Cu(II)-Regulated On-Site Assembly of Highly Chemiluminescent Multifunctionalized Carbon Nanotubes for Inorganic Pyrophosphatase Activity Determination

A novel signal-on chemiluminescence (CL) assay for pyrophosphatase (PPase) activity determination was innovatively developed based on the Cu(II)-regulated on-site assembly of highly chemiluminescent Cu(II), N-(aminobutyl)-N-(ethylisoluminol) (ABEI), gold nanodot, and chitosan multifunctionalized carbon nanotubes (Cu(II)/ABEI–Au/cs–CNTs). First, ABEI-functionalized gold nanodots (ABEI–Au) were assembled on the surface of chitosan-modified carbon nanotubes (cs–CNTs) via the reduction of HAuCl4 with ABEI in a cs–CNT suspension to form ABEI–Au/cs–CNTs. Then, it was found that the catalyst Cu(II) can be selectively, efficiently, and quickly adsorbed onto ABEI–Au/cs–CNTs via the high-affinity interactions between Cu(II) and cs–CNTs to form novel hybrid nanomaterials Cu(II)/ABEI–Au/cs–CNTs. The CL intensity of Cu(II)/ABEI–Au/cs–CNTs was enhanced by about 2 orders of magnitude compared with that of ABEI–Au/cs–CNTs. Furthermore, it was found that in the presence of pyrophosphate ions (PPi), PPi could coordinate with Cu(II) to form a stable PPi–Cu(II) complex and block the assembly of Cu(II)/ABEI–Au/cs–CNTs. After the addition of PPase, PPase could catalyze the hydrolysis of PPi into Pi and release Cu(II) from the PPi–Cu(II) complex. The released free Cu(II) could trigger the on-site assembly of highly chemiluminescent Cu(II)/ABEI–Au/cs–CNTs, resulting in an enhanced CL intensity. The enhanced CL intensity had a good linear relationship with the activity units of PPase ranging from 0.025 to 0.5 U, with a detection limit of 9 mU. The method was employed to monitor the PPase inhibitor efficiently. Cu(II)/ABEI–Au/cs–CNTs with excellent CL may also find more applications in the development of novel CL analytical methods.