Luminol-labeled gold nanoparticles for ultrasensitive chemiluminescence-based chemical analyses

We report a study on chemiluminescence-based chemical analyses using luminol molecules covalently attached to 10 nm diameter gold nanoparticles (GNPs). Chemiluminescence (CL) has been systematically studied under two schemes by varying the concentrations of luminol-labeled GNPs and [Fe(CN)6]3− catalyst, respectively. The CL signal of luminol-labeled GNPs is enhanced by 5 to 10 times compared to the bulk luminol solutions of the same concentration. The log–log plot of the CL signal versus the number of luminol-labeled GNPs suspended in a standard 96-well plate shows two characteristic linear curves with distinct slopes across eight orders of magnitude variation in the GNP quantity (from 1.82 × 102 to 1.82 × 1010 GNPs per well). The detection limit represented by the cross-point of these two curves can reach down to ∼6.1 × 105 GNPs per well (corresponding to 1.0 × 10−14 M GNP and 2.4 × 10−11 M equivalent luminol concentration). The attachment of luminol molecules to GNP nano-carriers allows a large amount of luminol to be placed in a greatly reduced volume (or area) toward developing miniaturized CL sensors. We have demonstrated this by preloading dried luminol-labeled GNPs in homemade microwell arrays (with a volume of ∼12 μL per well). A linear log–log curve can be obtained across the full range from 1 × 103 to 1 × 1010 GNPs per microwell. The CL signal was detectable with as few as ∼1000 GNPs. We have further applied this microwell method to the detection of highly diluted blood samples, in both intact and lysed forms, which releases Fe3+-containing hemoglobin to catalyze luminol CL. The lysed blood sample can be detected even after a 108 fold dilution (corresponding to ∼0.18 cells per well). This ultrasensitive CL detection method may be readily adapted for developing various miniaturized multiplex biosensors for rapid chemical/biochemical analyses.