Article pubs.acs.org/JPCC
Cite This: J. Phys. Chem. C XXXX, XXX, XXX−XXX
Close Spectroscopic Look at Dye-Stained Polymer Microbeads Daniel Kage,†,§ Linn Fischer,†,∥ Katrin Hoffmann,† Thomas Thiele,‡ Uwe Schedler,‡ and Ute Resch-Genger*,† †
Federal Institute for Materials Research and Testing (BAM), Division Biophotonics, Richard-Willstätter-Str. 11, D-12489 Berlin, Germany ‡ PolyAn GmbH, Rudolf-Baschant-Str. 2, D-13086 Berlin, Germany § Department of Physics, Humboldt-Universität zu Berlin, Newtonstr. 15, D-12489 Berlin, Germany ∥ Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, D-12489 Berlin, Germany S Supporting Information *
ABSTRACT: Dye-stained micrometer-sized polymer beads are important tools in the life sciences with applications in biomedical, biochemical, and clinical research. Here, bead-based assays are increasingly used, for example, in DNA sequencing and the detection of autoimmune diseases or pathogenic microorganisms. Moreover, stained beads are employed as calibration tools for fluorescence microscopy and flow cytometry methods with increasing complexity. To address the requirements concerning the relevant fluorescence features, the spectroscopic properties of representative polymer beads with diameters ranging from about 1 to 10 μm stained with varying concentrations of rhodamine 6G were systematically assessed. The observed dependence of the spectral properties, fluorescence decay kinetics, and fluorescence quantum yields on bead size and dye loading concentration is attributed to different fluorescence characteristics of fluorophores located in the particle core and near-surface dye molecules. Supported by the fluorescence anisotropy measurements, the origin of the observed alteration of fluorescence features is ascribed to a combination of excitation energy transfer and polarity-related effects that are especially pronounced at the interface of the bead and the surrounding medium. The results of our studies underline the need to carefully control and optimize all parameters that can affect the fluorescence properties of the dye-stained beads. dye-stained fluorescent calibration beads are used which should ideally closely match in size and fluorescence properties with the samples to be analyzed.19−22 Suitable beads, which must exhibit a very narrow size distribution, a homogeneous and persistent dye staining, and functional groups for subsequent biofunctionalization, can be produced either by the steric incorporation of hydrophobic encoding fluorophores during bead manufacturing23 or by the loading of premanufactured beads with organic dyes using swelling procedures.24,25 Despite their ever-increasing use, the reproducible preparation of dye-encoded carrier and calibration beads still presents a challenge,12,13 as this requires a precise control of the dye loading concentration per bead for all intensity-based codes and control of the spectral position, shape, and width of the emission spectrum of the fluorescent microbeads. The latter is important for avoiding or minimizing spectral cross talk for integral fluorescence detection. Hence, parameters like the influence of the microenvironment on the optical properties of the dyes such as their typical polaritydependent absorption and emission spectra, molar absorption
1. INTRODUCTION Microbead-based technologies have meanwhile developed into versatile and broadly used tools for highly parallelized quantitative multiparametric analyses1−3 and for the detection and quantification of nucleic acids and proteins in the life sciences and medical diagnostics.4 Typical applications range from DNA hybridization assays over solid-phase polymerase chain reactions to DNA sequencing and the detection of autoimmune diseases5 and pathogens.6 Among the many advantages of the bead-based assays are their high throughput and the ease of automation. Moreover, they can be read out with the existing instrumentation like flow cytometers initially developed for the analysis of cells,7 or alternatively, with a microscope using microtiter plates.5,8 The use of fluorescence methods for the readout of beadbased assays requires dye-encoded carrier beads, with the spectral or intensity codes signaling the surface chemistry or target-specific ligands at the bead surface.9−12 Also, novel encoding strategies, utilizing the parameter fluorescence lifetime, are emerging13−15 that encompass additional parameters to be controlled. Moreover, fluorescence techniques like flow cytometry and fluorescence microscopy require regular instrument calibration and performance validation for comparable and quantitative measurements.16−18 Here, typically © XXXX American Chemical Society
Received: March 15, 2018 Revised: May 15, 2018
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DOI: 10.1021/acs.jpcc.8b02546 J. Phys. Chem. C XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry C coefficients, and fluorescence quantum yields as well as dye− dye interaction must be considered. There exist several spectroscopic studies of stained nanometer-sized polymer beads26,27 and organic dyes embedded into polymer films.28−31 To our knowledge, however, no comprehensive studies on the optical spectroscopic properties of micrometer-sized beads are available, particularly because of the strong scattering of microparticle dispersions which renders the fluorescence measurements very challenging. The existing studies focus typically on bead preparation and application and barely address the optical properties.2,24,25,32,33 To underline the challenges of fabricating polymer beads with precisely tailored fluorescence properties, we systematically