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Chemical and optical characterisation of
nanomaterials in biological systems
EMRP project NEW03
Deliverable D3.1.1
Confirmation of the agreed suitable fluorescent and
quantum yield standards for WP3
based on
Activity 3.1.1
BAM will identify suitable spectral fluorescence standards and working quantum yield
standards to be used for the subsequent activities in WP3 i.e. fluorescence
measurements and relative quantum yield measurements of fluorescent
nanomaterials FM1-FM4 (D1.3.4) and standards in aqueous media and biological
serum, to be used by BAM, PTB, and NPL.
Main partner:
BAM (Ute Resch-Genger)
Other partners:
Delivery Date:
November 2012
Delivery Type:
NanoChOp, WP3, Deliverable D3.1.1
Page 1
The aim of WP3 is to establish measurement techniques for the traceable optical
characterisation of fluorescent nanomaterials, in particular for the key fluorometric parameter
fluorescence quantum yield (QY). In addition, the determination of parameters controlling the
signalling behaviour of fluorescent nanoparticles will be assessed, e.g. the number of
fluorophores per particle, the number of selected surface functionalities or the number of
proteins adsorbed onto the fluorescent nanomaterials FM1-FM4.
In order to achieve traceable fluorescence measurements and QY measurements of the
fluorescent nanomaterials FM1-FM4 suitable spectral fluorescence standards and working
QY standards are necessary. The spectral fluorescent standards are required to establish
spectral correction functions for the emission spectrometers (used to measure relative QY
and other nanoparticle parameters such as number of dyes, surface functionalities or
adsorbed protein based on fluorescence intensity measurements of transparent dye
solutions) as well as for the integration sphere setups (used to measure absolute QY of
transparent and scattering chromophore systems). The QY standards are needed as
reference standards for the relative QY measurements and can be used to validate the
absolute QY measured with the integration spheres.
Choice of suitable spectral fluorescence standards
Quantitative fluorescence measurements can only be performed correctly if the relative
spectral responsivity of the used fluorescence instrument is known. The spectral responsivity
of a fluorescence measuring device depends mainly on the spectral responsivity (sensitivity)
of its detection system, consisting of the monochromator gratings and the photodetector
(mostly photomultiplier tubes, PMTs) and on the wavelength-dependence of the optical
components (lenses and polarizers). In order to measure the relative spectral responsivity
either physical transfer standards (calibrated light sources) or chemical reference materials
(fluorescence dyes with known emission intensities over their whole emission spectra) can
be used. By comparing the measured spectra of the physical or chemical standard with the
known “real” (certified) spectra, a spectral instrument correction function can be created.
Within the NanoChOp project, chemical reference materials will be used, because they are
less expensive, easier to handle, and therefore better suitable for the broad community of
fluorescence spectroscopists than physical transfer standards. For the tasks of the
NanoChOp project, spectral fluorescent standards will be used to either establish spectral
correction functions for all emission spectrometers and the integration sphere setups used.
The Federal Institute for Materials Research and Testing (BAM) has already developed and
certified a set of five spectral fluorescence standard dyes that are commercially available as
Spectral Fluorescence Standard Kit from Sigma Aldrich and BAM. This kit provides a simple,
flexible, and traceable calibration tool for the determination and control of the relative
spectral responsivity of fluorescence instruments. The kit contains the five spectral
fluorescence standard dyes F001 to F005 supplied as powders ready for use, the solvent for
their dissolution (ethanol, spectroscopic grade), the respective BAM certificate and a CD with
the BAM software LINKCORR which can be used to create the instrument correction
NanoChOp, WP3, Deliverable D3.1.1
Page 2
function from the measured and certified fluorescence emission spectra. The Spectral
Fluorescence Standard Kit is optimized for fluorescence spectrometers and can be
employed for a broad variety of measurement geometries. With proper consideration of the
underlying measurement principle, it can be adapted to the calibration of other types of
fluorescence instruments. The working principle of the Spectral Fluorescence Standard Kit is
shown in Figure 1. This kit was recently assessed in an inter-laboratory comparison of four
national metrology institutes (i.e. NIST, NRC, PTB and BAM) that was organized by BAM
(Resch-Genger et al., Anal. Chem. 2012, 84, 3889-3898) and employed in a study on the
comparison of emission spectra from field laboratories (Resch-Genger et al., Anal. Chem.
