TITLE: Chemiluminescence of coelenterazine catalyzed by cyclodextrins as a luminescence reference standard for luminometers 
AUTHORS: Misha   Koksharov

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bioluminescence and chemiluminescence are widely used in sensitive detection methods in biomedical sciences and analytical chemistry. A limitation of this type of measurements is that luminometers and platereaders do not directly quantify absolute quantum output of the reaction but report "relative luminescence units" (RLU) which are specific for a given instrument and reaction vessel design. At the same time, there are no simple and convenient luminescence reference standards that would have been universally available, so results (RLU measurements) reported by different instruments and laboratories usually cannot be directly compared.</div><div class = "text-block">I have found that cyclodextrins - which are often used to solubilize coelenterazine (CTZ) analogs and other compounds in water buffers - catalyze a weak chemiluminescence of CTZ (and its analogs). Chemiluminescence of 20 µM CTZ in the presence of 10 mM β-cyclodextrin or 10 mM trimethyl-β-cyclodextrin in the 50 mM Na-phosphate buffer (pH 7.40) can be used as a simple and convenient reference standard to define and compare RLU readings obtained by different instruments. This system is composed of only small molecules of a defined chemical composition which are not expensive and available in high purity from multiple suppliers making this system convenient for the general use as a luminescence reference standard.</div></div>

[STEPS]
?. Prepare for the experiment.
?. Bring the reference assay solutions (with 10 mM β-CD and/or with 10 mM TMCD) to room temperature.
?. Turn on the luminescence plate reader or luminometer, warm it up and make it ready for the measurements.
?. Take out a small (20-40 μl) aliquot of 15 mM CTZ in propylene glycol from  and allow it to reach   in the dark.
-80 °C
0 Room temperature
The 15 mM CTZ stock of the correct concentration should give an absorbance of 0.150 at λ=431-435 nm upon a 1:1000 dilution into absolute ethanol or methanol  (ε=10000 L⋅mol−1⋅cm−1).
?. [Preparation of the luminescence reference mixture]
Prepare a tube to make a desired volume of the reference luminescence solution.1.5-2 ml tubes can be used for smaller volumes and 12 ml tubes can be used for larger volumes.For example, add 1.5 ml 10 mM cyclodextrin assay solution to a 1.5-2 ml tube or add 3 ml to a 12 ml tube.
?. [Preparation of the luminescence reference mixture]
Calculate the amount of the 15 mM CTZ stock required to obtain the 20 μM final solution in the assay mix.For example, use 2 μl of stock solution for the 1.5 ml volume and 4 μl for the 3 ml volume.
The presence of 18 mM propylene glycol (obtained as a result of the 1:750 dilution of the CTZ stock made in propylene glycol into a water-based assay buffer) does not affect the chemiluminescence values when compared with a vehicle-free 20 μM CTZ solution. The latter can be obtained by evaporating the required μl volume of a CTZ stock made in ethanol and its re-dissolution in the cyclodextrin assay buffer by a thorough mechanical mixing.
?. [Preparation of the luminescence reference mixture]
Place the required small volume of the 15 mM CTZ stock above the level of the buffer as several micro-drops (on the tube walls or under the cap).
?. [Preparation of the luminescence reference mixture]
Close the tube and mix by vigorous vortexing and shaking.Make sure that CTZ is fully solubilized.
In the presence of 10 mM β-CD or TM-β-CD the 20 μM concentration of CTZ is nominally soluble and should go into solution after a rapid vortex mixing. If using a solid CTZ residue, e.g. after evaporation (rather than micro-droplets in PG or other solvent), then a thorough mechanical mixing may be required to bring it into solution even in the presence of cyclodextrins.In the absence of cyclodextrins or other solubilizers (e.g. serum) in water buffers, 10-20 μM CTZ is in the supersaturated concentration range at pH 7.40 (above the thermodynamic solubility), so some yellow crystals can occasionally be observed if mixing was not rapid enough.
?. [Preparation of the luminescence reference mixture]
Quickly dispense the final assay mix into the microplate wells or tubes which will be used for luminescence measurements.
?. [Luminescence measurements]
Start to measure luminescence within the 5-10 min after mixing CTZ with the assay solution, because chemiluminescence will slowly decay.Chemiluminescence signal can be averaged for the first 10-20 min for TMCD and 30 min for β-CD.The chemiluminescence signal slowly decays (as CTZ is slowly oxidized) but can be considered stable for practical purposes during the first 10-20 min with 10 mM TMCD and 30-50 min for 10 mM β-CD (see the Figure below).
Chemiluminescence of CTZ with TMCD is about 20 times higher than with β-CD making TMCD a more convenient luminescence standard.The two reference solutions can be used to complement each other as two independent reference standards with the different light output.With not very sensitive instruments, the chemiluminescence with β-CD can be not very far from the detection limits making in some cases its use problematic. β-CD is available at low prices and from a wider number of suppliers compared to TMCDA cheaper RMCD (randomly methylated β-CD) can be used in place of TMCD for routine reference measurements, since it leads to a similar light output. However, it must be initially referenced against TMCD/β-CD because this randomly methylated compound can differ considerably lot to lot.
How the signal varies depending on differences (i.e. uncertainties and errors) in the composition of the assay mix:the signal depends on CTZ concentration with a slightly less than proportional or semi-saturating response which depends on the type of cyclodextrin (see the Guidelines section)the response is less than proportional relative to the cyclodextrin concentration (see the Guidelines section)changing pH by -0.20 and +0.20 units from pH 7.40 results in approximately 17% decrease or 20% increase in chemiluminescence, respectively.
?. [Luminescence measurements]
Use the measured luminescence values (usually, the relative light units per second, RLU/sec) as a reference standard to calibrate the RLU values of the instrument relative to other instruments or to variations in the values generated on the same instrument at different dates due to changes in its operation.If the same reference standard is measured on another instrument, then the RLU values produced by these instruments can be directly compared by calculating an appropriate conversion coefficient.The Table 1 below shows the reference values obtained for the Biotek HTX platereader in this work.
Examples of reference values that can be obtained.  PlateVolume per wellProbe10 mM CDAveraging time, minLuminescence,
RLU/sec
(corrected for the background)
Background, RLU/sec
(uncorrected)
PMT gain = 13524-well CytoOne
(transparent)0.5Bottom-CD3038 ± 414 ± 3 (*a)
4.4 ± 1.7 (*b)TMCD10932 ± 5696-well Nunc
(black walls, 
clear thin bottom)
(with lid on top)0.1Bottom-CD2070 ± 78 ± 2TMCD101325 ± 27Top-CD2018.1 ± 3.34.8 ± 1.3TMCD10319 ± 1496-well CulturePlate, OptiPlate
(white non-transparent)
(no lid on top)0.1Top-CD30140 ± 84.1 ± 1.4TMCD102670 ± 450.2-CD30231 ± 11TMCD104338 ± 59The chemiluminescence of these reference solutions can also be used to assess and reference the sensitivity of imaging instruments. Below is an example of a weak chemiluminescence of these solutions captured on Azure 600 imager (Azure Biosystems, USA). A transparent CytoOne 24-well plate contained 500 μl samples in the following wells: TMCD reference solution in A1-A3, β-CD reference solution in D4-D6, a plain buffer in C1-C3 and all other wells were left empty. The plate was imaged for 300 sec using the chemiluminescence mode with the 3x3 pixel binning. The instrument obtains 32-bit tiff images which have an intensity range of white from 0 (black) to 65535. The image in the panel "A" was saved as jpeg file without any adjustments, while in the panel "B" the intensity was trimmed to the 63742-65535 range to highlight the observed chemiluminescence.