The hosts are already resigned to playing next season in Serie B, but they put in a stellar performance to down the champions after the former Italy international struck.
The home side found themselves down to 10 men after just 12 minutes as Amadou Diawara received two yellow cards, but it took a penalty to secure the win for the Rossoneri.
The striker hit the winner in a dramatic finish in Florence as the visitors moved 12 points clear at the top and will be crowed champions if Napoli fail to win at Roma on Monday.
The Blues fell behind to Christian Eriksen's excellent early strike but goals from Pedro and Victor Moses ensured that Spurs' wretched run at Stamford Bridge continues.
The reigning champions will fancy their chances of progressing to the semi-finals after picking up an away win against the Basque side in Thursday's first-leg clash.
The visitors came from behind to earn a remarkable victory at Camp Nou, in a result that simultaneously dents the hosts' title hopes and boosts those of Atletico Madrid.
The Nerazzurri took the lead through Ivan Perisic but were unable to hold onto their lead in a game that ended in a stalemate – a result that benefits neither team.