2012, 84, 3899-3907).
Figure 1. Measured (dotted lines) and corrected (solid lines) emission spectra of the BAMcertified spectral fluorescence standard dyes F001-F005 (left). The deviations of the
measured instrument-dependent spectra from the instrument-intependent (corrected)
spectra are mainly due to different detector responsivities at different emission wavelengths
(dotted line, right). The quotients Q of the measured and corrected emission spectra of the
standard dyes F001-F005 can be used to create an instrument correction function (solid line,
right) using the LINKCORR program, which is provided with the Spectral Fluorescence
Standard Kit.
Choice of suitable quantum yield standards
Fluorescence quantum yield (QY) measurements can be either performed relative to a
standard with known QY or absolutely by using an integration sphere setup. For absolute QY
measurements no standard is needed. However, QY standards can be used to validate the
measurements performed with an integration sphere, e.g. by validating the data assessment
procedures. In most laboratories a relative measurement using the same excitation
wavelength for sample and standard is performed, because of its simplicity, low cost and
high sensitivity. Thereby a common fluorescent dye such as a rhodamine or coumarin dye
with known fluorescence QY is used as standard to which the QY of the sample is measured
The most error-prone steps in relative QY measurements are the correction of the measured
emission spectra for the instrument specific spectral responsivity (see above) and the
reliability of the QY value of the standard that is typically taken from the literature. The
problem is that these literature QY values can differ from reference to reference by several
NanoChOp, WP3, Deliverable D3.1.1
Page 3
percent and are often controversially discussed even for long known dye classes such as
coumarins. Brouwer listed in 2011 (Pure Appl. Chem. 2011, 83, 2213-2228) the QY values
of several different organic fluorescent dyes that are commonly used as QY standards.
These values differ and range e.g. for quinine sulfate from 0.52 ± 0.02 to 0.60 ± 0.02 (both in
0.05 M H2SO4), and for fluorescein from 0.91 ± 0.05 to 0.95 ± 0.03 (both in 0.1 M NaOH).
As the uncertainty of the QY value of the standard also determines the uncertainty of the
measured relative QY of the sample, suitable QY standards are necessary, of which the QY
are known with high precision and low uncertainty. In order to validate QY of different
organic fluorescent dyes commonly used as QY standards, the BAM group measured the
QY of several dyes using two relative and one absolute fluorometric QY method as well as
photo-acoustic spectroscopy (Würth et al., Anal. Chem. 2011, 83, 3431-3439; Würth et al.,
Talanta 2012, 90, 30-37; Würth et al., Anal. Chem. 2012, 84, 1345-1352) and calculated
complete uncertainty budgets for the obtained QY values. The investigated dyes were e.g.
quinine sulfate (QS) in 0.105 M HClO4, coumarin 153 (C153) in ethanol, fluorescein 27 (F27)
in 0.1 M NaOH, rhodamine 6G (R6G) in ethanol, and rhodamine 101 (R101) in ethanol. The
obtained QY values for these dyes are summarized in Table 1.
Table 1. QY values determined with relative measurements using the same (relative
methods 1) or different (relative method 2) excitation wavelengths for sample and standard,
as well as with an integration sphere setup (absolute method) compared to QY values from
literature references. For the relative methods 1 and 2, the QY value of 0.90 for R101
determined by with the absolute method was used.
method 1
method 2
0.587 ± 0.037
0.599 ± 0.040
0.633 ± 0.035
0.514 ± 0.031
0.538 ± 0.037
0.521 ± 0.029
0.40, 0.26, 0.58
0.824 ± 0.049
0.804 ± 0.054
0.785 ± 0.045
0.893 ± 0.053
0.894 ± 0.058
0.897 ± 0.050
0.900 ± 0.050
0.81, 0.86,
0.91 ± 0.0513
0.95, 0.94,
1.00 ± 0.0513
The emission spectra of the five dyes QS, C153, F27, R6G and R101 cover the whole visible
spectrum from ca. 400 nm to 700 nm and are thus suitable for both green emitting dyes
(used to label the silica nanoparticles RM1 to produce FM1/2) and red emitting dyes (used to
stain polystyrene nanoparticles to produce FM3/4). This will be tested in 2013.
NanoChOp, WP3, Deliverable D3.1.1
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