Spectroscopic Characterization of Coumarin-Stained Beads

Article pubs.acs.org/ac

Spectroscopic Characterization of Coumarin-Stained Beads: Quantification of the Number of Fluorophores Per Particle with Solid-State 19F-NMR and Measurement of Absolute Fluorescence Quantum Yields Alexandra Huber,† Thomas Behnke,‡ Christian Würth,‡ Christian Jaeger,*,† and Ute Resch-Genger*,‡ †

Structural Analysis Division and ‡Biophotonics Division, BAM Federal Institute for Materials Research and Testing, Department 1, Richard Willstaetter Strasse 11, D-12489 Berlin, Germany S Supporting Information *

ABSTRACT: The rational design of nano- and micrometersized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative (19F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF3 group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods.

F

characterization of their application-relevant features ranging from bead size and charge over dye (analyte) content per particle and type and number of surface functionalities to fluorescence quantum yields and brightness values.1,8,21−35 The latter quantities that are closely linked to the number of fluorophores per bead, determine the size of the accomplishable fluorescence output, and hence, the achievable sensitivity and amplification. Hence, they present straightforward tools for the comparison of different fluorescent reporters and their optimization and, thereby, the prediction of assay performance.31−33,35 Moreover, they play a significant role for the increasingly desired standardization of fluorescence measurements involving particle systems like flow cytometry and fluorescence microscopy. Currently, this standardization relies solely on the relative MESF system (MESF, molecules of equivalent soluble fluorophore) comparing signals of dissolved and encapsulated dyes to relate the relative fluorescence intensities of the beads to the concentration of a reference fluorophore in solution. 18,36 This circumvents the quantification of the number of fluorophores

luorescent reporters and labeling reagents are essential components for the fluorometric detection of analytes widely employed in a variety of areas including biomarker analysis and monitoring, drug screening, proteomic, and genomic studies, as well as food and environmental analysis.1−7 One of the fastest growing research fields is the use of functional nanomaterials as a new generation of fluorescent labels, probes, and sensors.1,8−14 In addition, micrometer-sized particles are gaining importance as analytical platforms for fluorescence assays enabling, for example, the large-scale analysis of biological systems using fluorescence microscopic techniques, flow cytometry, or microfluorometry for signal detection.15 The great potential of these materials has meanwhile paved the road for new assays with enhanced sensitivity, multiplexing capabilities, and sample throughput as well as for multimodal imaging strategies of disease-related biomarkers in vitro and in vivo. Simultaneously, there is an ever increasing market for calibration and reference beads for the characterization and performance validation of fluorescence microscopes and flow cytometers and as fluorescence intensity standards.16−20 At the core of these applications are simple and versatile strategies for the preparation of highly emissive nanometerand micrometer-sized particles with precise control of bead size, surface chemistry, and optical properties and for the © 2012 American Chemical Society

Received: January 8, 2012 Accepted: March 8, 2012 Published: March 8, 2012 3654

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Table 1. Quantification of C153-Loading of the PSP Calculated from the NMR Data and Optical Dilution Studies batch

concentration of applied dyea [mol/L]

applied amount of C153a [mmol/mol]

total mass of PTFEb [mg]

total mass of batchb [mg]

ACF3/CF2 area batch averageb

C153/PSb [mmol/mol]

C153/PSc [mmol/mol]

B-0.2 B-0.1 B-0.02

1.0 × 10−3 5.0 × 10−4 1.0 × 10−4

3.47 1.74 0.35

0.1101 0.1169 0.0878

49.5116 58.6274 58.0681

0.68 ± 0.03 0.56 ± 0.03 0.103 ± 0.005

2.16 ± 0.1 1.08 ± 0.06 0.19 ± 0.01

2.26 ± 0.18 0.96 ± 0.08 0.19 ± 0.02

a

Concentration of applied dye and its amount taken from the swelling solution used for PSP staining with C153 (see Materials and Instrumentation section). bFrom 19F-NMR measurements. cFrom the absorption spectra of solutions of dissolved beads using the previously determined molar absorption coefficient in THF in the presence of 0.1 wt-% PS.

