Biomolecular Detection Device

Abstract

Disclosed herein is a biomolecular detection device (1) for analyzing a cell, vesicle or a cellular or vesicular component, comprising an evanescent illuminator with an optical coupling unit configured for generating an evanescent field from coherent light (L) with a predefined wavelength on a first surface of the evanescent illuminator. The first surface of evanescent illuminator comprises a template nanopattern (5), containing a coherent arrangement of a plurality of predetermined lines along which membrane recognition elements for a binder structure (82) of a transmembrane protein (81), preferably a laterally diffusible transmembrane protein, of the cell, vesicle or the cellular or vesicular component (8) are arranged. The membrane recognition elements (53) are configured to bind the binder structure (82) of the transmembrane protein (81) for forming a transmembrane nanopattern within the cell, vesicle or the cellular or vesicular component (8) based on the template nanopattern (5) of the evanescent illuminator, such that light of the evanescent field is scattered by the cell, vesicle or the cellular or vesicular component (8) bound to the membrane recognition elements (53). The predetermined lines are arranged such that light scattered by the cell, vesicle or cellular or vesicular components (8) bound to the membrane recognition elements (53) constructively interferes at a predefined detection site (7) with a difference in optical path length that is an integer multiple of the predefined wavelength of the coherent light (L).

Claims

1. A biomolecular detection device for analyzing a cell, vesicle, or a cellular or vesicular component, comprising an evanescent illuminator with an optical coupling unit configured for generating an evanescent field from coherent light (L) with a predefined wavelength on a first surface of the evanescent illuminator, the first surface of the evanescent illuminator comprising a template nanopattern, containing a coherent arrangement of a plurality of predetermined lines along which membrane recognition elements for a binder structure of a transmembrane protein of the cell, vesicle, or the cellular or vesicular component are arranged, wherein the membrane recognition elements are configured to bind the binder structure of the transmembrane protein for forming a transmembrane nanopattern within the cell, vesicle, or the cellular or vesicular component based on the template nanopattern of the evanescent illuminator, such that light of the evanescent field is scattered by the cell, vesicle, or the cellular or vesicular component bound to the membrane recognition elements, and wherein the predetermined lines are arranged such that light scattered by the cell, vesicle or cellular or vesicular components bound to the membrane recognition elements constructively interferes at a predefined detection site with a difference in optical path length that is an integer multiple of the predefined wavelength of the coherent light (L).

2. The biomolecular detection device according to claim 1, wherein the evanescent illuminator comprises a carrier with a planar waveguide arranged on a surface of the carrier and an optical coupler as the optical coupling unit for coupling coherent light (L) of a predefined wavelength into the waveguide such that the coherent light propagates through the planar waveguide with an evanescent field of the coherent light (L) propagating along a first surface of the planar waveguide and wherein the first surface of the planar waveguide comprising the template nanopattern.

3. The biomolecular detection device according to claim 1, wherein the evanescent illuminator is a total internal reflection system configured for providing a beam of coherent light (L) at the predetermined wavelength and at a predetermined angle onto the first surface of the evanescent illuminator by means of the optical coupling unit, optionally by a prism.

4. The biomolecular detection device according to claim 1, wherein the membrane recognition elements are antibodies being specific to at least the binder structure of the transmembrane protein, or wherein the membrane recognition elements contain an electrophile moiety for establishing a covalent bond with the binder structure of the transmembrane protein.

5. The biomolecular detection device according to claim 1, wherein the plurality of predetermined lines comprises curved lines with a curvature configured such that light of the evanescent field scattered by the cell, vesicle, or the cellular or vesicular component bound to the membrane recognition elements interferes at the predefined detection site.

6. The biomolecular detection device according to claim 1, wherein the first surface of the evanescent illuminator comprises a cell adhesive.

7. The biomolecular detection device according to claim 1, wherein at least one cell, vesicle, or cellular or vesicular component is bound via the binder structure of the transmembrane protein to the membrane recognition elements.

8. The biomolecular detection device according to claim 1, wherein the predetermined lines are separated from each other by areas devoid of membrane recognition elements and wherein the areas devoid of membrane recognition elements are configured to inverse an optical modulation, which is induced by the binding of a structural recognition element to the binder structure of the transmembrane protein, such that the signal obtained from binding of the structural recognition element to the binder structure of the transmembrane protein is provided in a different operating window of the biomolecular detection device, wherein the different operating window is at an intensity close to zero.

