Molecular recognition based agglomeration of quantum dot bioconjugates for multiplexed antigen detection

Abstract

Sensitive and quantitative detection of proteomic biomarker panels is expected to significantly improve early diagnosis and therapy monitoring for cancers and other diseases. We developed a novel in-vitro diagnostics approach to multiplexed biomarker detection based on molecular recognition mediated nanoparticle self-assembly. The nanoparticles self-assemble into agglomerates via a rapid, single step, fluid phase reaction. The individual nanoparticles and agglomerates can be discriminated based on their light scattering properties and by other analytical techniques. Cadmium selenide quantum dots conjugated to polyclonal antibodies self-assemble to form quantum dot agglomerates in the presence of antigens, including angiopoietin-2, vascular endothelial growth factor A, and human immunoglobulin G. The reaction mixtures were characterized by one or more analytical techniques including dynamic light scattering, electrical sensing zone method (Coulter counter), and flow cytometry to characterize and quantify the self-assembled agglomerates. The size distribution of the quantum dot agglomerates was estimated to be between 500 nm and 4 microns. The individual components of the agglomerates are significantly smaller. Measured by dynamic light scattering, the quantum dot-antibody conjugates have a hydrodynamic diameter of 45 nm, while the antigens are 5 to 10 nm in diameter. We demonstrated quantitative and sensitive detection using flow cytometry of the candidate cancer biomarkers vascular endothelial growth factor A and angiopoietin-2. Non-multiplexed detection of the antigens was demonstrated with 50 femtomolar sensitivity limit. Multiplexed detection, using two quantum dot populations with distinct emission spectra of vascular endothelial growth factor A and angiopoietin-2 was demonstrated in the physiologically relevant picomolar concentration range. The kinetics of the self-assembly process were examined by measurements of the angiopoietin-2 mediated self-assembly of quantum dots over time, revealing a sigmoidal process. Antigen concentration modulates the slopes and inflection times of the sigmoidal kinetics curves. Further refinements to improve the sensitivity and specificity of this novel proteomic biomarker detection technique may improve the screening, diagnostics, and therapy response monitoring for cancers and other diseases. This approach to studying nanoparticle self-assembly may also provide a valuable tool for understanding the fundamental characteristics of nanoscale particle agglomeration.