were recorded at a Larmor frequency of 376.31 MHz and MAS rotation frequencies of 27 kHz. The 19F 90° pulse length was 2.8 μs. 19F chemical shifts were referenced to CFCl3 via the CF2 resonance of a PTFE (δ(19F) = −121.9 ppm) standard. The r.f. carrier was set to −90 ppm equaling the center between the isotropic shift of the CF3 group of C153 at −62 ppm and the CF2 signal of PTFE. For the 19F-NMR experiments, a repetition time of 10 s was chosen (longest T1 = 1.65 s for the CF2 group of the intensity standard PTFE). Only for the samples containing the smallest amount of C513 of about 0.02% (batch B-0.02, see SI, Table 1S), the repetition time was 5 s; hence, a T1 correction factor of 1.05 had to be taken into account for the CF2 area values. 1024 scans were accumulated for samples of batches B-0.1 and B-0.2, whereas 16 k scans were required for samples of batch B-0.02. More details like T1 measurements, 19F sample background subtraction procedure, and MAS sideband intensity correction are described in the SI. All spectra were processed using the BRUKER Topspin software package including the manual integration routine. Preparation of NMR samples. For each batch (see, e.g., Table 1), a large amount of C153-loaded PSP was dried at 50 °C overnight under vacuum, yielding minimum 50 mg of dried C153-loaded PS beads. To a precisely known mass of these beads determined with a calibrated Sartorius S4 microbalance (maximum load of 120 mg; uncertainty U(R) = 0.08 μg + 1.61 × 10−4 × R with R being the mass shown on the scale display), subsequently, a precisely known mass of PTFE beads (serving as intensity standard), that is, about 0.1 mg were added yielding the C153-PSP-PTFE batches studied (see Table 1). Special care was taken to achieve optimum homogenization of the C153 bead-PTFE batches by rigorous stirring of the beads. For the preparation of the NMR samples requiring weighing of the rotor (mass of ∼270 mg), a Sartorius BP211D balance (maximum load of 80 g; U(R) = 0.008 mg +2.92 × 10−6 × R) was used, see also SI. Optical Spectroscopy. Absorption Measurements. Absorption measurements of transparent dye solutions were carried out with a Cary 5000 UV−vis-NIR spectrophotometer from Varian, Inc. Relative Fluorescence Measurements. Fluorescence measurements for the validation of the absence of reabsorption effects for the absolute integrating sphere measurements and for the determination of fluorescence quantum yields Φf of selected samples relative to a standard were performed with a calibrated Spectronics Instruments 8100 spectrofluorometer equipped with Glan Thompson polarizers in the excitation and emission channel in a 0°/90° standard measurement geometry. The excitation polarizer was set to 0° and the emission polarizer to 54.7°.44 The resulting emission spectra were corrected for the spectral responsivity of the fluorometerś emission channel.44 C153 in ethanol was used as quantum yield

per bead and the challenging measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent particles. This encouraged us to develop new methods for the nondestructive determination of the number of dye molecules per bead and the characterization of the signal-relevant optical properties of emissive particles in suspension using quantitative solid-state nuclear magnetic resonance (NMR) spectroscopy,37,38 and a new integrating sphere setup custom-designed for spectrally resolved measurements of emission, absolute fluorescence quantum yields, transmission, and reflectance, of transparent and scattering systems.39,40 On the basis of the results obtained for 1000 nm-sized carboxylated polystyrene (PS) particles (PSP) stained with varying amounts of coumarin 153 (C153) containing a CF3 group, an new SI-traceable concept for the nondestructive quantification of analytes in and on beads and the determination of brightness values of scattering fluorophore-stained nanometer- and micrometersized beads is proposed.

