Development of a Cell Free Homocysteine Biosensor to Assess Folate Deficiency

Folate, commonly known as vitamin B9, is a critical micronutrient in human nutrition that plays an essential role in DNA, RNA, and protein synthesis. Folate deficiency can increase the risk of cancer, cardiovascular disease, neurological disease, and birth defects. Therefore, it is important to have an accurate and prompt method for testing folate levels. In clinical settings, folate levels are measured through testing the concentration in red blood cells (RBC) or serum. Both of these biomarkers are susceptible to false negative results. Additionally, the methods used for testing these biomarkers must be performed in a laboratory using power and expensive equipment, both of which are not easily accessible across the globe. As a result, access to necessary information about one’s health is limited to those who can afford it. The Biomarkers of Nutritional Development (BOND) project named folate as one of six micronutrients that warrant immediate attention in biomedical research due to their importance in public health.

Alternatively, plasma homocysteine can be used as a biomarker for folate deficiency, resulting in a new, low-cost, and minimal-equipment method for detecting folate. Homocysteine undergoes a process in the body, remethylation, wherein folate is required to convert it to methionine. Consequently, high homocysteine levels are correlated with low folate levels. Measuring levels of homocysteine in blood plasma is easier than processing RBCs, and leads to more accurate results than serum, which makes this idea very attractive.

We propose a homocysteine biosensor in a cell-free system that uses the interaction between homocysteine and an Escherichia Coli transcription factor: MetR. Cell free reactions are becoming increasingly popular in synthetic biology. They allow us to synthesize proteins outside of the cell, which produces faster results, while also being less complicated than whole-cell systems.The cell-free process achieves this by giving us direct access to the inner contents of the cell, removing the barrier of a cell membrane. In our sensor, the homocysteine/MetR interaction drives expression of the reporter gene, Green Fluorescent Protein (GFP), alerting us of the patient’s homocysteine levels. Through extensive testing we have proven that our proposed biosensor detects homocysteine concentrations within the physiologically relevant range.