Photothermal readout of surface-arrayed proteins: attomole detection levels with gold nanoparticle visualization

Transverse photothermal beam deflection (tPBD) is used to detect and quantify proteins arrayed on slides. The slides are "read" using an argon-ion excitation source. Optical absorption cross-sections of most proteins are too small for submonolayer coverages to produce thermal gradients of sufficient magnitude for detection using tPBD. Thus, surface-arrayed proteins are stained using mercaptoalkanoic acid coated gold nanoparticles (maa-AuNP). The large optical cross-sections of AuNP combined with electrolyte-induced AuNP aggregation afford a highly sensitive method for protein detection. Following maa-AuNP staining, the tPBD signal varies linearly with the amount of protein (Neutravidin) spotted on the slide surface: from 0.001 to 1.0 monolayer of protein. In a single 0.7 mm diameter array spot, the tPBD detection limit is 33 amol of Neutravidin or fewer than 55 protein molecules per microm2. Despite the nonspecific nature of interactions between maa-AuNP and proteins, significant variations in protein staining efficacy are observed. The factors controlling staining are not elucidated in detail, but there is a correlation between protein pI and protein staining. Proteins with pI approximately 6 are more effectively visualized by maa-AuNp than are more acidic or more basic proteins. The influence of AuNP diameter and mercaptoalkanoic acid chain length on protein staining and selectivity is investigated. The results demonstrate that AuNP staining coupled with tPBD detection constitutes a sensitive and practical method for probing protein arrays.