(513d) Cell-Free Systems for Equipment-Free Quantitation of Micronutrients in Human Serum

Micronutrient deficiencies cause hundreds of thousands of deaths each year, and challenges associated with current diagnostic methods preclude treatment in affected areas. Cell-free transcription-translation (TX-TL) systems, which have been effectively used in infectious disease diagnostics, could enable the development of field-friendly micronutrient tests. However, to date, TX-TL diagnostics have been limited to diseases with nucleic acid biomarkers. Deviations in levels of other biomarkers (such as ions, metabolites, and proteins) are indicators of many diseases (including micronutrient deficiencies), but distinguishing between different levels, rather than the presence or absence of these molecules, is a major challenge. We have developed a TX-TL diagnostic tool that reliably detects physiologically relevant concentrations of a micronutrient in human serum. The test requires no equipment to interpret and can be readily adapted to detect other analytes.

We demonstrated that a micronutrient-responsive protein works in TX-TL systems, and we developed standards so that the color of the test reaction can be matched to an array of reference reactions run with the same sample matrix. In the developed test, the micronutrient-responsive regulator controls transcription of an enzyme that cleaves a substrate to a colored dye. To account for changes in assay output over time and serum matrix effects, we designed standard reference reactions to be run in parallel with the sample. Standard reactions have saturated micronutrient concentrations and varying amounts of transcriptional regulator, so the amount of regulator, rather than the amount of micronutrient, controls the colorimetric output. Regulator concentrations in the standards are correlated with micronutrient concentrations in the test reaction, and the correlation is reproducible across lysate batches and perturbations in reaction conditions.

To better enable equipment-free quantitation, we designed the test to utilize the array of colors that correspond with different degrees of substrate cleavage. Interestingly, in serum the visible spectrum of the colored product changes, and it is difficult to distinguish between intermediate reaction states. Interaction of the dye with serum proteins causes this spectrum shift, and small molecules can displace the colored product and shift back the spectrum. At high concentrations, these molecules also quench the reaction, enabling users to preserve test results for potential later analysis.

The developed test is the first TX-TL assay for small molecules in a complex matrix and is a significant step towards a low-cost, field-deployable test for micronutrient deficiencies. This platform can also be readily expanded to detect other analytes of interest.