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MATERIALS AND INSTRUMENTATION Materials and Sample Preparation. Carboxyl-functionalized polystyrene particles with diameters of 1000 nm were purchased from Kisker Biotech GmbH. All particles were ultrasonically treated prior to use. Coumarin 153 (batch number 029303) was obtained from Fluka GmbH and used without further purification. Its purity was determined via HPLC analysis (see Supporting Information, SI). All solvents (tetrahydrofuran (THF), dibutylether (BOB), and ethanol (EtOH)) that were of spectroscopic grade, were purchased from Sigma-Aldrich Co. and were used as received. For the quantitative determination of the average number of C153 molecules per PS mass with 19F-NMR, polytetrafluoethylene (PTFE) beads with typical diameters of 1000 nm from Sigma Aldrich Co. were employed as standard. Preparation of C153-Stained Beads. C153 was dissolved in THF in different concentrations ranging from 1 × 10−3 mol/L to 1 × 10−4 mol/L (see Table 1, batches B-0.2, B-0.1, and B-0.02). Dye loading of the PSP was performed by a previously described swelling procedure.41−43 Ten milliliters of a dyecontaining solution were added to 60 mL of an aqueous suspension of the PSP (0.5 weight-% (wt-%)) with the degree of dye loading being controlled by the amount of C153 applied (Table 1). After 30 min of swelling, 80 mL water were added and the occasionally shaken suspension was centrifuged (5000 g for 10 min). The accordingly separated PSP were washed twice with bidistilled water followed by resuspension in bidistilled water in an ultrasonic bath. Instrumentation. 19F-NMR Measurements. Solid-state NMR experiments were performed with a Bruker Avance DSX 400 spectrometer (widebore magnet) with a remodeled 2.5 mm double-resonance probe at a magnetic field strength of 9.4 T. The 19F Magic Angle Sample Spinning (MAS) NMR spectra 3655

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BOB and ethanol, the absorption spectra of dye solutions of different dye concentrations, originating from at least two stock solutions, were measured for dye concentrations ranging from 5 × 10−6 mol/L to 8 × 10−6 mol/L. In all cases, 1 cm-quartz cuvettes (Hellma GmbH) were used. For the preparation of the bead suspensions for the optical measurements, the washed C153-stained PSP were suspended in bidestilled water, resulting in a PSP concentration of 0.1 wt-%. These suspensions were then transferred to conventional 1 cm-quartz cells. Determination of the Number of Dye Molecules Per Bead via a Photometric Dissolution Method. For each dye loading concentration applied (see Table 1), a previously dried amount of C153-loaded PSP of known mass from the same batch as used for the 19F-NMR studies was dissolved in THF, resulting in a 0.1 wt-% PS/THF solution. The absorption spectra of these THF solutions were measured and the average amount of dye incorporated into 3 mg of PSP employed for the dye loading studies was calculated from the absorbance measured at the dye’s longest wavelength absorption maximum, using the Beer−Lambert law and the previously determined ε value of the dye in THF containing 0.1 wt-% of dissolved PS. The relative amount of encapsulated dye equals the quotient of the applied and the actually incorporated amount of fluorophore molecules. This procedure was validated for different fluorophores by the determination of the recovery rate of the applied dyes over a broad concentration range and elemental analysis measuring the sulfur content of beads dyed with a fluorophore equipped with sulfonate groups. The average number of C153 molecules per particles was then calculated from the experimentally determined (average) amount of incorporated dye and the number of particles in 3 mg of PSP. The latter was calculated as the quotient of the volume from the applied amount of polystyrene and the volume of a 1000 nm-sized PSP assuming spherical particles with a density of 1050 kg/m 3 . The particle size was provided by the manufacturer and previously controlled before and after dye loading using DLS for a broad variety of fluorophores loaded into these beads using similar swelling procedures.41,42 Safety Considerations. Proper safety procedures for handling, storage, and disposal of the organic dyes should be observed.

