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Colorimetric multiplexed immunoassay using specific aggregation of antigenic peptide-modified luminous nanoparticles
Toshihiro Ihara1, Yasunori Mori, Takaaki Imamura
1Department of Applied Chemistry and Biochemistry, Graduate School of Science and Technology, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan. toshi@chem.kumamoto-u.ac.jp
Insights
This study introduces a novel one-step immunoassay for detecting multiple antibodies simultaneously. The system uses color-coded nanospheres to differentiate antibodies, enabling rapid and multiplexed detection in a single sample.
Area of Science:
- Biotechnology
- Nanotechnology
- Immunology
Background:
- Multiplexed immunoassays are crucial for simultaneous detection of multiple analytes.
- Current methods can be complex and time-consuming.
- Development of rapid, single-step multiplexed assays is needed.
Purpose of the Study:
- To develop a rapid, one-step multiplexed immunoassay for simultaneous antibody detection.
- To utilize luminous nanospheres for distinct signal generation.
- To enable differentiation of multiple antibodies in a single reaction vessel.
Main Methods:
- Immobilization of antigenic peptides (FAK, c-Myc, alpha-catenin) onto 40 nm luminous nanospheres.
- Functionalization of nanospheres with cationic or anionic pentamer tails.
- Mixing of antibody samples with differentially color-coded nanospheres.
- Observation of aggregate formation and emission color for antibody identification.
Main Results:
- Successful immobilization of peptides onto nanospheres.
- Distinct red and green emissions for FAK/c-Myc and c-Myc/alpha-catenin peptide-conjugated spheres, respectively.
- Formation of antibody-specific aggregates with distinct colors (red, green, yellow) upon mixing.
- Demonstration of multiplexed detection of anti-FAK, anti-c-Myc, and anti-alpha-catenin antibodies in one step.
Conclusions:
- The developed nanosphere-based immunoassay allows for rapid, one-step multiplexed detection of antibodies.
- Aggregate color differentiation provides a simple mechanism for identifying multiple targets simultaneously.
- This system offers a promising platform for advanced diagnostic applications.
Abstract:
A rapid immunoassay capable of detecting specific antibodies in one-step procedure is described. Antigenic peptides with cationic (KKKKC) or anionic (DDDDC) pentamer tail were immobilized on luminous nanospheres of 40 nm diameter (Ø) through cystamine and bifunctional linker molecules under various conditions. The numbers of each peptide anchored to a sphere were 5.0 x 10(2) and 0.8-3.8 x 10(3), respectively. A mixture of the antigenic peptides of FAK and c-Myc was immobilized to the spheres with red emission, while that of c-Myc and alpha-catenin was likewise to green spheres. Multiplexed immunoassay was easily achieved by adding the antibodies to a mixed dispersed solution of these spheres under appropriate conditions. Anti-FAK and anti-alpha-catenin antibodies formed aggregates with red and green emissions, respectively. On the other hand, the anti-c-Myc antibody formed aggregates emitting a yellow light. This system enabled us to differentiate three antibodies in one vessel from the definite differences in aggregate color.
