Dataset Information

Electrokinetic Mixing for Improving the Kinetics of an HbA1c Immunoassay.

ABSTRACT: The efficiency of the diagnostic platforms utilizing ELISA technique or immunoassays depends highly on incubation times of the recognition elements or signaling molecules and volume of the patient samples. In conventional immunoassays, long incubation times and excess amounts of the recognition and signaling molecules are used. The technology proposed here uses electrokinetic mixing of the reagents involved in a sandwich immunoassay based diagnostic assay in electrode-enabled microwell plates in such a way that the incubation times and volumes can be reduced substantially. The integration of the electrodes at the bottom of the conventional microwell plates ensures that the motions of the liquid flows in the wells can be controlled through the application of high frequency AC current along these electrodes. The strategy to generate chaotic mixing by modification of standard multiwell plates, enables its use in high throughput screening, in contrast to microfluidic channel-based technologies that are difficult to incorporate into conventional plates. An immunoassay for detection of glycated hemoglobin (HbA1c) that can reveal a patient's average level of blood sugar from the past 2-3 months instead of just measuring the current levels and thereby constitutes a reliable diabetes monitoring platform was chosen as a pilot assay for technology demonstration. The overall incubation time for the assay was reduced by approximately a factor of five when electrokinetic mixing was employed. Furthermore, when the quantity of the reagents was reduced by half, almost no distinguishable signals could be obtained with conventional immunoassay, while electrokinetic mixing still facilitated acquisition of signals while varying concentration of the glycated hemoglobin. There was also a substantial difference in the signal intensities especially for the low concentrations of the HbA1c obtained from electrokinetic mixing assisted and conventional immunoassay when the quantity of the reagents and incubation times were kept constant, which is also an indication of the increase in bioassay efficiency. The electrokinetic mixing technique has the potential to improve the efficiency of immunoassay based diagnostic platforms with reduced assay time and reagent amounts, leading to higher throughput analysis of clinical samples. It may also open new avenues in point of care diagnostic devices, where kinetics and sampling size/volume play a critical role.

SUBMITTER: Yasun E

PROVIDER: S-EPMC6934526 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Electrokinetic Mixing for Improving the Kinetics of an HbA1c Immunoassay.

Yasun Emir E Trusty Travis T Abolhosn Rania W RW Clarke Nigel J NJ Mezić Igor I

Scientific reports 20191227 1

The efficiency of the diagnostic platforms utilizing ELISA technique or immunoassays depends highly on incubation times of the recognition elements or signaling molecules and volume of the patient samples. In conventional immunoassays, long incubation times and excess amounts of the recognition and signaling molecules are used. The technology proposed here uses electrokinetic mixing of the reagents involved in a sandwich immunoassay based diagnostic assay in electrode-enabled microwell plates in ...[more]

PMID: 31882622

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Project description:To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.

Dataset Information

Electrokinetic Mixing for Improving the Kinetics of an HbA1c Immunoassay.

Publications

Electrokinetic Mixing for Improving the Kinetics of an HbA1c Immunoassay.

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