standard previously characterized by us absolutely with a commercial integrating sphere setup (Φf = 0.521 ± 0.029).40 Absolute Fluorescence Quantum Yields. Absolute Φf values of C153 in BOB, ethanol, and encapsulated in PSP (aqueous suspensions of C153-PSP), see Table 1, that equal the number of emitted photons (Nem) per absorbed photons (Nabs), were determined with a new custom-designed integrating sphere setup.45 This setup consists of a xenon lamp coupled to a single monochromator, a six inch Spectraflect-coated integrating sphere (Labsphere GmbH) coupled with a quartz fiber to an imaging spectrograph (Shamrock 303i, Andor Inc.) and a Peltier cooled thinned back side illuminated deep depletion charge coupled device (CCD array; Andor DU420A BRDD), and a reference detector. The sample or blank (i.e., the pure solvent) in a conventional 1 cm-quartz cell was mounted into the center of the integrating sphere and the excitation light was focused into the middle of the sample. The absolute fluorescence quantum yield was calculated from the measured spectrally corrected signals of the blank (ICB) and the sample (ICS) according to ref 40. The accuracy of the spectral correction was previously controlled by comparison of the corrected emission spectra of a set of emission standards determined with the integrating sphere setup with the corresponding spectra obtained with a calibrated spectrofluorometer.45 With our custom-designed instrument design and the procedures employed for calibration and data analysis, we could accomplish measurement uncertainties 100 nm.53 Alternatively, destructive approaches are used such as bead dissolution53−55 or dye extraction,53,56 followed by photometric dye quantification in solution. To evaluate the suitability of quantitative NMR spectroscopy (qNMR) for this purpose, we determined the number of coumarin 153 molecules in strongly scattering 1000 nm-sized carboxylated polystyrene particles stained with three different dye concentrations (Table 1).42,43 This common laser dye was chosen as it contains fluorine atoms, here a CF3 group (Figure 1), like

Figure 1. Chemical structure of C153.

many other analytically relevant fluorophores including certain fluoresceins, rhodamines, cyanines, and BODIPY dyes1,57,58 that enables the use of very sensitive 19F-NMR spectroscopy, exploiting the large gyromagnetic magnetic ratio and the 100% natural abundance of 19F. Moreover, C153 is known to be not very prone to dye aggregation and self-quenching which is favorable for the subsequent optical studies. For the 19F-NMR measurements, a known amount of PTFE beads, serving as intensity standard, was added to a known amount of each of the three C153 bead batches varying in C153 dye content (Table 1), the beads were carefully homogenized, each C153-PSP-PTFE batch was split into five fractions/samples (see SI, Table 1S), and the 19F-NMR spectra of each sample were independently measured as highlighted in Figure 2 and detailed in the SI (see, e.g., SI Table 2S). At first for representative samples from batches B-02 and B-0.02 containing the highest and smallest amount of C153 (Table 1), suitable measurement conditions for quantitative 19F-NMR were determined as described in the next section. Then, the NMR signal area ratio of the CF3 and CF2 resonances (referred to as ACF3/CF2) was determined for each sample of the C153PSP-PTFE batches B-0.2, B-0.1, and B-0.02, averaged for each batch to minimize measurement uncertainties due to possible sample inhomogeneities caused by addition of only a very small amount of PTFE standard to each PSP batch (see SI), and used for the calculation of the molar concentration of C153 per mole PS or per bead PS (thereby considering PSP size, see Materials and Instrumentation section). Measurement Conditions for 19F-NMR with C153-PSP. For the use of solid-state 19F-NMR to determine the concentration of C153 dye molecules per PS molecules and the number of C153 molecules per bead, the following conditions must be fulfilled: (i) the repetition delay for the 90° pulse must exceed five times the longest T1 relaxation time of the 19F resonances used for quantification and (ii) all MAS sideband intensities must be included into the integration procedure and the 19F probe background must be removed (see SI). The 19F MAS NMR spectra of samples from batches B-0.2

Ny nx A = x × ny Ay Nx

(2)

with nY and nx equaling the number of analyte and standard molecules and Nx and Ny the number of nuclei generating the corresponding NMR signals, respectively. Equation 2 can be rewritten, expressing the number of analyte molecules per gram sample (negligible masses of analyte C153 and standard PTFE as compared to PS; known chemical composition of and known molar mass Msample) yielding eq 3. nx nsample

= ACF3/CF2 ×

Ny Nx

×

my Msample · M y msample

(3)

In eq 3, ACF3/CF2 is the 19F-NMR area ratio Ax/Ay, Nx = 3 (three fluorine atoms per CF3 group per C153 molecule), Ny = 2 (CF2 as repeat unit of PTFE), my = mPTFE (mass of standard PTFE added), My = MPTFE (= 50.01 g/mol taking CF2 as repeat unit), and Msample = MPS (= 104.05 g/mol). The masses of each sample msample of the batches B-02, B-0.1, and B-0.02 are summarized in Table 1S in the SI. The resulting C135 concentrations including their uncertainties are given in Table 1. A detailed description of the uncertainty determination is included in the SI. As control and validation of our new qNMR strategy, the number of dye molecules per bead was determined also photometrically with a simple and versatile dissolution method (Figure 2, right scheme, and Table 1). Moreover, it was validated by comparing the ratio of the dye concentrations resulting for batches B-0.2, B-0.1, and B-0.02 with the ratios of the absorption coefficients μa of these bead suspensions as detailed in the next section. The relative standard deviation of the former method was determined to 8% by dissolving seven different batches of stained particles independently prepared. As follows from Table 1, the data obtained with 19F-NMR 3657

