Development of simultaneous refractive index and fluorescence detection platform using high-speed capillary electrophoresis

2021 ◽  
Author(s):  
Prabhavie Madushanie Opallage ◽  
Robert Dunn ◽  
Miyuru De Silva
Keyword(s):  
Refractive Index ◽  
Capillary Electrophoresis ◽  
Fluorescence Detection ◽  
High Speed

scholarly journals Dual Detection High-Speed Capillary Electrophoresis for Simultaneous Serum Protein Analysis and Immunoassays

2021 ◽  
Author(s):  
Prabhavie Opallage ◽  
Miyuru De Silva ◽  
Robert Dunn
Keyword(s):  
Refractive Index ◽  
Capillary Electrophoresis ◽  
Human Serum ◽  
Serum Protein ◽  
High Speed ◽  
Serum Proteins ◽  
Numerical Aperture ◽  
Excitation Source ◽  
Detection Zone ◽  
Dual Detection

Abstract Serum protein electrophoresis (SPE) and immunoassays are important tools used clinically to diagnose disease. SPE separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is usually followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of both SPE measurements and quantifying immunoassays, simultaneously. The HSCE uses a 10 cm long total length separation capillary (50 μm i.d., 80 μm o.d.) with an 8 cm length-to-detector for rapid analysis times. This is important for throughput in clinical settings and for quantifying immunoassays, where antigen-antibody complexes continually dissociate once injected into the non-equilibrium conditions of the separation capillary. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays), simultaneously. Light scattered back towards the excitation source measures refractive index changes using back-scatter interferometry (BSI), while fluorescence is collected from below the capillary with a high numerical aperture objective. To validate the dual detection HSCE approach, SPE and immunoassays are measured from human serum samples pre-incubated with fluorescein and an anti-fluorescein monoclonal antibody. We show that the BSI signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. Elevated pH is used in SPE to ensure all proteins are charged and to reduce protein adsorption to the capillary walls. The high pH, however, also reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification, but loss of resolution in the SPE. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.

Sign in / Sign up

Export Citation Format

Share Document