Fluorescence-based assays conquered the benches of life science labs due to many advantages: ﬂuorophores are often non-toxic, ﬂuorescent dyes are affordable and can be measured multiple times. The intensity of ﬂuorescent dyes is linearly related to their concentration and signiﬁcantly many assays are available. Not only did the use of ﬂuorescence assays per se increase but in particular the use of red ﬂuorescent dyes emitting in the red and infrared range of light has risen.
What drives the shift to red dyes?
Multiplexing. Research evolves fast and requires many and exact data in a short time. Thus, multiple assays are run in parallel to detect several biological aspects in one sample. For instance, expression of a ﬂuorescent reporter protein can be referenced to cell viability. However, luminophores and ﬂuorophores measured in the same sample need to be spectrally separated for efﬁcient signal separation. Many traditional assays use bright dyes emitting in the green range (AlexaFluor®488, FITC, GFP). Fluorescent dyes that are combined with such assays are often red-shifted. Similarly, red ﬂuorescent dyes are required for multiplexing with luminescence. Luciferases emit light over broad wavelength ranges, often up to 500-600 nm (Fig. 1). Separation of luminescent and ﬂuorescent signals is only possible when combining it with red ﬂuorescent dyes emitting ﬂuorescence over 650 nm.
Autoﬂuorescence reduction in cellular and complex samples. In complex biological samples with living cells, many unwanted ﬂuorescent components exist that interfere with the signal of the ﬂuorescent dye of interest (auto-ﬂuorescence): aromatic amino acid side chains of ﬂuorescent proteins, NAD(P)H, ﬂavin or phenol red occupy the light spectrum from the UV to the red range. Fluorescent molecules are mainly found in cellular or complex samples such as serum and plasma.
Using ﬂuorophores emitting further in the red range (>650 nm), like nile red or texas red prevents auto-ﬂuorescence. Another method circumvents auto-ﬂuorescence in two ways: long lifetime ﬂuorophores detected when auto-ﬂuorescence already faded and the detection of ﬂuorescence intensity in the red. This principle is popular in homogenous assay formats that measure molecule interactions by FRET: homogenous time-resolved Förster resonance energy transfer (HTRF®, Cisbio). A long-lifetime ﬂuorophore is used as donor and transfers energy to a red acceptor ﬂuorophore only if this is found in proximity.
The use of red ﬂuorescent dyes is inevitable when measuring cells or multiple assays at once. Thus, we tested the performance of the CLARIOstar Plus with dedicated detectors in measuring the red ﬂuorescent dye AlexaFluor® 647 and compared HTRF measurements performed with standard and dedicated detectors (PMT).
Materials & Methods
AlexaFluor 647 Testing
Testing of the CLARIOstar Plus was performed with the red ﬂuorescent dye AlexaFLuor® 647, a bright ﬂuorophore that is excited around 640 nm and emits at 680 nm (Fig. 2).
- black 384 sv microplate (Greiner #784076)
- CLARIOstar Plus (BMG LABTECH)
- AlexaFluor 647 (ThermoFisher Scientiﬁc)
- PBS (Biochrom #L1825)
The red ﬂuorescent dye was diluted to standards of 100 nM; 20 nM; 4 nM; 0.8 nM and 0.16 nM in PBS. Eight replicates (20 µl) and 85 PBS blanks (20 µl) were pipetted into the plate for determination of the detection limit. Fluorescence of the red ﬂuorescent dye was measured on the CLARIOstar Plus using the following instrument settings.
|Fluorescence intensity, top optic, end point
|Dedicated red-shifted PMT
|Number of flashes
Detector comparison for HTRF measurements
For comparison of standard and dedicated detector in HTRF measurements, a Cisbio kit based on Eu Cryptate donor (emission 620 nm) and XL665 (emission 665 nm) acceptor was used.
- white 96 well half area microplate (provided by Cisbio)
- CLARIOstar Plus (BMG LABTECH)
|Time resolved fluorescence, dual chromatic
|Dedicated red-shifted PMT and standard PMT
|Ex. Ex TR
Dichroic: LP TR
Em 1: 665-10,
Em 2: 620-10
|60 µs, Time: 400 µs
|Number of flashes
Results & Discussion
A standard curve of AlexaFluor647 was measured in ﬂuorescence to determine its detection limit on the CLARIOstar Plus (Fig. 3). The standard curve from 100 nM down to 0.16 nM of the red ﬂuorescent dye resulted in a highly linear correlation as indicated by an R² of > 0,9999. Calculation of the detection limit according to IUPAC standard resulted in a low limit of 0.8 pM (Table 1).
Table 1. Limit of detection calculation of the red ﬂuorescent dye AlexaFluor647 measured on the CLARIOstar Plus
|Standard deviation of the blank (and %CV)
|Slope of linear regression
|Limit of detection - LOD (3* SDBI/Slope)
The high sensitivity is not limited to red ﬂuorescent dyes. The LOD of 0.15 pM for FITC (data not shown) shows that the dedicated PMT is a suitable solution for multiplexed detection of red and green ﬂuorophores.
Testing the dedicated red-shifted detection of the CLARIOstar Plus for HTRF measurements revealed improvements in assay window, measurement stability and conse-quently assay quality. The deltaF is a measure of the assay window which relates the increase in FRET ratio of positive controls to the negative control. This measure was highly improved using the dedicated detector for red ﬂuorescent dyes as compared to the standard detector (Fig. 4) high positive control +17%, low positive control +15 %). As additionally the %CV, a measure of signal stability, of the blank was reduced using the dedicated detector the complete assay quality was improved. The Z’ value is indicative of assay quality and includes signal stability as well as distance between positive and negative controls. HTRF assays with low signals greatly beneﬁt from the novel detector as Z’ is improved from 0.27 to 0.66 (143 %). HTRF assays with high signals display very high quality with a Z’ > 0.9, irrespective of the detector (Fig. 4).
Due to increased use of cell-based assays and multiplexing, ﬂuorophores emitting in the red and far-red are on the rise. Here, we demonstrate the suitability of the CLARIOstar Plus to detect red ﬂuorescent dyes. It displays high sensitivity with a low LOD for the dye AlexaFluor647 (0.8 pM) and signiﬁcantly improved HTRF measurements. This makes the CLARIOstar Plus a reliable device for complex samples and multiplex measurements.