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Figure 2. Scheme of the spectroscopic characterization of the C153-stained beads with 19F-NMR (left) and optical spectroscopy (right) as used for the quantification of the dye molecules per bead and the determination of the absolute fluorescence quantum yields and the absorption coefficients of suspensions of the fluorescent beads in suspension.

the bead suspensions prevented the use of conventional spectrophotometers and spectrofluorometers. This is main reason why such measurements with beads with sizes exceeding about 10 nm have been only very rarely reported in the literature.33−36,41,52,56,59,60 As follows from Figure 4 (left panel) and Table 2, the absorption spectra of the C153-stained beads that are barely affected by dye concentration, indicating the absence of aggregate formation and electronic interactions between the dye molecules, are red-shifted compared to the spectrum of C153 in BOB and reveal a considerable loss in vibronic structure. However, they are blue-shifted compared to the absorption spectrum of C153 in ethanol and H2O/EtOH. This suggests a more polar environment of the C153 molecules in the PSP as found in BOB although this trend is not reflected by the emission spectra. The absence of dye aggregation and electronic interactions is also confirmed by the excellent

spectroscopy and our dissolution method match within their stated uncertainties, thereby underlining the suitability of qNMR for the characterization of beads and the quantification of their analyte content. Characterization of the Signal-Relevant Optical Properties of C153-Stained PSP. To study the influence of PSP encapsulation on the signaling behavior of the C153loaded beads, we determined the absorption and corrected emission spectra and the absolute fluorescence quantum yields of dilute aqueous suspensions of the dye-stained beads from batches B0.2, B-0.1, and B-0.02 and compared the results to the corresponding data obtained for C153 in apolar BOB, showing a comparable polarity as plain PS, and in ethanol as well as in water containing 0.48 vol-% ethanol (H2O/EtOH) acting as model systems for a polar and protic dye environment. These measurements were performed with a new custom built integrating sphere spectrometer45 as the strong scattering of 3658

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Table 2. Spectroscopic Properties of C153 in BOB, EtOH, and H2O/EtOH (Water Containing 0.48 vol-% Ethanol), and Incorporated into PSP (0.1 wt-% Bead Suspensions in Water) dye

matrix

C153 C153 C153 C153 C153 C153

BOB EtOH H2O/EtOH PSP (B-0.02) PSP (B-0.1) PSP (B-0.2)

λabsa λemb [nm] [nm] 401 423 427 410 410 410

470 530 556 477 480 488

Stokes shift [cm−1] 3661 4773 5433 3426 3557 3898

number of absolute fluorophores per particle Φf 0.92 0.54 0.104c 0.96 0.95 0.94

1.2 × 1010 6.0 × 1010 1.4 × 1011

a

Longest wavelength absorption maximum. bEmission maximum. The Φf of C153 in the water/ethanol mixture was obtained relative to C153 in ethanol; all other Φf values were measured absolutely with the integrating sphere setup. The number of fluorophores per particle was calculated from the photometrically determined dye concentration after dissolving the C153-stained beads (Table 1), an average particle size of 1000 nm, and the particle concentration (see Materials and Instrumentation section). c

Figure 3. 19F MAS NMR spectra of samples of batches B-0.2 (highest amount of C153), with the asterisks marking MAS spinning sidebands of the CF2 signal: 2048 scans were accumulated.

