Preparation and Applications of a Chemiluminescent Film Deposited on a Well Plate: Focusing on 96-Well Technology

In this study, polydopamine (PDA) was employed for the first time as a coupling intermediate for the immobilization of a chemiluminescent (CL) film containing both the catalyst (zeolitic imidazolate framework-67, ZIF-67) and CL substance (N-(4-aminobutyl)-N-ethylisoluminol, ABEI) in the wells of a 96-well plate. The prepared CL film showed good responses to H₂O₂, with a limit of detection (LOD) of 1.2 nM, as well as to acetylcholinesterase (AChE) by employing a cascade enzymatic reaction, and it could be applied for the screening of AChE inhibitors. More interestingly, a decreased CL signal was observed when the CL film was incubated with gentamicin (GEN), which is a kind of aminoglycoside (AG). Mechanism studies indicated that the CL responses of the CL film to GEN should be attributed to the competitive binding of GEN with cobalt ions in the CL film, resulting in the loss of catalytic centers in the ABEI-H₂O₂ CL reaction and a subsequent decrease of CL signals. The CL film exhibited good sensitivity for the detection of AGs such as GEN, neomycin (NEO), streptomycin (STR), kanamycin (KAN), and amikacin (AMI), with LODs of 2.4 nM, 7.8 nM, 2.4 nM, 9.6 nM, and 3.0 nM, respectively. The proposed heterogeneous sensing platform could be employed for the detection of AGs in different dosage forms and complex matrices such as natural water, serum, and milk, possessing advantages including high sensitivity, good specificity, and high efficiency.