9. (canceled)

10. A method of detecting molecular interactions associated with cells, vesicles, or cellular or vesicular components, comprising: providing the biomolecular detection device according to claim 1; applying a cell or vesicle to the membrane recognition elements, wherein the cell or the vesicle comprises a membrane and at least one transmembrane protein with an extracellular or extravesicular binder structure, optionally where at least one transmembrane protein is laterally diffused along the membrane; aligning the al least one transmembrane protein of the cell or the vesicle according to the template nanopattern of the first surface of the evanescent illuminator, such that a transmembrane nanopattern is formed in the membrane of the cell or the vesicle, wherein the transmembrane pattern corresponds at least partially to the template nanopattern of the first surface of the evanescent illuminator; generating a beam of coherent light at a predefined beam generation location relative to the plurality of predetermined lines, the beam of coherent light having a predefined wavelength and being incident on the membrane recognition elements with the bound transmembrane protein in a manner that diffracted portions of the incident beam of coherent light constructively interfere at the predefined detection site relative to the plurality of predetermined lines with a difference in optical path length that is an integer multiple of the predefined wavelength of the coherent light to provide a signal representative of the membrane recognition elements with the transmembrane protein of a cell, vesicle, or cellular or vesicular component bound thereto at the predefined detection site; and measuring the signal representative for the membrane recognition elements with the transmembrane protein of a cell, vesicle, or cellular or vesicular component bound thereto.

11. The method according to claim 10, further comprising the step of comparing the measured signal representative of the membrane recognition elements with the transmembrane of a cell, vesicle, or cellular or vesicular component bound thereto, with an unbound signal representative of only the membrane recognition elements.

12. The method of claim 10, wherein a cell is applied to the membrane recognition elements and wherein before generating the beam of coherent light the cell is modified such that only parts of the cell membrane remain on the biomolecular detection device.

13. The method according to claim 10, wherein the binder structure of the transmembrane protein is specific to an antibody being arranged along the predefined lines of the evanescent illuminator of the biomolecular detection device.

14. The method according to claim 10, wherein a protein of interest of the biomolecular interaction comprises a high-mass moiety.

15. The method according to claim 10, wherein additionally, optionally simultaneously, a fluorescent and/or bioluminescent signal is recorded.

16. A method for generating a transmembrane nanopattern within a cell, vesicle, or cellular or vesicular component, the method comprising: providing the biomolecular detection device according to claim 1; applying a cell or a vesicle is applied to the membrane recognition elements, wherein the cell or the vesicle comprises a membrane and at least one transmembrane protein with an extracellular or extravascular binder structure; optionally laterally diffusing at least one transmembrane protein along the membrane; and binding the extracellular binding structure of the at least one transmembrane protein to any of the membrane recognition elements and aligning the at least one transmembrane protein according to the template nanopattern of the first surface of the evanescent illuminator, such that a transmembrane nanopattern is formed in the membrane of the cell or the vesicle, wherein the transmembrane pattern corresponds at least partially to the template nanopattern of the first surface of the evanescent illuminator.

17. The method according to claim 16, wherein the binder structure of the transmembrane protein is specific to an antibody being arranged along the predefined lines of the evanescent illuminator of the biomolecular detection device.

18. The method according to claim 12, wherein after aligning the transmembrane protein and forming of the transmembrane nanopattern, the cell is modified such that only parts of the cell membrane remain on the biomolecular detection device.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0137] FIG. 1 shows a schematic representation of a biomolecular detection device according to a first embodiment of the invention;

[0138] FIG. 2 shows a schematic cross-sectional view of a biomolecular detection device according to another embodiment of the invention;

[0139] FIGS. 3a and 3b show a membrane of a cell and an enlarged view of a waveguide of a biomolecular detection device according to an embodiment of the invention;

[0140] FIGS. 4a to 4c show a signal obtained by a biomolecular detection device according to an embodiment of the invention during the generation of a nanopattern within a cell, vesicle or cellular or vesicular component.

[0141] FIGS. 5a and 5b show a signal obtained from applying HEK293 cells to a biomolecular detection device according to the invention and further stimulating and inhibiting β.sub.2ARs.

[0142] FIGS. 6a and 6b show a signal obtained from applying HEK293 cells to a biomolecular detection device according to the invention and further applying an off-target stimulant (FIG. 6a) as well as a positive control with an on-target stimulant (FIG. 6b).