correlation between the ratio of the amount of PSPincorporated dye determined photometrically for batches B-0.2 and B-0.1 (2.26 (mmol/mol)/0.96(mmol/mol) = 2.35; see Table 1) and the ratio of the corresponding absorption coefficients of the 0.1 wt-% aqueous bead suspensions, determined at 412 nm with the integrating sphere setup (μa(B-0.2)/μa(B-0.1) = 2.44). In addition, this underlines the suitability of the number of incorporated fluorophores as a measure for signal intensity in the absence of dye−dye interactions. As follows from the right panel of Figure 4, the emission spectrum of the beads from batch B-0.02 closely resembles that of C153 in BOB and is strongly blue-shifted compared to C153 in ethanol. The slight red shift in emission observed for beads from batch B-0.2 points to reabsorption caused by the higher degree of dye loading.39 Because of the considerable Stokes shift of C153 of about 50 nm in PSP (3661 cm−1, see Table 2), these effects are only small. This is also reflected by the absolute fluorescence quantum yields (absolute Φf) of the different C153-stained PSP reaching values of 0.94−0.96 (Table 2) that are barely affected by dye concentration and closely resemble the Φf value of 0.92 obtained for C153 in BOB. Correction of the measured absolute Φf values of the bead suspensions for reabsorption

effects39 yields Φf values close to 1.0. These very high Φf values also emphasize the absence of fluorescence quenching dye−dye interactions within the C153-stained PSP.25,31 Moreover, they underlines the fluorescence enhancing role of the PSP, protecting polarity-sensitive C153 from fluorescence quenching water molecules. When comparing the Φf values of suspensions of C153-stained PSP to the Φf of C153 in polar and protic ethanol (Φf = 0.540 ± 0.016) and in H2O/EtOH (C153 is insoluble in water; Φf = 0.104), see Table 2, this yields PSP encapsulation-induced fluorescence enhancement factors of 1.75 and ∼9.0, respectively. Enhancement factors in the order of 2−10 have been reported also for other fluorescent nanomaterials like silica or calcium phosphate nanoparticles loaded with organic dyes.32−34,54 A common measure for the size of the accomplishable fluorescence output and thus, the achievable sensitivity and signal amplification, is the brightness. For molecular systems, the brightness is defined as the product of the molar decadic absorption coefficient ε(λex) at the excitation wavelength (λex) and Φf for single photon processes1 or the product of the peak two-photon absorption cross section and Φf for two photon

Figure 4. Normalized absorption (left) and normalized corrected emission spectra (right) of C153 in BOB (solid squares) and EtOH (solid circles), and the corresponding spectra of suspensions of 1000 nm-sized C153-loaded PSP (0.1 wt-% bead suspension; open circles, batch B-0.02, that is, lowest C153 concentration; open squares, batch B-0.2, that is, highest C153 concentration) measured with an integration sphere setup; excitation was at 415 nm. The absorption spectrum of the beads from batch B-0.02, closely matching that of batch B-0.2, was omitted for clarity reasons. 3659

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processes.61 For multichromophore systems like nanometersized beads with diameters in the order of ∼10 nm, the brightness per bead is often calculated by multiplying the molecular brightness ε(λex) × Φf with the number of encapsulated fluorophores per bead, assuming, for example, similar ε values of the dyes in the beads and in solution as well as the absence of concentration-dependent dye aggregation and electronic interactions between the dye molecules in the beads and neglecting scattering.25,32,42,52,54,62 Although we can exclude the former effects for our C153-stained PSP, the latter cannot be neglected for 1000 nm-sized beads. Moreover, differences in local electric field and possible influences of resonant enhancement from the particles can play a role as was only recently reported for 16 nm-sized nanoparticles.52 Hence, especially for larger beads with sizes exceeding several ten nanometers, the determination of brightness values per bead can be highly erroneous using conventional spectroscopic instrumentation and simplified procedures. A more reliable approach currently assessed by us can be the measurement of absolute fluorescence quantum yields and absorption coefficients of particle suspensions with an integrating sphere setup. In the case of our C153-stained particles, the strong increase of the μa values of the C153-PSP suspensions with increasing C153 loading concentration, in conjunction with the almost dye concentration-independent fluorescence quantum yields of these beads (Figure 4 and Table 2), accounts for the observed linear increase in brightness with increasing number of dye molecules per particle. With this respect, our C153-PSP suspensions present almost ideal systems. For other dyes with a more pronounced tendency to form nonemissive aggregates and a smaller Stokes shift,31,63 the dye loading concentration yielding maximum bead brightness needs to be optimized experimentally, thus searching for a particle size- and matrixdependent balance between the increase in particle absorption cross-section with increasing dye loading concentration and the concentration-dependent reduction in dye fluorescence quantum yield.

