Fluorescence Measurements on Functionalized

Polymer Surfaces—Problems
and Troubleshooting
KATRIN HOFFMANN, RENATE MIX, UTE RESCH-GENGER, AND JOERG F. FRIEDRICH
Federal Institute for Materials Research and Testing (BAM), Div. I.5, Berlin, Germany

Plasma-chemically tailor-made polymer surfaces are of ever-increasing importance to control

surface properties in material science, as well as for (bio)analytical and biomedical applications.
For the characterization of such systems, sensitive fluorescence techniques are attractive tools. To
underline the potential and drawbacks of these strategies, this article addresses different problems
that complicate fluorometric analysis. To overcome some of these limitations, such as nonspecific
adsorption of unreacted fluorescent probes, we discuss potential troubleshooting, including the
use of a chromogenic and fluorogenic pyrylium dye for the detection of amino functionalities at
polypropylene surfaces.

Key words: fluorescence spectroscopy; polymer functionalization; surface labeling; pyrylium label

Introduction surface functions is complicated by different factors,8,11

Functionalization of polymer surfaces with OH,
NH 2 , or CHO groups by, for example, plasma chem-
ical modification allows control of the hydrophilicity,
as well as the adsorption and wetting properties of
polymeric surfaces and provides the basis for the at-
tachment of bio- and sensor molecules.1,2 Crucial for
all applications of these tailor-made polymeric materi-
als is the characterization of the type and the density of
reactive functional groups at the surface. Commonly
used surface-sensitive techniques such as X-ray photo-
electron spectroscopy and attenuated total reflection
Fourier transform infrared spectroscopy are expen-
sive, provide only the elemental composition of a 5-
to 7-nm-thick surface layer, or are limited to a surface
layer with a thickness of 1.6–2.5 µm3 ; they often fail
at low concentrations of surface functionalities. Thus,
the application of straightforward labeling techniques
in combination with simple fluorometric methods be-
comes attractive.4
Despite the obvious potential of fluorometry, there
have been only a few examples reported for the charac-
terization of surface functionalities on (bio)analytically
relevant supports.4–10 Fluorometric characterization of
Address for correspondence: Dr. Katrin Hoffmann, Federal Institute
for Materials Research and Testing (BAM) I.5 Bioanalytics, Richard-
Willstätter-Str. 11, D-12489 Berlin-Adlershof, Germany. Voice: +030-
8104-5878; fax: +030-8104-5005.
katrin.hoffmann@bam.de
Ann. N.Y. Acad. Sci. 1130: 28–34 (2008).

C 2008 New York Academy of Sciences.
doi: 10.1196/annals.1430.015 28
including nonspecific adsorption,8 an inhomogeneous
dye distribution or penetration of dye molecules into
polymers,12–14 dye–dye and dye–matrix interactions,
and the sensitivity of the fluorophore’s spectroscopic
properties to the microenvironment.25 Because the
potential of fluorometry is still under debate, this
report discusses the possibilities of and general re-
quirements on the fluorometric characterization of
dye-labeled surface functionalities, exemplary for
plasma-functionalized polypropylene (PP) films. The
results obtained with a traditional dansyl label that
does not reveal binding-induced changes of its spec-
troscopic properties with those of a chromogenic and
fluorogenic pyrylium dye are compared.
Results and Discussion
Plasma Chemical Modification
An elegant approach to introduce functional groups
into chemically inert polymer surfaces is the appli-
cation of plasma processes, thus forming anchoring
sites for chemical grafting of different labels or other
functional molecules. For the presented spectrofluoro-
metric characterization of PP films (100-µm thickness;
Goodfellow, Cambridge, U.K.), we used two different
routes for surface modification. The first approach is
generating 10–14 OH groups/100 C atoms on the
PP surface by means of an oxygen plasma, followed
by a wet-chemical reduction process. Another promis-
ing approach is the plasma-initiated polymerization
of monomers carrying functional groups. With this
Hoffmann et al.: Fluorescence Spectroscopy on Polymer Surfaces 29

FIGURE 1. Labeling reactions. (A) Labeling of OH groups at a PP surface with a dansyl fluorophore
by using a diisocyanate spacer. (B) Labeling of surface amino groups with the blue Py-1 chromophore,
yielding a red PP conjugate. (In color in Annals online.)