[0143] FIGS. 7a and 7b show a schematic representation of a biomolecular detection device according to another embodiment of the invention.

[0144] FIGS. 8a and 8b show a schematic representation of a biomolecular detection device according to another embodiment of the invention.

DESCRIPTION

[0145] FIG. 1 shows a biomolecular detection device 1 according to an embodiment of the invention. The biomolecular detection device 1 comprises an evanescent illuminator comprising in this embodiment a carrier 2 with a surface on which planar waveguide 3 is arranged. The detection device further comprises optical coupler 4 for coupling coherent light L of a predefined wavelength into planar waveguide 3 such that coherent light propagates through the planar waveguide with an evanescent field of the coherent light propagating along a first surface of planar waveguide 3. The first surface of the planar waveguide is the surface facing away from carrier 2, i.e. the surface which is visible in FIG. 1. Besides optical coupler 4, the first surface of the waveguide 3 comprises template nanopattern 5, which contains a plurality of predetermined lines along which membrane recognition elements are arranged (not shown in FIG. 1). In general, a light source 6 is employed for providing an incoming beam of coherent light L towards optical coupler 4. Optical coupler 4 couples the coherent light L into planar waveguide 3 upon which coherent light L propagates in the direction of the arrow shown in FIG. 1 towards template nanopattern 5. If the membrane recognition elements are bound to a cell, vesicle or cellular or vesicular component, light is scattered and due to the predefined lines, which are arranged such that light scattered by the cell, vesicle or cellular or vesicular components bound to the membrane recognition elements constructively interferes at a predefined detection site 7. In the particular embodiment shown, the predetermined lines are curved lines having a curvature configured such that light of the evanescent field scattered by the cell, vesicle or the cellular or vesicular component bound to the membrane recognition elements interferes at the predefined detection site 7. The distance between each of the membrane recognition elements of template nanopattern 5 and detection site 7 is referred to as the optical path length.

[0146] FIG. 2 illustrates a schematic cross-section of a biomolecular detection device 1 shown in FIG. 1 through template nanopattern 5 along the propagation direction of the light L through planar waveguide 3. Device 1 contains carrier 2 with waveguide 3 arranged on its surface. Arranged on top of the first surface of planar waveguide 3 is living cell 8. Furthermore, template nanopattern 5 comprises ridges 51 and grooves 52. Ridges 51 are areas along which membrane recognition elements are arranged. Grooves 52 are areas which do not contain any membrane recognition elements. Thus, a binder structure of a transmembrane protein of cell 8 can only bind to nanopattern 5 at a corresponding ridge. It should be noted that the widths of the cell, nanopattern, waveguide and carrier do not provide any indication of their actual widths or the width ratios.

[0147] FIG. 3a shows an enlarged schematic representation of a biomolecular detection device of FIG. 2 directly after a cell 8 has been applied to the first surface of the waveguide. The cell comprises transmembrane proteins 81 with extracellular binder structure 82. In the particular embodiment shown, the transmembrane protein is a G-protein coupled receptor (GPCR). The template nanopattern of the waveguide comprises predetermined lines with ridge 51 comprising membrane recognition elements 53. As can be seen, binder structures 82 of the transmembrane proteins cannot yet interact with membrane recognition elements 53.

[0148] In the lower portion of FIG. 3b, the GPCRs were laterally diffused within the membrane, thereby enabling binding of the extracellular binder structure to the membrane recognition elements. As a result, each bound GPCR is locked at a specific position within the membrane. The overall arrangement of all GPCRs bound to membrane recognition elements is a nanopattern which corresponds to the template nanopattern of the waveguide. In other words, the template nanopattern of the waveguide has been transformed into the cell. The upper portion of FIG. 3b illustrates binding of ligand 86 to the extracellular portion of the nanopattern formed in the membrane (i). The attachment of the ligand entails a mass increase, which provides an increase of the measured signal. Binding of ligand 86 leads to an intracellular interaction. G-protein signaling is affected by ligand binding, in which the Ga subunit 83, Gβ subunit 84 and Gγ subunit 85 subunit are released. This release causes a mass decrease, which triggers a decrease of the observed signal (ii). Receptor desensitization requires recruitment of cytosolic protein 87, which again causes a mass increase and thus triggers an increase of the observed signal. Due to the absence of cross-sensitivity, direct information about the mass of the complex at a specific point in time can be obtained.

