Fig. 2

APEX signal amplification and multiplexed profiling. a Step-by-step APEX transmission spectral changes. We performed a series of operations, namely antibody conjugation (anti-CD63) onto the sensor, exosome binding, enzyme labeling and enzymatic deposition, and monitored the resultant spectral shifts. While the enzyme labeling did not cause any significant changes, the deposit formation led to ~400% signal enhancement (****P < 0.0001, n.s. not significant, Student’s t-test). b Comparison of APEX signal amplification and optical deposit area coverage. The increase in area coverage was determined by scanning electron microscopy (SEM) analysis (****P < 0.0001, Student’s t-test). All data were normalized to that before the signal amplification. Inserts (right) show SEM images of sensor-bound exosomes, before and after APEX amplification. c Finite-difference time-domain simulations with back illumination. The APEX sensor design, but not the gold-on-glass design, enables the generation of enhanced electromagnetic fields through back illumination. Back illumination minimizes direct incident light exposure on the enzyme activity (which occurs on the sensor’s top surface). Arrows indicate the direction of incident illumination. d Real-time sensorgrams of APEX amplification kinetics. Different concentrations of the optical substrate (3,3′-diaminobenzidine tetrahydrochloride; high: 1 mg ml⁻¹, low: 0.01 mg ml⁻¹) were used to monitor the amplification efficiency. All data were normalized against negative controls, performed with IgG isotype control antibodies. e Comparison of the detection sensitivities of APEX, ELISA, and western blotting. The APEX detection limit (dotted line), before and after amplification, was determined by titrating a known quantity of exosomes and measuring their CD63 signal. f Specificity of APEX assays for measuring target proteins. Assays were developed for amyloid β (Aβ42), amyloid precursor protein (APP), alpha-synuclein (α-syn), close homolog of L1 (CHL1), insulin receptor substrate 1 (IRS-1), neural cell adhesion molecule (NCAM), and tau protein. All assays demonstrated specific detection. Heat map signals were assay (row) normalized. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in (a, b, d, and e). Source data are provided as a Source Data file