Cofactor sensing proteins for metabolic assays at point-of-care

Miniaturized blood analysis promises a substantial improvement for the process of healthcare. However, most current blood analysis methods were developed in the context of centralized laboratories and there is a lack of robust detection technologies suitable for miniaturized analysis. Here, we exploited protein engineering to develop bioluminescent sensors for the reduced nicotinamide adenine dinucluotide phosphate (NADPH) and the oxidized nicotinamide adenine dinucluotide (NAD+), which are key to the quantification of many metabolic assays. The bioluminescent sensors bypass the limitations of conventional detection methods such as absorbance-, fluorescence- or electrochemistry-based assays and feature improved sensitivity and selectivity. Hence, we further developed point-of-care test papers for the management of phenylketonuria that can quantify phenylalanine with 0.5 ÎŒL of non-separated blood using a digital camera. Similarly, point-of-care test papers were also developed for glucose, glutamate, latactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase. The NAD+ level in bodily fluids and live cells are of interest for metabolic studies related to age-induced diseases. Here, we utilized the sensor to enable rapid in vitro and in cellulo assessment of NAD+ levels in a format compatible with high throughput screenings. This technology could further enable accurate and easy-to-use quantification platforms for a large number of clinically-interesting analytes for metabolic studies.