If there is presence of water you can expect an interaction between free silanol groups from silica and any molecule that can establish van der Waals forces. You need to completely remove DTT before adding the fluorescent dye.

If I understand correctly, you observe fluorescence on the slide that is devoid of thiol (I assume that it is a slide that has been simply cleaned and activated, no silane, no DTT ?). Hence, you can exclude the fact that DTT plays a role and it is more likely your washing procedure after aging with the F5M solution that must be modified. How did you wash the slide ?

Dear Omar and Juliette, yep, I agree, that it is critically important to remove all DTT, which was unspecifically bound on the silica surface. But these is a problem, that just washing with huge amount of water did not result in all DTT removing. I tried different solvents, like ethanol, acetone, DMSO. No good results :( Moreover, if I treat silica with F5M (without treatment before with DTT), I also can not remove all F5M by washing with water, ethanol or DMSO.

You discribe your support as silica (not as glass). Could you clarify what it is and what treatment do you perform on it before adsorption of F5M ?

Dear Juliette, silica, in general, is silicon dioxide (SiO2) (in short: https://www.britannica.com/science/silica), glass contains c.a. 75-80% of silica and other is metal oxides. Thereby, from the point of chemical modification, surface chemistry of silica and glass should be very similar. The steps of silica modification, which I did, are common and are:

1) Cleaning in izopropanol and aceton in ultrasonic bath

2) Cleaning and activation by Piranha (3:1 H2SO4:H2O2)

3) Washing with high extent of DI water

4) Soaking in acetone 2 h at room temperature (normally it should be mercaptosilane immobilization, but I excluded it for control experiment)

5) Washing with acetone

6) Keeping in the oven at 110 oC 2 h (normally it should be silane annealing, but I excluded it for control experiment)

7) Treatment with 10 mM DTT in PBS buffer 30 min at 37 oC (normally it should be silane disulfide bridges removing)

8) Washing by

a) DI water

b) DMSO

c) PBS

under stirring 30 min

9) Treatment with 3.5 microM F5M in 20 mM Na(H)PO4 buffer with 8 mM EDTA 2 h at room temperature (normally it should be silane labeling: F5M interraction with mercapto-group of silane)

10) Washing by

a) DI water

b) DMSO

c) PBS

under stirring 30 min

11) Treatment with 10 mM KOH solution during overnight at room temperature (normally it should be hydrolysis of siloxane bonds between silane and silica, which result in F5M-silane release into solution)

12) Removing of treated silica and fluorescence measurements of obtained solution.

So, steps 8 and 10 did not result in good removing of DTT and F5M, thereby I saw the solution fluorescence in the end. And I need to optimize steps 8 and 10, which should result into no fluorescence at the step 12.

So your blank experiment is not on a pure silica slide (it may contain adsorbed DTT). Did you observe an adsorption of F5M on the slide after step 6 ? It would help you to determine if the "incorrectly" performed washing step is after treatment with DTT or after adsorption of F5M.

Concerning the nature of your silica slide, my question was rather related to the presence of pores in the substrate (I happen to know the difference between glass and silica :-) ). Regarding their surface properties, some glasses -depending on their composition- may have higher PZC than silica, but as adsorption and washing are performed at pH>7, it should not make any difference.

For the removal of DTT you could increase slightly the pH (at pH=9, most of DTT will be deprotonated and bear a negative charge, as the surface charge of the silica slide is also negative, it would favor its desorption). Alternatively if DTT desorption is definively a difficult step, you could perfom the curing step under neutral atmosphere (N2 or vacuum to avoid the formation of S-S bridge)