Direct detection of inorganic ions and underivatized amino acids in seconds using high-speed capillary electrophoresis coupled with back-scatter interferometry

Silva, Miyuru De; Opallage, Prabhavie M; Dunn, Robert C.

doi:10.1039/d0ay02218g

Cited by 10 publications

(21 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The HSCE approach uses a short length capillary (10 cm) for rapid separation times. 59,60 The capillary has an ultra-thin wall (15 µm) which, as shown previously, efficiently dissipates heat into a surrounding thermal paste and mitigates the effects of Joule heating at high field strengths. This combined with a short total length enables rapid separations on the seconds time scale.…”

Section: Introductionmentioning

confidence: 85%

“…The custom-designed HSCE is similar to that recently described. 59 The separation takes place in a 10 cm long total length fused silica capillary ( 50 The HSCE is supported on a Zeiss Axiovert 100 TV inverted fluorescence microscope. For the BSI signal, the 488 nm line from an argon-krypton laser (Coherent, Innova 70, Santa Clara, CA) is focused into the detection zone of the capillary.…”

Section: Experimental Apparatusmentioning

confidence: 99%

See 1 more Smart Citation

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

Opallage¹,

Silva²,

Dunn³

2021

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Introductionmentioning

confidence: 85%

Section: Experimental Apparatusmentioning

confidence: 99%

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

Opallage¹,

Silva²,

Dunn³

2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The custom-designed HSCE is similar to that recently described 74 . The separation takes place in a 10 cm long total length fused silica capillary (50 μm i.d., 80 μm o.d.…”

Section: Methodsmentioning

confidence: 99%

Dual detection high-speed capillary electrophoresis for simultaneous serum protein analysis and immunoassays

Opallage

Silva

Dunn

2022

Sci Rep

Self Cite

View full text Add to dashboard Cite

Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is 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 simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index 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. The high pH reduces protein adsorption but 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. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.

show abstract

“…Within the operating detection limits, the BSI signal senses all RI changes in the composition of the BGE due to analyte or system zones. The sample plug, therefore, is easily measured in the BSI signal and used previously to quantify the EOF and convert analyte migration times to electrophoretic mobilities [42,43].…”

Section: Introductionmentioning

confidence: 99%

Sample plug induced peak splitting in capillary electrophoresis studied using dual backscattered interferometry and fluorescence detection

Silva

Dunn

2023

Electrophoresis

Self Cite

View full text Add to dashboard Cite

The appearance of unexpected peaks in capillary electrophoresis (CE) is common and can lengthen the time of method development as assay conditions and experimental parameters are varied to understand and mitigate the effects of the additional peaks. Additional peaks can arise when a single-analyte zone is split into multiple zones. Understanding the underlying mechanism of these phenomena, recognizing conditions that favor its presence, and knowing how to confirm and eliminate the effect are important for efficient method optimization. In this study, we examine how the overlap of analyte zones with the sample plug can lead to peak splitting. This is explored experimentally using dual detection CE, which enables both the sample plug and analyte zones to be independently and simultaneously measured from the same detection volume. Simulations performed via COMSOL Multiphysics confirm the origin of the splitting and help guide experiments to reduce and eliminate the effect. Our findings show that this peak splitting mechanism can arise in separations of both small and large molecules but is, especially, prevalent in separations of slowly migrating macromolecules. This effect is also more prevalent when using a short length-to-detector, as is commonly found in microfluidic applications. A simple diffusion-less model is introduced to develop strategies for reducing peak splitting that avoids modifying the apparatus, such as by lengthening the separation length, which can be difficult. Decreasing the sample plug length and slowing the electroosmotic flow can both reduce this effect, which is confirmed experimentally.

show abstract

Direct detection of inorganic ions and underivatized amino acids in seconds using high-speed capillary electrophoresis coupled with back-scatter interferometry

Abstract: A short length, ultra-thin wall separation capillary combined with back-scatter interferometry enables amino acid separation and detection in seconds.

Cited by 10 publications

References 59 publications

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

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

Dual detection high-speed capillary electrophoresis for simultaneous serum protein analysis and immunoassays

Sample plug induced peak splitting in capillary electrophoresis studied using dual backscattered interferometry and fluorescence detection

Contact Info

Product

Resources

About