contain CFx (x = 1,2,3) groups, for example, to enhance their photostability, this new approach to analyte quantification is expected to be of considerable general interest. For example, the presented approach can be easily extended to other common chromophores containing fluorine atoms, such as certain fluoresceins, rhodamines, cyanines, or BODIPY dyes. This can also pave the road for multimodal labels, for example, for surface analysis that can be read out with NMR and optical spectroscopy, as well as X-Ray photoelectron spectroscopy (XPS) in the case of fluorine substituents. Simultaneously, this opens new routes for the metrological traceability of fluorescence methods, providing an elegant way to link fluorescence signals to the amount of substance via a primary method. Concerning the rational design of new labels for such applications, CF3 groups (for the analyte and the reference standard) are expected to yield optimum results due to their small anisotropy of the chemical shift compared to other F-substituents, yielding less rotational side bands and enabling a straightforward evaluation of measured intensities. Applications of qNMR currently investigated by us with this respect are ranging from the quantification of other fluorine-containing dyes encapsulated in or bound to beads over the use of C13labeled ligands and reporters for studies of the surface chemistry of nano- and micrometer-sized particles to the rational design of fluorescent reporters for combined NMR and fluorescence as well as XPS studies using, for example, multifluorine-substituted fluorescent reporters to improve the limited sensitivity of F-NMR in the lower ppm region. In addition, we present a new strategy for the reliable characterization of the signal-relevant optical properties of scattering suspensions of fluorescent materials like beads with sizes exceeding a few ten nanometers using a new customdesigned integrating sphere setup for spectrally resolved measurements of emission, absolute fluorescence quantum yields, transmission, and reflectance. Whether this approach, in conjunction with the metrological traceability offered by qNMR, can present an alternative to the presently employed relative MESF concept and may enable the assignment of absolute brightness values to beads remains to be shown. With this respect, we are currently performing systematic studies of absolute fluorescence quantum yields and absorption coefficients of suspensions of differently sized beads loaded with varying amounts of fluorophores from common dye classes varying in the size of their Stokes shift and their aggregation tendency.

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CONCLUSION AND OUTLOOK Quantitative NMR spectroscopy can be exploited for the nondestructive determination of the concentration of analyte molecules in carrier systems like nanometer- and micrometersized beads as has been shown for 1000 nm-sized polystyrene particles loaded with various amounts of coumarin 153 containing a CF3 group. Contrary to nondestructive optical measurements of beads, this method is not affected by dye−dye interactions and dye aggregation and not restricted to very small nanometer-sized beads only. Main advantages of 19FNMR for this purpose compared to other NMR techniques are its high sensitivity due to its large gyromagnetic ratio and its 100% natural abundance. With an improved experimental setup using, for example, larger rotor diameters and background-free NMR probes and fluorescent reporters equipped with a higher number of CF3 groups such as found, for example, in certain commercial xanthenes dyes or newly developed cyanine dyes, we envision a limit of detection for a reliable concentration determination of analyte molecules in the nmol/g (sample) range (i.e., nmol analyte molecules per g (sample/matrix)) with 19 F-NMR. Moreover, with other reference standards than PTFE, which contain less fluorine atoms per molecular unit, the complicated weighting procedure of the samples can be facilitated. As many application-relevant molecules like certain polymers, drugs as well as laser dyes and fluorescent reporters

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ASSOCIATED CONTENT

S Supporting Information *

Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 30 8104 1131 (C.J.); +49 30 8104 1134 (U.R.-G.). Fax: +49 30 8104 1131 (C.J.); +49 30 8104 1157 (U.R.-G.). E-mail: [email protected] (C.J.); ute.resch@ bam.de (U.R.-G.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.H., T.B., and C.W. contributed equally. We gratefully acknowledge financial support from the Federal Ministry of 3660

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Analytical Chemistry

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Economics and Technology (BMWI-22/06; 17/07). We would also like to thank Mrs. M. Spieles and Mrs. A. Hoffmann for technical assistance.

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dx.doi.org/10.1021/ac3000682 | Anal. Chem. 2012, 84, 3654−3661