Semisynthetic sensor proteins enable metabolic assays at the point of care

Abstract

Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, light-emitting sensor proteins for use in paper-based metabolic assays. The metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color in the presence of the reduced cofactor, enabling metabolite quantification with the use of a digital camera. The approach makes any metabolite that can be oxidized by the cofactor a candidate for quantitative point-of-care assays, as shown for phenylalanine, glucose, and glutamate. Phenylalanine blood levels of phenylketonuria patients were analyzed at the point of care within minutes with only 0.5 microliters of blood. Results were within 15% of those obtained with standard testing methods.

A Biomimetic Plasmonic Nanoreactor for Reliable Metabolite Detection

“…The spectrum showed characteristic bands of 3‐MPBA at 415 (C–S stretching), 785 (C–H out‐of‐plane bending), 998 (C–C in‐plane bending), 1023 (C–H in‐plane bending), 1076 (C–C in‐plane bending coupled with C–S stretching), 1558 (nontotally symmetric ring stretching), and 1576 cm −1 (totally symmetric ring stretching). [ 6c,18g ] After incubation in H 2 O 2 , the SERS spectrum of 3‐MPBA‐functionalized MOSF@GNPs was in good agreement with that of 3‐hydroxythiophenol (3‐HTP) (Figure 1F), indicating the transformation of boronate to phenol (Figure 1E). The new peaks at 882 and 1589 cm −1 were ascribed to ring stretching and totally symmetric ring stretching of 3‐HTP.…”

“…By contrast, enzyme‐based bioassays, where the presence of analytes is optically or electrochemically detected through the highly specific and efficient enzymatic reaction, provide novel opportunities for detection metabolite biomarkers. [ 6 ] These approaches show enough figures of merits regarding device portability, speed, sample consumption and cost. However, the inherent instability of enzyme at elevated temperature, in circumstance containing digestive enzymes or chemical chelates, and when storing the enzyme long‐term represents a longstanding yet largely unsolved challenge that leads to poor reproducibility and accuracy in enzyme‐based metabolite analysis.…”

“…at the micromolar level. [ 6c ] Despite their utility, the previous SERS sensors execute their functions by enzymes dissolved in bulk solution, suffering from activity instability. A multifunctional nanoreactor that allows continuous robust enzymatic recognition and catalysis of target metabolites in complex biological samples, while working as a sensitive and user‐friendly optical biosensor, would be desirable and highly useful.…”

Chemogenetic Control of Nanobodies

“…The new termini of this cpDHFR are located in an active site loop of wild-type DHFR. Furthermore, cpDHFR is partially unfolded in the absence of its cofactor nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR inhibitors such as trimethoprim (TMP) 8,9 . TMP is a clinically approved anti-bacterial drug that has excellent cell and tissue permeability and is not toxic for mammalian cells.…”

Luciferase‐Induced Photouncaging: Bioluminolysis

“…[5] Compared to conventional fluorescence imaging, bioluminescence imaging is ultrasensitive,s hows high signal-to-noise ratios,a nd does not cause phototoxicity.B ioluminescence resonance energy transfer (BRET) thus attracted significant attention in the study of dynamic processes in biological systems such as protein-protein interactions,signaling events,and metabolite or drug sensors. [6][7][8][9][10][11][12] Thev ast majority of BRET applications have focused on using the photons generated by al uciferase as as ignal reporting on an event. More recently,t here has been as hift to extend the utility of BRET from ar eporter function to an effector function.…”