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A biosensor for measuring NAD+ levels at the point of care


The cellular level of nicotinamide adenine dinucleotide (NAD+), through its different functions, affects cellular metabolism and signalling1,2,3. A decrease in the NAD+ content has been associated with various pathologies and physiological aging4,5, while strategies to boost cellular NAD+ levels have been shown to be effective against age-related diseases in many animal models6. The link between decreased NAD+ levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD+, ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD+ levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD+. This NAD+-dependent colour change enables the use of the biosensor in paper-based assays in which NAD+ is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD+ levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD+-dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.

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Fig. 1: NAD+ sensor and assay design.
Fig. 2: NAD+ sensor performance in paper-based assays.
Fig. 3: NAD+ sensor for kinetic measurements.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data for Figs. 13 and Extended Data Figs. 39 are provided with the paper. Plasmids of the sensors are available on AddGene through No.135627 (NAD-Luc), 135628 (NAD-cpLuc) and 135629 (NAD-cpLuc2).


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This work was supported by the Max Planck Society, the Swiss National Science Foundation and EPFL.

Author information

Authors and Affiliations



Q.Y. and K.J. conceived this study. Q.Y., N.P., L.X., C.G., S.F., D.B., L.P., E.K., J.A. and K.J. performed experiments, provided samples, performed data analysis and contributed to manuscript writing.

Corresponding author

Correspondence to Kai Johnsson.

Ethics declarations

Competing interests

K.J. is inventor on a patent application filed by EPFL. Patent number: US10221439B2; Sensors, methods and kits for detecting nicotinamide adenine dinucleotides. J.A. is a consultant to Mitobridge. All other authors declare no conflict of interest.

Additional information

Peer review information Primary Handling Editor(s): Ana Mateus; Christoph Schmitt.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Improving sensor performance using cpNLuc.

The distance between the active site (glowing blue ball) and the fluorophore Cy3 is shortened when using cpNLuc as the BRET donor, resulting in a higher energy transfer efficiency in the closed state of the sensor.

Extended Data Fig. 2 Cofactor binding site of wild-type hSPR.

Residues contributing to cofactor binding and randomized for improving NAD+ binding affinity of the BRET sensor are highlighted in yellow. The selected mutant NAD-cpLuc2 carries the SPR mutation A41D, R42L and R17K. The SPR used for the generation of sensor NAD-Luc and NAD-cpLuc contains the mutation A41D and R42W to switch the cofactor sensitivity from NADP+ to NAD+. SPR PDB ID = 4HWK.

Extended Data Fig. 3 Titration of NAD-cpLuc and NAD-cpLuc2.

Titration curve of a, NAD-cpLuc and b, NAD-cpLuc2 with various cofactors and biosynthetic precursors of NAD+. Error bars represent ± SD of n = 3 independent measurements.

Source data

Extended Data Fig. 4 Modularity of BRET sensor components.

a, Cartoon of the sensor using TLuc as BRET donor. b, Performance of the sensor using TLuc as BRET donor. The resulting sensor showed a smaller signal response compared to the cpNLuc-based sensor. c, Cartoon of the sensor using HaloTag (Halo) as the self-labeling protein. d, Performance of the sensor using HaloTag for attaching fluorescent sulfamethoxazole derivative Halo-PEG11-Cy3-SMX. The structure of Halo-PEG11-Cy3-SMX and its synthesis is described in the section “SYNTHESIS OF COMPOUNDS”. Error bars represent ± SD of n = 3 independent measurements.

Source data

Extended Data Fig. 5 NAD+ quantification by test paper for samples prepared with and without centrifugation step.

a, cell lysate, b, homogenized mice liver and c, spiked human blood. Error bars represent ± SD of n = 3 independent measurements.

Source data

Extended Data Fig. 6 Stability of test paper emission ratio during 5 min measurement.

The ratio was monitored by camera after spotting samples with known NAD+ concentrations onto the test paper. The stable ratio indicates a minimal sample evaporation and signal drift. The experiment was repeated three times independently with similar results.

Source data

Extended Data Fig. 7 Measurement of enzymatic activities in plasma.

a, The measurement of spiked ALT in plasma. b, The measurement of spiked AST in plasma. Error bars represent ± SD of n = 3 independent measurements.

Source data

Extended Data Fig. 8 Stability of test paper under room temperature storage.

The stability is evaluated by measuring a, total light intensity, b, Ro and Rc, and c, c50. The values were given as mean ± SD of n = 3 independent measurements.

Source data

Extended Data Fig. 9 Effect of 1 U/mL alcohol dehydrogenase (ADH from S. cerevisiae) on sample NAD+ stability.

The natural level of ADH (1 U/mL) converted no more than 20% of the NAD+ in the sample treated using the current method compared to a method using 50 mM ADH inhibitor (4-methylpyrazole hydrochloride). Error bars represent mean ± SD of n = 3 independent measurements.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, and Methods

Reporting Summary

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Yu, Q., Pourmandi, N., Xue, L. et al. A biosensor for measuring NAD+ levels at the point of care. Nat Metab 1, 1219–1225 (2019).

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