[0149] FIGS. 4a and 4b illustrate an ideal output signal obtained by using a biomolecular detection device as described herein. In FIG. 4a, the measurement has started and a constant response is obtained, as the binder structures of the transmembrane proteins are not yet bound to the membrane recognition elements of the waveguide. When binding occurs, the measured signal increases until it reaches a constant value (i.e. when all possible binder structures are bound to a membrane recognition element, see FIG. 4b). FIG. 4c shows an experimentally obtained signal obtained by binding β.sub.2ARs (beta-2 adrenergic receptors) in HEK293 cells with a fused autoreactive SNAP tag protein to the molecular recognition elements of the nanopattern of the biomolecular detection device. In this particular embodiment, the SNAP tag protein is bound to a BG-NH.sub.2 derivative which is bound to the waveguide. As can be seen, after around 80-100 min, a constant signal is observed.

[0150] FIG. 5a shows the real time formation of a nanopattern and further the response obtained from stimulating and inhibiting the employed HEK293 cells. With reference to FIG. 5a, the first increase of the measured signal between 0 and 150 min corresponds to the formation of the nanopattern within the cell membrane. At 150 min, the cultivation has been changed to assay buffer resulting in a decrease of the signal. FIG. 5b shows an enlarged view of the dashed area in FIG. 5a. At around 200 min, the cells were stimulated with 1 μM isoproterenol, which resulted in an increase of the measured signal. Competitive inhibition of isoproterenol with 10 μM ICI 155.881 at around 225 min lead to a decrease of the signal and partial recovery of the initial state. This measurement proves that the obtained signal is a consequence of the observed intracellular interaction and not just the result of a free SNAP tag bound to the molecular recognition element.

[0151] FIG. 6a shows the signal obtained upon stimulation of the HEK293 cells with 5 μM NECA to activate the off-target adenosine receptors. Due to slight variations in the initial measured signal intensity, the mass increase at the receptor, relative to the initial mass is depicted. However, with no contribution from off-target receptors this mass increase corresponds to the molecular weight increase relative to the molecular weight of the immobilized receptor complex (assuming all receptors were activated). The results show that no significant signal change is observed upon activation of the adenosine receptor with NECA. In contrast, a control experiment with 1 μM isoproterenol results in the signal increase which has already been shown in FIG. 4c. In summary, these results show that indeed no cross sensitivity of any off target interactions is observed and that these interactions do not influence the measurement result. As explained above, the reason for the absence of cross sensitivity is that the adenosine receptors are not bound to the molecular recognition elements and therefore no scattered light is received at the detection site. In fact, receptors that are distributed randomly, scatter light in an incoherent way and thus do not participate in this focusing effect. Similarly, morphological changes to the cell body are incoherent in their nature and therefore do not contribute to the measured signal either. This is in sharp contrast to DMR and other label-free assays that record both specific and nonspecific molecular interactions as well as morphological changes to the cell. As a consequence, cellular morphology can not only answer the question if GPCRs are activated but also shed light on the temporal occurrence and progression as well as mass of the molecules involved.

[0152] FIGS. 7a and 7b show a biomolecular detection device 1′ according to an embodiment of the invention. Device 1′ comprises an evanescent illuminator 2′ with an optical coupling unit 4′ configured for generating an evanescent field 9′ from coherent light with a predefined wavelength of a light source 6′ on a first surface of the evanescent illuminator 2′. The first surface of the evanescent illuminator 2′ comprises template nanopattern 5′ containing a coherent arrangement of a plurality of predetermined lines along which membrane recognition elements for a binder structure of a transmembrane protein 81 of cell 8′. As can be seen, by establishing a chemical bond between the membrane recognition element and the laterally diffusible transmembrane proteins 81, the nanopattern 5′ of the evanescent illuminator 2′ is transposed into the cell as a transmembrane nanopattern. In the embodiment in FIG. 7a, light source 6′ and detection unit 7′ are physically separate components, while in the embodiment shown in FIG. 7b, they are integral part of evanescent illuminator 2′.

