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Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay
Published on: September 7, 2018
Self-contained microelectrochemical immunoassay for small volumes using mouse IgG as a model system
Zoraida P Aguilar1, Walter R Vandaveer, Ingrid Fritsch
1Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701, USA.
Insights
This study presents a novel microelectrochemical immunoassay capable of analyzing minuscule sample volumes. The developed assay offers rapid detection of analytes like mouse IgG, demonstrating potential for ultra-small volume biological analysis.
Area of Science:
- Electrochemistry
- Immunoassay Development
- Microfluidics
Background:
- Traditional immunoassays often require larger sample volumes, limiting their application in certain research areas.
- Microfabricated devices offer potential for miniaturization and increased sensitivity in analytical techniques.
- Enzyme-linked immunosorbent assays (ELISA) are widely used but can be time-consuming and require significant reagent volumes.
Purpose of the Study:
- To develop a self-contained microelectrochemical immunoassay utilizing ultra-small sample volumes.
- To demonstrate the assay's capability for rapid detection of mouse IgG using a sandwich-type format.
- To evaluate the performance of the microfluidic device for sensitive electrochemical detection.
Main Methods:
- Fabrication of a microfluidic chip with individually addressable microelectrodes within a microcavity.
- Covalent attachment of primary antibodies to a gold recessed microdisk electrode (RMD) using self-assembled monolayers (SAMs).
- Electrochemical detection of enzymatically generated species via cyclic voltammetry using a gold nanoband electrode.
Main Results:
- Successful development of a microelectrochemical immunoassay using only 1 microL of antigen and secondary antibody-enzyme conjugate.
- Achieved rapid detection of enzymatically generated species in less than 30 seconds.
- Demonstrated high sensitivity with detection limits for mouse IgG as low as 56 fM (9 pg/mL) and for p-aminophenyl phosphate (PAPR) as low as 4.4 nM (6.4 ng/mL).
Conclusions:
- The developed microelectrochemical immunoassay is highly efficient, requiring minimal sample and reagent volumes.
- The proximity of electrodes to the antibody-modified surface enhances detection sensitivity and speed.
- The device shows promise for ultra-small volume analysis, potentially down to picoliter volumes.
Abstract:
A self-contained, microelectrochemical immunoassay on the smallest volumes reported to date (1 microL for the antigen, 1 microL for the secondary antibody-enzyme conjugate, and 200 nL for the electrochemically detected species) has been developed using mouse IgG as a model system in a sandwich-type enzyme-linked immunosorbant assay, which takes less than 30 min to both complete the assembly of immunoassay components onto the antibody-modified surface and detect enzymatically generated species (excluding time for electrochemical cleaning of electrodes). These studies demonstrate the advantage of the close proximity of electrodes to modified surfaces and their application in the analysis of small volumes. Using a 50 microm diameter x 8 microm deep cavity with individually addressable electrodes on a microfabricated chip, the primary antibody was selectively and covalently attached at a gold, recessed microdisk (RMD) at the bottom of the microcavity to the free end of SAMs of either 11-mercaptoundecanoic acid or 11-mercapto-1-undecanol using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. Nonspecific adsorption to the surrounding material, polyimide, of the microcavity device was eliminated. Electrochemical desorption was used to confine the immunoassay activity at the RMD. Enzymatic conversion of the substrate p-aminophenyl phosphate top-aminophenol is detectable in less than 30 s using cyclic voltammetry at a gold, tubular nanoband electrode, which is on the wall of the microcavity and immediately adjacent to the modified RMD. A third electrode, also within the region of the microcavity, served as the pseudoreference/auxiliary electrode. Calibration curves obtained for 1-microL solutions of 5-100 ng/mL of IgG and for 200 nL-solutions of 5 microM to 4 mM of PAPR gave detection limits of 4.4 nM (6.4 ng/mL) or 880 fmol (129 pg) for PAPR and 56 fM (9 pg/mL) or 56 zmol (9 fg) for IgG. It is expected that the device may be suitable for analysis with volumes down to tens of picoliters.
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