Label-free affinity assays by rapid detection of immune complexes in submicrometer pores.

Abstract

We present herein a method that uses a submicrometer pore to detect and characterize immune complexes consisting of proteins such as staphylococcal enterotoxin B (an agent with bioterrorism potential) and polyclonal antibodies. The assay is rapid, label free, requires no immobilization or modification of the antibody or antigen, and achieves single-aggregate sensitivity by monitoring changes in electrical resistance when immune complexes pass through the submicrometer pore. Adopting a recently developed nanofabrication technique based on a femtosecond-pulsed laser made it possible to fabricate pores with conical geometries and diameters as small as 575 nm. These pores allowed sensing of immune complexes that consisted of 610–17 300 proteins and detection of proteins at concentrations as low as 30 nm. Monitoring the passage of individual immune complexes enabled determination of the size distribution and the growth of these complexes. This method senses immune complexes (and potentially other molecules or nanoparticles that can be induced to form specific assemblies) in solution, and the antibody or antigen to be detected can be present in complex media such as serum. Owing to the small footprint and simple detection scheme, submicrometer pore-based sensing of specific complexes may enable portable or high-throughput immunoassays for diagnostics and biodefense. Coulter counting, which monitors the transient change in resistance (resistive pulse) that occurs when a particle passes