[0153] FIGS. 8a and 8b show an alternative embodiment of a biomolecular detection device 1″ according to the invention. The biomolecular detection device 1 comprises an evanescent illuminator which in these particular embodiments is a total internal reflection system configured for providing a beam of coherent light at the predetermined wavelength from light source 6″ and at a predetermined angle onto the first surface of the evanescent illuminator by means of the optical coupling unit 4″. The optical coupling unit in these embodiments is a prism. In the embodiment shown in FIG. 8a, the evanescent illuminator comprises an index matching medium 11″ such as index matching oils, DMSO, glycerol, water mixtures, hydrogels, etc., and a carrier slide 12″ which contains the template nanopattern 5″. Alternatively, as shown in FIG. 8b, the evanescent illuminator can be devoid of index matching medium 11″ and a carrier slide 12″. In this case, the nanopattern 5″ is directly provided on a first surface of the optical coupling unit 4″, i.e. the prism.

Example

Methods and Materials

[0154] Cell culture medium DMEM High Glucose (4.5 g/l) with L-Glutamine (BioConcept, Switzerland), Lipofectamine® 2000, Opti-MEM® I (1×) Versene 1:5000 (1×), hank's balanced salt solution (HBSS), Zeozin™ was purchased from Life Technologies Europe (Zug, Switzerland). HEPES was from GERBU Biotechnik GmbH (Heidelberg, Germany), Fetal Bovine Serum (FBS) was purchased from Sigma-Aldrich Chemie GmbH (Buchs SG, Switzerland). G418 was from InvivoGen (San Diego, USA), Tissue Culture Flasks from VWR International GmbH (Dietikon, Switzerland), Biofil® Tissue culture plate 24 wells were from Axon Lab AG (Baden-Dättwil, Switzerland). Corning Costar sterile black 96 well plates, clear bottom, TC treated, Poly-D-Lysine coated were from Vitaris AG (Baar, Switzerland), Custom coated CulturPlate-96, White Opaque 96-well Microplate, Sterile and Tissue Culture Treated and ViewPlate-96, White 96-well Microplate with Clear Bottom, Sterile and Tissue Culture Treated were from Perkin Elmer (Schwerzenbach, Switzerland). TPP 6-well tissue culture plates were from Faust Laborbedarf AG (Schaffhausen, Switzerland) Coelenterazine 400a, Deep Blue C (DBC) was purchased from Cayman Chemical (Ann Arbor, Mich., United States). The GRGDSPGSC-(DBCO) peptide was custom synthesized by LifeTein, LLC (Somerset, N.J., USA). BG-GLA-NHS was obtained from BioConcept Ltd. (Alschwil, Switzerland). Azido-PEG4-NHS was obtained from Jena Bioscience (Jena, Germany). The PAA-g-PEG-NH-PhSNPPOC copolymer, used as a biocompatible coating, was provided by SuSoS. Isoproterenol hydrochloride and formoterol hemifumarate was purchased from Tocris Bioscience (Bristol, UK). ICI 118,551 hydrochloride, Fluorescein-O′-acetic acid and all other chemicals were purchased from Sigma-Aldrich Chemie GmbH (Buchs SG, Switzerland). Thin-film optical waveguides with a 145 nm Ta.sub.2O.sub.5 layer were obtained from Zeptosens with the in and out coupling gratings covered with a 1 μm thick layer of SiO.sub.2 by IMT Masken and Teilungen AG (Greifensee, Switzerland).

Expression Constructs

[0155] Beta-Arrestin 2-mPlum: mPlum coding sequence was amplified by PCR (Phusion polymerase, Finnzymes) and transferred to a pcDNA3 expression vector containing the beta-arrestin2 coding sequence.

[0156] Beta-Arrestin 2-GFP: Beta-arrestin 2 coding sequence was amplified by PCR (Phusion polymerase, Finnzymes) and transferred to a pEGFP (Clonetech) expression vector.

Cell Lines and Cell Culture

[0157] A HEK293 cell line stably expressing the SNAP-beta2-adrenergic receptor (referred to as SNAP-β2AR) was purchased from Cisbio (Codolet, France). HEK293 stably overexpressing All HEK293 cells were cultured in DMEM supplemented with 10%-v/v fetal bovine serum and 600 μg/ml G418 with 5% CO2 at 37° C.

