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Intensity interrogation near cutoff resonance for label-free cellular profiling.

Nazirizadeh Y, Behrends V, Prósz A, Orgovan N, Horvath R, Ferrie AM, Fang Y, Selhuber-Unkel C, Gerken M - Sci Rep (2016)

Bottom Line: We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate.For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength.The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

View Article: PubMed Central - PubMed

Affiliation: Byosens GmbH, 20357 Hamburg, Germany.

ABSTRACT
We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

No MeSH data available.


Related in: MedlinePlus

Validation experiments of the intensity based readout with G protein-coupled receptor (GPCR) assays.(a) Real-time response of A431 cells to bradykinin dose variations. (b) Dose response curve with duplicates of maximum response to bradykinin. The EC50 value derived from this curve is 0.45 nM. (c) Comparison of real-time response of A431 cells to bradykinin at ambient (23 °C) and physiological (37 °C) conditions. The maximum response and the curve shape show differences. (d) Whole cell real-time response of A431 cells to protease-activated receptor-2 activating peptide SLIGRL dose variations. (e) Dose response curve with duplicates of maximum response to SLIGRL resulting in an EC50 value of 3.1 μM.
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f3: Validation experiments of the intensity based readout with G protein-coupled receptor (GPCR) assays.(a) Real-time response of A431 cells to bradykinin dose variations. (b) Dose response curve with duplicates of maximum response to bradykinin. The EC50 value derived from this curve is 0.45 nM. (c) Comparison of real-time response of A431 cells to bradykinin at ambient (23 °C) and physiological (37 °C) conditions. The maximum response and the curve shape show differences. (d) Whole cell real-time response of A431 cells to protease-activated receptor-2 activating peptide SLIGRL dose variations. (e) Dose response curve with duplicates of maximum response to SLIGRL resulting in an EC50 value of 3.1 μM.

Mentions: The intensity-based interrogation method was also validated by GPCR experiments with bradykinin and protease-activated receptor-2 (PAR2) activating peptide SLIGRL. Bradykinin is an agonist for the bradykinin B2 receptor and activates both Gαq and Gαs pathways in A431 cells18. Results showed that bradykinin triggered a robust, dose-dependent intensity signal in A431 when assayed at ambient temperature (Fig. 3a), with a half maximal effective concentration (EC50) of 0.45 nM (Fig. 3b). The shape of the biosensor response curve, kinetics and EC50 obtained were all highly comparable to the results reported with a spectral interrogation reader18. Interestingly, compared to ambient conditions, bradykinin triggered an intensity signal having faster kinetics, greater amplitude and an additional shoulder when the cells were assayed under physiological conditions (37 °C) (Fig. 3c). This behavior was also observed in previous experiments19 and emphasized the importance of experiments under physiological conditions. The PAR2 agonist SLIGRL also triggered a robust, dose-dependent intensity signal in A431 cells (Fig. 3d), with an EC50 of 3.1 μM (Fig. 3e). Again, the shape, kinetics and EC50 obtained were all comparable to previous results measured with a spectral interrogation reader20.


Intensity interrogation near cutoff resonance for label-free cellular profiling.

Nazirizadeh Y, Behrends V, Prósz A, Orgovan N, Horvath R, Ferrie AM, Fang Y, Selhuber-Unkel C, Gerken M - Sci Rep (2016)

Validation experiments of the intensity based readout with G protein-coupled receptor (GPCR) assays.(a) Real-time response of A431 cells to bradykinin dose variations. (b) Dose response curve with duplicates of maximum response to bradykinin. The EC50 value derived from this curve is 0.45 nM. (c) Comparison of real-time response of A431 cells to bradykinin at ambient (23 °C) and physiological (37 °C) conditions. The maximum response and the curve shape show differences. (d) Whole cell real-time response of A431 cells to protease-activated receptor-2 activating peptide SLIGRL dose variations. (e) Dose response curve with duplicates of maximum response to SLIGRL resulting in an EC50 value of 3.1 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834563&req=5

f3: Validation experiments of the intensity based readout with G protein-coupled receptor (GPCR) assays.(a) Real-time response of A431 cells to bradykinin dose variations. (b) Dose response curve with duplicates of maximum response to bradykinin. The EC50 value derived from this curve is 0.45 nM. (c) Comparison of real-time response of A431 cells to bradykinin at ambient (23 °C) and physiological (37 °C) conditions. The maximum response and the curve shape show differences. (d) Whole cell real-time response of A431 cells to protease-activated receptor-2 activating peptide SLIGRL dose variations. (e) Dose response curve with duplicates of maximum response to SLIGRL resulting in an EC50 value of 3.1 μM.
Mentions: The intensity-based interrogation method was also validated by GPCR experiments with bradykinin and protease-activated receptor-2 (PAR2) activating peptide SLIGRL. Bradykinin is an agonist for the bradykinin B2 receptor and activates both Gαq and Gαs pathways in A431 cells18. Results showed that bradykinin triggered a robust, dose-dependent intensity signal in A431 when assayed at ambient temperature (Fig. 3a), with a half maximal effective concentration (EC50) of 0.45 nM (Fig. 3b). The shape of the biosensor response curve, kinetics and EC50 obtained were all highly comparable to the results reported with a spectral interrogation reader18. Interestingly, compared to ambient conditions, bradykinin triggered an intensity signal having faster kinetics, greater amplitude and an additional shoulder when the cells were assayed under physiological conditions (37 °C) (Fig. 3c). This behavior was also observed in previous experiments19 and emphasized the importance of experiments under physiological conditions. The PAR2 agonist SLIGRL also triggered a robust, dose-dependent intensity signal in A431 cells (Fig. 3d), with an EC50 of 3.1 μM (Fig. 3e). Again, the shape, kinetics and EC50 obtained were all comparable to previous results measured with a spectral interrogation reader20.

Bottom Line: We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate.For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength.The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

View Article: PubMed Central - PubMed

Affiliation: Byosens GmbH, 20357 Hamburg, Germany.

ABSTRACT
We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

No MeSH data available.


Related in: MedlinePlus