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I`m trying to immobilize an antibody onto the glass surface by means of GPTOMS (glycidoxypropyl trimetoxy silane). Using the same protocol (silanization in toluene) I get different results any time, what are key points to check?

Looking through literature on immobilization, I see a lot of articles where authors use APTES (aminopropyl triethoxy silane) and only a few using GPTOMS, why? Epoxy group seems so atractive...

For toluene:

  1. Rince slides with water
  2. Ultrasonicate them in isopropyl alcohol for 3 min, dry them
  3. Put for 3 minutes into piranha solution
  4. Rinse with distilled water
  5. Treat with oxygen plasma for 3 min
  6. Slowly put slides into 2 or 4 % silane solution in toluene, incubate for 3 min
  7. Rince with toluene and isopropyl alcohol.
  8. Put them on dry heater (max temp. of ours is 97 C, most protocols say 110 C), incubate an hour

For ethanol:

  1. Rince slides with water
  2. Ultrasonicate them in isopropyl alcohol for 3 min, dry them
  3. Put for 3 minutes into piranha solution
  4. Rinse with distilled water
  5. Treat with oxygen plasma for 3 min
  6. Put slides slowly into 2% silane in ethanol solution (silane + alcohol + acetic acid (pH 4,5-5,5)), incubate 3 minutes
  7. Rince slides with ethanol
  8. Put them on dry heater (max temp. of ours is 97 C, most protocols say 110 C), incubate an hour
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  • $\begingroup$ At a guess, surface preparation and contamination before applying GPTOMS? Glass adsorbs monolayers from detergents, water and anything else around. $\endgroup$ Sep 22 '20 at 19:07
  • $\begingroup$ Please take a look at protocols - we actually clean the surface with everything avaliable... $\endgroup$
    – Hedgehog
    Sep 22 '20 at 19:16
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    $\begingroup$ That does not mean that there is no contamination! Heaters can give off VOC's, for example. Having worked in vapor deposition and in electron micraoscopy, I've seen how easily a surface can become contaminated. I never did get a perfect OsO4 stain... $\endgroup$ Sep 22 '20 at 19:21
  • $\begingroup$ Can you elaborate "I get different results any time, what are key points to check?" What is your criterion? $\endgroup$
    – M. Farooq
    Sep 22 '20 at 20:26
  • $\begingroup$ When Im trying to immobilize antibodies onto the surface, fluorescent-labeled or regular ones with further immobilization of the blood cells, I get results like "fluorescence"/"no flu..."/"only mild flu..." using the same protocol. With cells... they may cover all the surface, no matter if theres antibody immobilized or not. ANd may not cover at all. $\endgroup$
    – Hedgehog
    Sep 23 '20 at 6:17
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The triethoxysilane group has to hydrolyze first in order to bond with a silanol group of glass surface. It seems that you are cleaning the slides very well. However, I think your temperature is too low and your time of exposure is too short. The hydrolysis of ethyoxysilane group is slow.

Secondly, I guess you are posting questions on this topic for almost a year without much success. In such cases, it is always good to go back and try a control experiment and think about a new approach. The control experiment is that you try just neat glass slides and see if your antibody sticks to glass or not. Glass itself is a good adsorber. Probably you might get similar results of "fluorescence, no fluorescence and mild fluorescence". This will confirm that your silane binding procedure is doing nothing!

Second control test: Epoxy silane coated slides are commercially available. Why don't you try using them and see if your immobilization chemistry works on commercial slides or not. Schott makes commercial slides by this name: Nexterion® Slide E

Let me tell you how silanes are bonded onto silica. The reaction time is typically 10 hours under reflux conditions of toluene and other added solvents and catalysts! Sometimes a very very small amount of water is added to created polymer like bonding. There are at least several hundred papers on this topic. The surface area is silica is quite high so it is not a direct comparison with glass slides but still 3 min and room temperature is doing nothing!

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  • $\begingroup$ Could you please tell me, what is the correct temperature then? $\endgroup$
    – Hedgehog
    Sep 25 '20 at 6:52
  • $\begingroup$ I had mentioned reflux temperature around ~110 C of toluene. $\endgroup$
    – M. Farooq
    Sep 25 '20 at 13:38
  • $\begingroup$ From what I heard, its enough to prepare either acidified solution of silane in alcohol or toluene solution, sink slide into it, extract, rince, bake at about 110 degrees - and youll have coated surface... $\endgroup$
    – Hedgehog
    Sep 25 '20 at 17:05
  • $\begingroup$ Since you have already tried it and certainly enough it is not working. You will have to study multiple variables and optimize them. $\endgroup$
    – M. Farooq
    Sep 25 '20 at 17:08
  • $\begingroup$ Could you please tell me, how important humidity control and on what steps? $\endgroup$
    – Hedgehog
    Oct 3 '20 at 16:21

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