Luminescent sensor proteins for point-of-care diagnostics. Dr. Remco Arts Account Manager Promega Benelux

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1 Luminescent sensor proteins for point-of-care diagnostics Dr. Remco Arts Account Manager Promega Benelux

2 Point-of-care diagnostics Diagnostic tests that are performed close to the patient Fast Potentially more affordable Convenient for patient Attractive for third world application

3 Antibody diagnostics: why? Infectious disease Specific detection of antibodies can be used as a diagnosis Therapeutic drug monitoring Monoclonal antibodies are used in e.g. cancer therapy Different patients clear antibodies at different rates Pharmacokinetic differences between patients correlate with clinical outcome Dengue fever distribution In both cases, a point-of-care test is desirable

4 Traditional antibody detection: ELISA Classical assays: Slow, multi-step, require sophisticated equipment or skilled personnel Requirements for point-of-care diagnostics: Fast, easy to perform and interpret, affordable, applicable in a non-laboratory setting Our goal Develop technology that permits detection and quantification of antibodies directly in solution in a point-of-care compatible assay

5 AbSense: FRET-based antibody detection Golynskiy et al, ChemBioChem, 2010, 11,

6 Luminescent antibody sensors (LUMABS) Green emission Blue emission Requirements for donor luciferase: Small, easily folded, stable, cofactor independent, long and stable luminescent signal, bright Traditional luciferases (Renilla, Gaussia, Firefly..) are all limited in at least one respect

7 Published August 2012 Cited 347 times (as of April 17 th )

8 Hall et al., ACS Chem Bio, 2012, 7, NanoLuc

9 NanoLuc ticks all the boxes Small (19 kda) Stable Glow-type luminescence Extremely bright Hall et al., ACS Chem. Biol., 2012

10 NanoLuc and mneongreen should constitute efficient BRET pair NanoLuc mneongreen

11 Normalized intensity Normalized intensity Normalized intensity Increasing BRET efficiency using helper domains No helper domain Antiparallel leucine zipper SH3-proline rich peptide No antibody 1 nm antibody Wavelength (nm) Wavelength(nm) Wavelength(nm)

12 Emission Ratio (green/blue) NanoLuc enables pm detection limit Monovalent epitope affinity: 42 nm Overall sensor affinity: 83 pm [anti-hiv1-p17] (M) Limit of detection: 10 pm Target antibody: HIV1-p17 Not limited by NanoLuc brightness!

13 Normalized intensity Emission Ratio (green/blue) 100 pm sensor pm sensor Wavelength(nm) Time (min) No antibody 100 pm antibody 500 pm antibody 5 nm antibody

14 Exchanging epitopes allows different antibodies to be targeted HIV Influenza Dengue Cetuximab Trastuzumab Rituximab

15 Dengue virus 1 sensor response [Ab] (nm) Optimized buffer conditions 100 pm DEN1-LUMABS 50 μl reaction volume 0.25 μl Nano-Glo assay substrate K d = 1.67 ± 0.3 nm Nokia Lumia 920 ISO 3200 Exposure 4 s

16 Antibody detection in blood plasma using Android smartphone Arts et al., Analytical Chemistry, 2016 Target: Dengue virus 1 NS1 antibodies Medium: Blood plasma [Sensor]: 1 nm

17 Take home messages BRET sensors can be applied in blood plasma with minimal matrix effects Modular antibody detection strategy Readout compatible with smartphone

18 More NanoLuc at TU Eindhoven BRET sensors Any FRET sensor could be converted to a BRET sensor Emission at 460 nm -> exceptional compatibility with brightest fluorescent proteins

19 More NanoLuc at TU Eindhoven Cell imaging Bioluminescence vs. Fluorescence No photobleaching No autofluorescence Compatible with optogenetics NanoLuc in cell imaging No posttranslation modifications No cofactors required Framerate of several seconds for live cell imaging Aper et al, ACS Chem. Biol. 2016

20 More NanoLuc at TU Eindhoven Cell imaging Caspase activity can be measured in live HeLa cells Den Hamer et al., ACS Sensors, 2017

21 Wrapping up Bioluminescence beats fluorescence in Background signal, scattering, signal-to-noise, optogenetic projects, photobleaching NanoLuc is the luciferase of choice in BRET sensors, cell imaging, studying gene expression, protein-protein interactions, live animal imaging Excellent brightness, stability, no need for cofactors leading, low background signal Lowest limit of detection and the most reliable signal NanoLuc-based BRET sensors at TU Eindhoven Antibodies, Zn 2+, caspases, DNA, Zn 2+, creatinine, dinitrophenol, HIV1-p17 protein, ctni Further reading: Arts et al., Analytical Chemistry, 2016 Aper et al. ACS Chemical Biology, 2016 Arts et al., ACS Sensors, 2017 Den Hamer et al., ACS Sensors, 2016 Arts et al., Methods in Enzymology, 2017 Engelen et al., ChemComm, 2016

22 Acknowledgement Prof. Maarten Merkx Prof. Luc Brunsveld Martijn van Rosmalen Susann Ludwig Anniek den Hamer Stijn Aper Ilona den Hartog Stefan Zijlema Chemical Biology group Department of Biomedical Engineering

23 Caspase sensors using NanoLuc Den Hamer et al., ACS Sensors, 2017

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