strategy, NH 2 -terminated PP surfaces have been gen-
erated.12,15
Labeling of Surface Functionalities
Different synthetic concepts have been tested for
fluorophore labeling of surface functionalities,13,15
as well as to introduce spacer molecules.16 Con-
ventional isothiocyanate labels or amino-terminated
labels like dansyl fluorophores were covalently at-
tached by means of spacer molecules to OH func-
tions at the polymer surface by strategies adopted from,
for example, well-investigated polyurethane chemistry
(FIG. 1A).13 For the fluorometric characterization of
amino-functionalized PP surfaces, the chromogenic
and fluorogenic pyrylium dye Py-1 has been used,
which was recently introduced for the derivatization
of primary amino groups (FIG. 1B).17,18 For each series
of fluorescence measurements,19 blank or so-called ref-
erence samples were prepared by reaction of the non-
functionalized polymer film with the respective flu-
orescent label by using the same procedure as that
used to covalently attach fluorophores to the surface-
functionalized films.
Problems Inherent to Fluorescence
Measurements at Surfaces
Fluorescence techniques in conjunction with fluo-
rescent labels can be exploited to characterize sur-
face functionalities at low concentration levels in the
range between approximately 109 (on glass4 ) and 1012
(on polymer supports8 ) mol/cm2 . The reliable use of
these techniques, however, requires overcoming some
challenges.7 First, the observed fluorescence signal re-
sults from the bulk material because commonly used
steady-state fluorescence spectroscopy is not a surface-
sensitive technique.6 Second, further complications
can arise from background emission either from the
polymer support itself and/or from adsorbed (non-
linked) dye molecules. Furthermore, the environmen-
tal dependence of the spectroscopic properties of most
dyes and matrix–dye or dye–dye interactions result-
ing in fluorescence quenching must be taken into ac-
count.20 All these problems raise qualms about the
reliability of quantification relying on fluorescence
measurements after labeling reactions. Despite these
limitations, the unique sensitivity of fluorometry with
a limit of detection on the order of approximately
10−12 mol/cm2 for fluorophores on polymer surfaces,8
in conjunction with its comparative ease of use, renders
fluorescence techniques as attractive tools for analyz-
ing organically modified polymer surfaces, especially
when only monitoring of changes in surface group con-
centration is needed.
Effect of Spectral Correction:
Comparability of Data
In general and independent of the type of fluores-
cent samples (e.g., fluorophores in solution or fluores-
cent labels attached to polymer surfaces), the fluores-
cence signal is affected by both the fluorescent analyte
and instrumental effects. Relevant properties of ana-
lytes are the fluorophore’s absorptance at the excita-
tion wavelength and its fluorescence quantum yield.
These quantities, as well as the spectral shape and po-
sition of the fluorophore’s absorption, excitation, and
emission spectra, can strongly depend on the polarity
of the dye’s microenvironment, that is, on the solvent
or the matrix (e.g., polarity, proticity, pH, and viscos-
ity). This dependence complicates quantification from
measurements of relative fluorescence intensities. From
30 Annals of the New York Academy of Sciences

TABLE 1. Selected spectroscopic data (spectrally cor-

rected) of PP-DNS-Hy in different solvents
ET a / Stokes
Solvent kJ mol−1 ν̃a b s /cm−1 ν̃e m /cm−1 shiftb /cm−1

1.4-Dioxane 150.5 30 000 21 790 8210

DMFc 183.4 30 140 21 890 8250
DMSOd 188.4 30 090 22 055 8035
Propanol 203.2 30 010 21 990 8020
Ethanol 216.9 30 140 21 840 8300
Water 263.8 29 930 21 680 8250
Dry (air) —e 29 850 21 770 8080
a
E T , Dimroth and Reichardt’s polarity scale of solvents.23
b
The Stokes shift is calculated from the energetic difference
between the longest wavelength maximum of the fluorescence
FIGURE 2. Comparison of the measured uncorrected excitation spectra (ν̃a b s ) and the corresponding emission maxima
(gray) and the spectrally corrected (black) excitation (ν̃e m ).
c
(dashed lines) and emission (solid lines) spectra of a labeled DMF, dimethyl formamide.
d
PP film. The PP–Py-1 conjugate was excited at 503 nm, and DMSO, dimethyl sulfoxide.
e
the excitation spectrum was recorded at 602 nm. —, none.