Preparation of Sensor Chips

[0158] Thin-film optical waveguides were treated with a similar protocol as reported previously (Nat. Nanotechnology 2017, DOI: 10.1038/NNANO.2017.168). In short, waveguides were washed with 0.1% aqueous Tween 20, followed by ultrasound assisted washing in MilliQ water, Isopropanol and Toluene. The chips were then soaked in warm Hellmanex III for 1 min, thoroughly rinsed with MilliQ water and cleaned with highly oxidizing Piranha solution (H.sub.2SO.sub.4/H.sub.2O.sub.2 7:3) for 30 min. After excessive washing with MilliQ water, the chips were centrifuge dried at 800 rcf for 2 min and activated by oxygen plasma. After plasma treatment, the chips were immediately immersed in the PAA-g-PEG-NH-PhSNPPOC graft copolymer coating solution (0.1 mg/ml in 1 mM HEPES pH 7.4) for 60 min. To fully passivate the layer, the chips were washed with MilliQ water and ethanol and immersed in a 25 mM solution of methyl chloroformate in anhydrous acetonitrile containing 2 equiv. of N,N-diisopropylethylamine for 5 min. The coated chips were washed with ethanol and MilliQ water, and blow dried by a nitrogen jet. Prepared sensor chips were stored in the dark at 4° C. until further use.

Preparation of Template Nanopatterns

[0159] Template nanopatterns were prepared according to the standard reactive immersion lithography (RIL) process described (Nat. Nanotechnology 2017, DOI: 10.1038/NNANO.2017.168). Briefly, a copolymer-coated sensor chip was placed in a custom holder. The phasemask used to generate the nanopattern was aligned using an alignment help and the gap between the chip and phase mask was filled with a solution of 0.1%-v/v Hydroxyl amine in DMSO. The photolithographic exposure was conducted at 405 nm with a dose of 2000 mJ/cm.sup.2 in a custom-built setup. After illumination the chip was washed with isopropanol and MilliQ water and the activated ridges were functionalized with 1 mM amine reactive SNAP-tag substrate (BG-GLA-NHS), which is covalently bound by the SNAP-tag protein. In order to increase cell adhesion to the chip, remaining PhSNPPOC groups were removed by flood exposure. The free binding sites were then functionalized with the hetero biofunctional crosslinker azido-PEG4-NHS. Finally, the chip was incubated with an azide reactive aqueous solution of 0.5 mM GRGDSPGSC-(DBCO) overnight, washed with isopropanol and MilliQ water and dried with a jet of nitrogen.

Cell Measurements

[0160] SNAP-β.sub.2AR cells were grown to 60-80% confluency in T25 culture flasks, washed twice with warm phosphate-buffered saline (PBS), incubated with 1× Versene for 5 min and resuspended in cell culture medium. In order to decrease baseline signal contributions from non-functional cellular debris, the cells were centrifuged at 50 rpm for 1 min and resuspended in culture media two times sequentially. The cells were seeded to reach confluency on the waveguide in an incubation chamber containing 500 μl cell culture media. Cells were only seeded when viability exceeded 90%, as determined by a Countess automated cell counter (Invitrogen). Except for the real-time establishment of the transmembrane nanopattern, seeded cells were kept in a CO.sub.2 incubator at 37° C. for 2 h to allow cells adherence to the sensor chip (and covalent interaction of the SNAP-tag on the β.sub.2AR with the SNAP-tag substrate on the chip). The incubation chamber containing the cells was then washed twice with warm HBSS buffer (supplemented with 20 mM HEPES, pH 7.4) adjusted for DMSO and transferred to a modified F3000 ZeptoReader (Zeptosens) which was kept at 35° C. The biomolecular detection device was then allowed to temperature equilibrate inside the ZeptoReader for 5-10 min before performing the assay. For all assays, the signal was monitored for 7 min (baseline measurement) before careful substitution of the buffer with buffer containing the β.sub.2AR ligand and monitored for another 21 min thereafter. For the real-time establishment of the transmembrane nanopattern experiment, the measurement was performed in cell culture media supplemented with 20 mM HEPES, pH 7.4. Once the nanopattern was established, the culture media was carefully exchanged with HBSS buffer (supplemented with 20 mM HEPES, pH 7.4).

[0161] Typical instrument parameters for signal acquisition were as follows: one image every 10 s using the 635 nm laser with an integration time of 0.25-1 s depending on the intensity of the initial signal and a grey filter value of 0.001 in the illumination path of the ZeptoReader.

Data Analysis

[0162] For all assays, the square root of the raw signal was taken. The baseline was then fitted linearly and used to de-trend the signal. Data was then displayed as a fractional change compared to baseline.

[0163] For the BRET arrestin recruitment assay the concentration-response curves with area under the curve (AUC) vs. ligand concentrations were fitted using the nonlinear regression “log(inhibitor) vs. response (three parameters)” in Graph Pad Prism to calculate the pIC50 values.