Multiplexed monitoring refers to the detection and quantification of multiple biomarkers within a single assay. In the context of TOXBOX, the proposed approach allows for a comprehensive analysis of the inflammatory response triggered by exposure to a toxic agent. This is particularly valuable in safety assessments, as it reduces sample volume requirements, increases throughput capabilities and provides a high content of data critical for evaluation.
CSIC’s role in TOXBOX
One of CSIC’s main responsibilities in the TOXBOX project is the development of a multiplexed microarray for the simultaneous detection of several inflammatory markers, including cytokines, chemokines and other signalling molecules. The target molecules have been selected to represent different stages of the inflammatory process, which enables a more complete understanding of the inflammatory response, the potential synergistic effects among mediators, and the progression or severity of toxic effects.
Challenges in Developing a Multiplexed Platform for the Detection of Several Biomarkers
The development of a multiplexed platform for multiple biomarkers presents a series of challenges that must be carefully addressed to ensure analytical accuracy, sensitivity, and reproducibility.
One of the main challenges lies in the selection and optimization of a set of compatible biomarkers, ensuring that they do not interfere with each other. Cross-reactivity between analytes and cooperative interactions between antibodies can lead to a non-specific result.
Another critical aspect is the matrix effect of complex biological samples (e.g., blood, saliva, tears), which can also impact assay performance. Some components of these samples may interfere with binding interactions or signal transduction mechanisms, which would require incorporating sample treatment and further optimization of the reagents and buffers.
Moreover, achieving uniform sensitivity and limit of detection across all targets is particularly challenging, especially when markers vary significantly in abundance in the samples to be studied. Balancing this variability without compromising throughput or assay time is essential.
Future Work
Next steps will be related with analysis of samples from the different organoid-on-a-chip models proposed in the project, which will allow the consortium to have a preliminary profile of inflammatory molecules that can be generated or whose levels can be altered under a certain stimulus. In addition, the integration of the multiplexed microarray chip with microfluidics will be an essential work in the future development of the automated module.