studied the signal-relevant properties of the carrier and calibration beads of different sizes stained with different concentrations of a typical encoding dye. For this study, poly(methyl methacrylate) (PMMA) polymer microbeads with diameters ranging from about 1 μm to about 10 μm were chosen, stained with rhodamine 6G (Rh6G) sterically incorporated during the dispersion polymerization reaction. Rh6G has been chosen as it is a widely used, photostable fluorophore34 whose properties in a polymer matrix are expected to be representative of many other cationic dyes. The parameters and properties assessed included the influence of light scattering on measurements as well as particularly all fluorescence features like spectral shape and width of the emission spectra, fluorescence decay kinetics, quantum yields, and emission anisotropies which could affect the encoding strategies and instrument calibration. In addition, the spatial homogeneity of dye staining and the fluorescence decay kinetics were determined on a single-particle level using confocal laser scanning microscopy (CLSM) and fluorescence lifetime imaging (FLIM), thereby also addressing fluorescence lifetime multiplexing as an emerging approach to spectral and intensity encoding.13−15
2.2. Synthesis of Fluorescent Microbeads. Plain, dyestained polymer (PMMA) microbeads lacking reactive surface groups have been prepared using a dispersion polymerization technique,35,36 in which the dye is directly incorporated into the beads during particle formation. This guarantees a homogeneous distribution within the bead and largely prevents dye leaking in application-relevant aqueous environments containing, for example, surfactants or proteins. To obtain a narrow bead size distribution, a custom-made modified reaction chamber was employed to control the polymerization temperature and the rotation speed of the sealed polymerization flasks. The bead size can be controlled by parameters like monomer, stabilizer, and radical initiator concentration as well as the reaction temperature. A typical procedure for bead preparation is given in the following. Measures of 9.8 g PVP-K30 and 980 mg aerosol-OT were dissolved in 170 mL methanol. The mixture was transferred to the reaction flask containing 15 mL of destabilized methyl methacrylate, 8 mg of Rh6G, and 400 mg of AIBN. The sealed flask was placed in the preheated reaction chamber, in which the polymerization was performed at a rotating speed of 22 turns per minute at a temperature of 63 °C for 21 h. After cooling down to room temperature, the resulting bead suspension was poured into 600 mL water to precipitate the dye-stained PMMA beads. After decantation of the water, the fluorescent beads were washed several times with water and removed from the solution by centrifugation in 50 mL falcon tubes. These washing−centrifugation cycles were repeated until the supernatant did not contain methyl methacrylate. The nominal dye loading concentration used in the following sections is the molar concentration of the dye in the reaction volume. The effective dye loading concentration can differ from this value as will be discussed in section 3.5. The beads were classified according to their diameter into small (8 μm) beads. 2.3. Optical Spectroscopy. 2.3.1. Absorption Spectroscopy. The absorption spectra were acquired on a SPECORD 210 PLUS (Analytik Jena AG) dual beam absorption spectrometer. 2.3.2. Steady-State Fluorescence and Anisotropy Measurements. The fluorescence spectra were measured with calibrated spectrofluorometers FSP920 and FLS920 (Edinburgh Instruments Ltd.) using a 0°/90° geometry for the excitation and emission channel and magic angle37 polarizer settings to circumvent polarization effects. All emission spectra were corrected for the wavelength dependence of the spectral responsivity of the detection channel and all the excitation spectra for the wavelength dependence of the spectral radiant power reaching the sample.38 For the fluorescence anisotropy measurements, the instrument-characteristic G factor for data correction was determined along with the fluorescence measurements.37 The spectral band-pass for measuring the emission spectra was usually set to 6 nm. 2.3.3. Fluorescence Lifetime Measurements. The timeresolved measurements were performed with an FLS920 (Edinburgh Instruments Ltd.) lifetime spectrometer equipped with a Hamamatsu R38090U-50 (Hamamatsu Photonics K.K.) MCP-PMT and a Fianium Supercontinuum SC400-2-PP (NKT Photonics A/S) laser allowing for standard timecorrelated single-photon counting. The instrument response function, obtained with a LUDOX suspension, has a pulse width of about 250 ps and is mainly determined by the excitation pulse width. The repetition rate for excitation was set
2. EXPERIMENTAL SECTION 2.1. Materials. Methanol (99+%, extrapure), methyl methacrylate (99%, stabilized), polyvinylpyrrolidone K90 (average molecular weight of 1 300 000), dioctylsulfosuccinate, sodium salt (96%), and aluminum oxide (neutral for chromatography 50−200 μm, 60A) were purchased from Acros Organics NV, Belgium. Polyvinylpyrrolidone K30 (average molecular weight of 40 000) was obtained from Sigma Aldrich Chemie GmbH, Germany. Azobisisobutyrodinitrile (AIBN) was purchased from Molekula GmbH, Germany, and Rh6G from Merck KGaA, Germany, respectively. The spectroscopic grade acetone used for dissolving fluorescent beads was obtained from PanReac AppliChem ITW Reagents. Methyl methacrylate was destabilized with aluminum oxide prior to use. All other chemicals were used as received. The chemical structures of Rh6G and PMMA are displayed in Figure 1.