a much more pronounced dependence of their spec-

the instrument side, the excitation light intensity at
sample position and the wavelength- and polarization-
dependent spectral responsivity of the emission chan-
nel affect the shape and the intensity of the mea-
sured fluorescence spectra. This effect is highlighted in
FIGURE 2, which compares the measured instrument-
specific fluorescence spectrum of the pyrylium dye
Py-1, bound to plasma-chemically-generated surface
amino groups of PP films, and its analogue, corrected
for such instrument-specific effects. Accordingly, the
comparison of fluorescence spectra measured at dif-
ferent instruments or at different times requires the
removal of wavelength-dependent instrumental effects
from the raw emission data by spectral correction
procedures.21,22
Spectral correction becomes particularly important
if different polarizer settings are used. For example,
neglecting the polarization dependence of the spectral
responsivity of the emission channel can even result in
an apparent polarization dependence of the measured
spectra.22
Sensitivity of the Fluorophore’s Spectroscopic
Properties to Microenvironment Including
Surface Attachment
Almost all reactive dyes intended for the detection
of functional groups at surfaces are xanthene dyes
(e.g., fluoresceins, rhodamines). These chromophores
reveal a locally excited state–type emission, the spec-
troscopic properties of which are less sensitive to the
fluorophore’s microenvironment. Charge transfer dyes
such as dansyl chromophores, which are also com-
monly used for fluorophore labeling, however, reveal
troscopic features on microenvironment.23 Their sol-
vatochromic behavior can be used to study the accessi-
bility of small molecules24 and to characterize local po-
larity effects, which mainly determine the properties of
surface-modified polymers.3,25,26 To illustrate such ef-
fects for dansyl chromophores bound to PP surfaces, we
determined the microenvironment-dependent spectral
properties of the surface-attached fluorophore. For that
purpose, labeled PP films were immersed for 15 min
in the solvents listed in TABLE 1, and the fluorescence
spectra of the nondried films were recorded. Measur-
able, even though minor, solvent-dependent spectro-
scopic properties, shown in FIGURE 3, reveal the re-
sponse of the surface-attached dansyl chromophores
to changes in the polarity of the microenviron-
ment.
With the exception of polarity-probing stud-
ies of specific fluorophore–solvent interactions, the
environment-dependent emission properties must be
taken into account. Thus, intensive washing, and in
particular drying, procedures are required to guaran-
tee reproducible and comparable fluorescence data.
Despite careful sample preparation steps, the fluo-
rometric detection of surface functionalities on solid
supports is often hampered by an enhanced back-
ground signal due to nonspecifically adsorbed dye
molecules for fluorophores lacking binding-induced
spectroscopic changes. Hence, more suitable are so-
phisticated labels revealing changes in their absorp-
tion and fluorescence properties upon surface attach-
ment.6,8,27 This approach enables a clear distinction
between covalently linked and free, only adsorbed,
reporter molecules. Excellent examples for this design
Hoffmann et al.: Fluorescence Spectroscopy on Polymer Surfaces 31

FIGURE 3. Steady-state fluorescence emission spectra of a plasma-chemically-modified and dansyl-

labeled PP film impregnated with several solvents (left) and (right) shift of the emission maximum of the
film as a function of Dimroth and Reichardt’s polarity scale E T .23

of both the excitation and emission spectra,12 as illus-

FIGURE 4. Hypsochromic shifts of the excitation
(dashed lines) and the emission (solid lines) spectra of
Py-1 after its reaction with amino-terminated PP surfaces.
The emission of the PP–Py-1 conjugate (symbols) was ex-
cited at 503 nm, and the fluorescence of the nonreacted
PP-adsorbate (line) at 621 nm; the corresponding excitation
spectra were recorded at 602 and 665 nm, respectively.
concept are amino-sensitive pyrylium dyes, such as
Py-1.17,18 Because of the reaction of amino groups
in, for example, proteins, the Py-1 absorption and
emission maxima shift hypsochromically and the fluo-
rescence quantum yield of the very weakly emissive
pyrylium dye strongly increases.17,18 In our studies on
amino-functionalized solid PP supports, the labeling
reaction with Py-1 also induced significant blueshifts
trated in FIGURE 4.
Also, the formation of the PP–Py-1 conjugate is ac-
companied by a strong increase in emission intensity
(note the noisier signals in FIG. 4 of the normalized
emission spectra of the unmodified reference film).12
The drastic changes in energy and intensity of the
absorption and emission bands elegantly circumvent
the simultaneous excitation of nonspecifically adsorbed
dye molecules, thereby enhancing the signal-to-noise
ratio and reducing labor-intensive washing steps.
Reproducibility of Fluorescence Measurements
at Solid Supports
Despite improved fluorophore labeling and de-
tection approaches, fluorescence studies on solid
substrates are prone to reduced measurement repro-
ducibility compared with measurements of transpar-
ent dilute solutions taken using cuvettes. For the latter,
the reproducibility is commonly better than 2%, as
revealed in FIGURE 5A.21
For measurements of solid films with a routine flu-
orometer, however, a significantly lower reproducibil-
ity has been found.21 Use of traditional dansyl labels
yields an uncertainty of 15% despite tedious wash-
ing and sample preparation procedures (FIG. 5B).13
This uncertainty arises, most likely, from errors due to
sample positioning in front-face measurement geome-
tries and inhomogeneous functionalization/labeling,
as well as from sample-related parameters such as
scattering and reflections and difficulties of eliminating
32 Annals of the New York Academy of Sciences

FIGURE 5. Reproducibility of fluorescence measurements in solution compared with those on solid

polymer supports. Standard deviations (error bars) of repeatedly recorded emission spectra (uncorrected,
gray solid lines) of an organic dye in solution (A), of dansyl-labeled PP (excitation at 350 nm; B), and of
an amino-modified and Py-1–derivatized PP film (excitation at 503 nm, C).

