Fluorescence Cross-Correlation Spectroscopy as a Universal Method for Protein Detection with Low False Positives

Abstract

Specific, quantitative and sensitive protein detection with minimal sample preparation is an enduring need in biology and medicine. Protein detection assays ideally provide quick, definitive measurements that use only small amounts of material. Fluorescence cross-correlation spectroscopy (FCCS) has been proposed and developed as a protein detection assay for several years. Here, we combine several recent advances in FCCS apparatus and analysis to demonstrate it as an important method for sensitive, quantitative, information-rich protein detection with low false positives. The addition of alternating laser excitation (ALEX) to FCCS along with a method to exclude signals from occasional aggregates leads to a very low rate of false positives, allowing the detection and quantification of the concentrations of a wide variety of proteins. We detect human chorionic gonadotropin (hCG) using an antibody-based sandwich assay, and quantitatively compare our results with calculations based on binding equilibrium equations. Furthermore, using our aggregate exclusion method, we detect smaller oligomers of the prion protein PrP by excluding bright signals from large aggregates.