Was Mephisto aware that Yukio had Satan's blood in him after all?
I'm not sure if it's a plot hole but it's definitely a bit dubious. Yukio's body wasn't strong enough for Satan's powers, so they rejected him leaving him a pure human. 

But Satan is still his dad, so I guess he might still have inherited stuff other than demonic powers. Maybe that includes Satan's blood type, assuming Satan has blood.

There aren't really any answers in the manga, because the anime goes in its own direction after episode 16 or so, but to answer the question, yes Mephistopheles definitely knew he was the son of Satan but that he had no demonic powers, no he almost certainly wasn't talking about him when he said our youngest brother.