagnostic industry during the manufacture of lateral flow assays, and is believed to result from an unfolding of hydrophobic parts of the bio molecule due to the presence of a less polar solvent. It is thought that hydrophobic amino acids are brought closer to the protein surface resulting in increased surface area for hydrophobic interactions with the nitrocellulose. This technique may increase the risk of false positives, and assays will need to be performed to determine whether this step has any influence on the biological activity of the molecule.
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SCHOTT has changed the name of the Nexterion® nitrocellulose slides, what are now the differences between the slides?
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- Nexterion® Slide NC-D (D = "Dark" grey)
This slide was made with nitrocellulose containing a black pigment that reduced auto-fluorescence. This product has been discontinued.
- Nexterion® Slide NC-N (N = "Non-contact" printers)
This slide is identical to the Nexterion® Slide NC-W (W = "White") product. It was renamed as this nitrocellulose coating produces a superior performance with "non-contact" type printers. The product ordering codes remain the same.
- Nexterion® Slide NC-C (C = "Contact" printers)
This slide has membrane pads manufactured with the recently introduced "small pore" nitrocellulose. This nitrocellulose membrane contains a higher solids content, and has a more dense structure making it particularly well suited for use with "contact" type microarray printers. This surface works well with applications in which the probes are suspended in printing buffers that contain high concentrations of detergents, such as those used to array cell lysates.
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Why and how did SCHOTT decide that the Nexterion® Slide NC-N/C nitrocellulose pads should be 11 microns thick?
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There is a potential loss of fluorescence signal intensity from the labelled probes if they lie deep within the matrix of the nitrocellulose structure. It was determined empirically that 11 microns was the optimum thickness to use for nitrocellulose coated slides. To measure the effect of depth on signal intensity, 4 nL of 10µg/mL Cy-3 labelled anti mouse IgG was applied to a nitrocellulose membrane containing a light (photon) absorbing pigment. After spotting, analysis of the confocal signal intensity showed that fluorescence photons came from a maximum depth of 10 µm within the membrane. Fluorophors that lie below the surface do contribute to the overall signal, but with a reduced quantum yield the lower they are, due to light absorption within the membrane.
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Contact
SCHOTT Technical Glass Solutions GmbH
Otto-Schott-Strasse 13
07745 Jena
Germany
 | +49 (0)3641/681-4066 |
 | +49 (0)3641/681-4970 |
E-mail
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