Single Gold Nanoparticles Counter: An Ultrasensitive Detection Platform for One-Step Homogeneous Immunoassays and DNA Hybridization Assays

In this paper, we present for the first time a single gold nanoparticle counter (SGNPC) in solution based on the photon bursting in a highly focused laser beam (less than 1 fL) due to the plasmon resonance scattering and Brownian motion of gold nanoparticles (GNPs). The photon burst intensity of single 36 nm GNPs is several tens to hundreds times stronger than that of quantum dots (QDs) and organic dyes. The relationship between the photon burst counts and GNPs concentration shows an excellent linearity. The linear range is over 4 orders of magnitude, and the detection limit of GNPs (36 nm) is 17 fM. On the basis of this single nanoparticle technique, we developed an ultrasensitive and highly selective detection platform for homogeneous immunoassay and DNA hybridization assays using GNPs as probes, which were 2−5 orders of magnitude more sensitive than current homogeneous methods. We used this technology to construct homogeneous sandwich immunoassays for cancer biomarkers, such as carcinoembryonic antigen (CEA) and alpha fetal protein (AFP), and aptamer recognition for thrombin. The detection limits are 130 fM for CEA, 714 fM for AFP and 2.72 pM for thrombin. Our method was successfully applied for direct determination of CEA, AFP and thrombin levels in sera from healthy subjects and cancer patients. In homogeneous DNA hybridization detection, we chose methylenetetrahydrofolate reductase (MTHFR) gene as a target. This assay successfully distinguished DNA sequences with single base mismatches, and the detection limits for the target were at 1 fM level.