background fluorescence.22 To evaluate the effect of

the type of fluorescent label on the reproducibility of
emission measurements on polymer films, we anal-
ogously determined the standard deviation from the
variation of the signal intensities of repeated emission
records for amino-functionalized PP derivatized with
the chromogenic label Py-1. With this labeling strat-
egy, the standard deviation could be reduced to ap-
proximately 7%, as illustrated in FIGURE 5C. This im-
proved measurement uncertainty for Py-1–derivatized
films is most likely owing to the strong binding-induced
spectroscopic changes. For the excitation wavelength
of approximately 500 nm used, the surface emission is
always recorded against an almost dark background FIGURE 6. Monoexponentially fitted decrease in rela-
even if nonreacted, adsorbed fluorophores or dye tive integral fluorescence emission I t /I 0 of dansyl-labeled
molecules diffused into the polymer are still present, PP (-◦-) and an amino-modified PP film derivatized with Py-1
because the free pyrylium dye remains silent under (-•-) after subsequent Soxhlet extractions with ethanol.
these conditions.12,17

Nonspecific Adsorption of Fluorophores loss of the initial fluorescence signal of approximately

Fluorescence measurements of functionalized poly-
mer supports or other polymeric and porous materials
typically require extensive washing and extraction pro-
cedures to remove nonreacted dye molecules, particu-
larly for conventional labels. The importance of such
purification procedures follows immediately from the
50% for plasma-chemically-modified PP films reacted
with dansyl labels (FIG. 6, open circles) as exemplary de-
termined by Soxhlet extraction. For the chromogenic
and fluorogenic Py-1 fluorophore, however, we ex-
pected a constant signal independent of extraction
steps because here exclusively the reacted dye is spec-
Hoffmann et al.: Fluorescence Spectroscopy on Polymer Surfaces
troscopically addressed. Astonishingly, also for PP–Py-
1 conjugates, the integral emission decreased with the
number of extraction cycles (FIG. 6, solid circles),12
although only amino-conjugated pyrylium molecules
contribute to the fluorescence signal.
However, the much slower extraction kinetics indi-
cates a different reaction course for the degradation
of the Py-1 fluorescence compared with that of the
dansyl label. The measured effect suggests either the
presence of an additional unspecific source of amino
functionalities aside from the polymer-attached amino
groups, which is implausible, or certain instabilities of
the Py-1–reacted amino surface functionalities. The
latter is possibly not related to fluorophore labeling
but rather to different routes for plasma-chemically-
induced surface functionalization. Dansyl labeling was
performed on OH functions at the polymer back-
bone, whereas Py-1 modification was carried out on
NH 2 functions of plasma-deposited ppAAm layers.
Because low-molecular-weight polymer fragments are
not resistant to organic solvents,6 the decrease in rel-
ative emission to about 60% of the initial fluores-
cence intensity is ascribed to some chemical instabil-
ity of the plasma-deposited ppAAm layers, where Py-
1–labeled low-molecular-weight oligomeric structures
are partially detached from the polymeric substrate.
This assumption is supported by additional investiga-
tions by means of surface-sensitive X-ray photoelectron
spectroscopy.12
Conclusion
Fluorometric analysis of functionalized surfaces
labeled with organic chromophores is a promising,
simple, and sensitive technique for monitoring plasma-
chemically-introduced OH and NH 2 groups at PP sup-
ports. The reliability of the data requires proper con-
sideration of various factors that influence fluorescence
measurements. Here, the choice of an appropriate
fluorescent label also plays a significant role. The cor-
rection of the emission spectra for the spectral charac-
teristics of the instrument is essential to obtaining com-
parable, instrument-independent results.22 To improve
the reproducibility of fluorescence data, one must ac-
count for the sensitivity of the fluorophore’s spectro-
scopic properties to the microenvironment, and careful
sample preparation, washing, and particularly drying
procedures become mandatory. These procedures are
especially important for labeling protocols that use flu-
orescent reporters that do not reveal binding-induced
spectral properties, where nonreacted dye molecules
also contribute to the fluorescence signal. Even though
33
quantitative fluorescence measurements on solid sup-
ports are still hampered by all the above-mentioned
complicating factors, the derivatization with the chro-
mogenic and fluorogenic label strongly improves the
relative fluorometric characterization of surface amino
groups directly at solid supports.
Acknowledgment
Technical assistance by R. Decker and financial
support from Federal Ministry of Economics and La-
bor (BMWA) are gratefully acknowledged. We are in-
debted to Prof. O. S. Wolfbeis and Actif Motiv GmbH
for the generous supply of Py-1.
Conflict of Interest
The authors declare no conflicts of interest.
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