Figure 1. Rh6G (left) and PMMA (right). B
DOI: 10.1021/acs.jpcc.8b02546 J. Phys. Chem. C XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry C
Figure 2. Fluorescence excitation spectra (A, λfl,em = 600 nm) and emission spectra (B, λfl,ex = 500 nm) of plain, differently sized Rh6G-stained beads, with largely uniform nominal dye loading concentration of ≤223 μmol/L (bead concentration in the range of 0.05−1 mg/mL).
2.3.5. FLIM and Microscopy. CLSM and FLIM were performed with a FluoView 1000 confocal microscope (Olympus Deutschland GmbH) upgraded with a FLIM− fluorescence correlation spectroscopy upgrade kit (PicoQuant GmbH) using droplets of bead dispersions in water. The excitation wavelength was 488 or 485 nm for steady-state and time-resolved measurements, respectively. A dichroic mirror (DM405/488) was used to reflect the excitation light to the sample. The emission light was collected within the range of 500−600 nm (steady state) or with a 525 nm long-pass filter (time-resolved). For time-resolved measurements, a repetition rate of 20 MHz was used. Data acquisition and analysis were done with the TimeHarp 200 TCSPC PC board using SymPhoTime software (PicoQuant GmbH) as well as selfbuilt analysis scripts (GNU Octave).
to 10 MHz. To avoid artifacts because of rotational motion, magic angle polarizer settings were applied. The fluorescence lifetimes are either intensity-weighted decay times determined from the least-squares multiexponential decay fits to the data or have been calculated by means of eq 1.37 ∞
τ=
∫0 tI(t ) dt ∞
∫0 I(t ) dt
(1)
For time-resolved fluorescence emission spectra (TRES37,39), the fluorescence decay curves were measured at varying emission wavelengths. Subsequently, the obtained fluorescence decay curves were fitted and the time-dependent intensity at the respective emission wavelength was derived. For absorption, steady-state, and time-resolved fluorescence measurements, standard 1 cm quartz cuvettes (Hellma GmbH & Co. KG) were used. The bead suspensions were continuously stirred during the measurements. All measurements were carried out at room temperature (approx. 22 °C). As the optical spectroscopic measurements can be strongly influenced by the bead concentration, the measurement conditions had to be optimized. The bead concentration was adjusted to give sufficiently high signal intensities while avoiding the distortions of the measurement results by scattering and/or reabsorption (cf. Supporting Information, section S2). Scattering is mainly governed by particle size and particle number density (number of particles per mL solvent), whereas reabsorption effects are directly related to the dye concentration, and thus the particle mass concentration (milligram particles per milliliter of solvent). For the fluorescence measurements, unification of the bead concentration is not necessary as long as the bead number density or bead mass concentration is kept below the critical values (cf. Supporting Information, section S2). Thus, the bead mass concentration was kept between 0.05 and 1 mg/mL to achieve reasonable signal intensities while avoiding the distortions of the measurements. 2.3.4. Fluorescence Quantum Yields. The fluorescence quantum yields of the scattering bead dispersions were obtained absolutely with a C11347 (Hamamatsu Photonics K.K.) integrating sphere setup using the emission correction curve determined by the instrument manufacturer. Samples with unstained beads of similar size and bead concentration were used as blanks. All the measurements were done with instrument-specific long-neck quartz cuvettes. The data have been corrected for reabsorption effects with a routine built-in by the instrument manufacturer.40
3. RESULTS AND DISCUSSION 3.1. Steady-State Fluorescence Measurements Spectral Properties. We studied ensembles of plain beads with varying diameter and largely comparable dye loading concentration (within the limits of the synthesis procedure) by means of steady-state fluorescence spectroscopy under previously optimized measurement conditions (see section 2.3 and Supporting Information, section S2). The resulting fluorescence excitation spectra of the bead ensembles and a spectrum of Rh6G dissolved in water are displayed in Figure 2A. The given dye concentration refers to the dye concentration applied during synthesis (nominal dye loading concentration) as defined in section 2.2. Generally, the excitation spectra of the Rh6G-stained beads are slightly red-shifted with respect to the spectrum of the dye in aqueous solution. This is attributed to the polar or ionic interactions between the cationic dye and the carbonyl and carboxyl groups of the PMMA matrix. The magnitude of the shift agrees well with the absorption data of Rh6G in solution and in PMMA films.28 The small beads, here with diameters 3 μm. For small beads with diameters approximately
