Surface Analytical Study of Poly(acrylic acid)-Grafted Microparticles

Article pubs.acs.org/JPCC

Surface Analytical Study of Poly(acrylic acid)-Grafted Microparticles (Beads): Characterization, Chemical Derivatization, and Quantification of Surface Carboxyl Groups Paul M. Dietrich,*,† Andreas Hennig,† Markus Holzweber,† Thomas Thiele,‡ Heike Borcherding,‡ Andreas Lippitz,† Uwe Schedler,‡ Ute Resch-Genger,† and Wolfgang E. S. Unger† †

BAM Federal Institute for Materials Research and Testing, D-12200 Berlin, Germany PolyAn GmbH, Rudolf-Baschant-Strasse 2, D-13086 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: We report a surface analytical study of poly(methyl methacrylate) (PMMA) microparticles (beads) with a grafted shell of poly(acrylic acid) (PAA) with thicknesses up to 4 nm using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), time-offlight secondary ion mass spectrometry (ToF-SIMS), and nearedge X-ray adsorption fine structure (NEXAFS) spectroscopy. These polymer microparticles were analyzed before and after reaction of the surface carboxyl (CO2H) groups with 2,2,2trifluoroethylamine (TFEA) to gain a better understanding of methods with use of covalently bound probe molecules for surface group analysis. The results obtained with chemical derivatization XPS using TFEA are discussed in terms of surface quantification of reactive CO2H groups on these PAA-coated microparticles. A labeling yield of about 50% was found for TFEA-derivatized particles with amounts of surface-grafted CO2H groups of 99 μmol/g or more, which is consistent with predicted reaction yields for homogeneously dispersed PAA hydrogels.

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INTRODUCTION The use of micro- and nanometer-sized particles in the material, life, and medical sciences requires the controlled functionalization of the surface of these beads, thereby providing the basis for numerous applications ranging from biosensing and drug delivery to optoelectronics and solar energy conversion.1−13 One of the key requirements for the broad application, future development, and public acceptance of these materials is the precise knowledge of the chemical nature, surface concentration, and spatial distribution of the functional groups at the surface. This also includes a reliable quality control for largescale productions of these materials. Because of this enormous interest, various analytical methods have been devoted to the characterization of functionalized surfaces, e.g. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), solidstate NMR, isothermal titration calorimetry, conductometry, potentiometry, as well as fluorometric and colorimetric assays and fluorophore labeling.14,15 Some of these methods complement each other in terms of accessible concentration range and scope of application, while other combinations could be used for mutual confirmation of the obtained results. Our initial motivation for the present work was based on a previous exploratory study on the comparability of surface quantification methods.14 Therein, polymer microparticles made for bead array applications composed of a poly(methyl © 2014 American Chemical Society

methacrylate) (PMMA) core with a grafted shell of poly(acrylic acid) (PAA) of various concentrations were analyzed by different methods including conductometry, 13C solid-state NMR, fluorophore labeling with an amino derivative of fluorescein isothiocyanate (FL-A), a supramolecular binding assay, and two colorimetric assays. We now extend our surface analytical investigation of these polymer microparticles to scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The main advantage of these surface chemical analysis methods compared to, e.g., optical methods is a direct access to detailed information about the chemical nature of the surface species and the thickness of the grafted PAA shells on the PAA-coated microparticles.

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EXPERIMENTAL SECTION Materials. If not noted otherwise, all chemicals were purchased from Sigma-Aldrich (Steinheim, Germany), AppliChem (Darmstadt, Germany), J. T. Baker (Deventer, Netherlands), Carl Roth (Karlsruhe, Germany), or Merck (Darmstadt, Received: June 4, 2014 Revised: July 29, 2014 Published: August 8, 2014 20393

dx.doi.org/10.1021/jp505519g | J. Phys. Chem. C 2014, 118, 20393−20404

The Journal of Physical Chemistry C

Article

Gaussian, 30% Lorentzian) was used in combination with a Shirley background. Curve fittings of C 1s and O 1s core-level spectra were done according to previous reports.22,23 Elemental compositions from survey spectra are calculated by using peak areas normalized on the basis of acquisition parameters after a Shirley background subtraction, experimental sensitivity factors, and transmission factors provided by the manufacturer. Damage of polymer microparticles by X-ray radiation was not observed. Chemical Derivatization XPS (CD-XPS) Using TFEA. Scheme 1 illustrates the amidation reaction of surface carboxyl (CO2H) groups on PAA-coated microparticles with TFEA that was used for CD-XPS.16−18

Germany) and were of the highest purity available when used for analytical measurements. PMMA microparticles (diameter 6 μm) with varying amounts of PAA were individually prepared by PolyAn GmbH (Berlin, Germany) for this study. Gold substrates (Georg Albert PVD, Germany) were prepared by thermal evaporation of 30 nm of Au (purity 99.99%) onto polished single-crystal Si (100) wafers that had been precoated with a 9 nm titanium adhesion layer. Sample Preparation. Surface functionalization with 2,2,2trifluoroethylamine (TFEA)16−18 was performed according to an established protocol.14 A 10 mg sample of polymer microparticles was washed into 660 μL reaction buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.0) by repeated cycles (>5) of centrifugation, removal of supernatant, and refilling of reaction buffer. Subsequently, 50 μL of 400 mM TFEA hydrochloride in reaction buffer was added. The reaction was started by adding 80 μL of 100 mg/mL (0.52 M) 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride salt (EDC) freshly dissolved in 4 °C cold water. The total reaction volume was 800 μL; final conditions were 12.5 mg/mL polymer microparticles, 25 mM TFEA, and 52 mM EDC. After 3 h of reaction time at room temperature, the microparticles were washed into 1 mL 0.1 M borate buffer, pH 9.0. Microparticle samples for SEM, XPS, and NEXAFS measurements were prepared by drop casting several drops (1−10 μL) of aqueous microparticles suspensions (10 mg/mL) on glass or gold-coated silicon substrates and were allowed to dry overnight in a closed Petri dish. That was repeated until the substrate was covered homogeneously. PAA reference films for XPS and NEXAFS were prepared by spin coating (4000 rpm, 30 s) of a 3% (w/w) aqueous PAA solution on a 1 × 1 cm2 piece of silicon or gold-coated Si wafer. Scanning Electron Microscopy (SEM). A Zeiss Gemini Supra40 with Schottky field emitter as a cathode has been used. High magnifications enabling fair observation of particles with sizes even smaller than 10 nm have been attained by means of a “high-resolution” electron detector called In-Lens. This type of detector is built in the electron column of the SEM and collects only SE1 electrons, i.e. those secondary electrons being emitted at the laterally small impact place of the primary electrons on the sample surface. This type of imaging is very sensitive to the nanomorphology of the sample surface, i.e. high-resolution top observation. Particle diameters were obtained from SEM images with use of ImageJ software.19 X-ray Photoelectron Spectroscopy (XPS). XPS measurements were carried out with an AXIS Ultra DLD electron spectrometer manufactured by Kratos Analytical, UK. XPS spectra were recorded with use of monochromated Al Kα excitation at a pass energy of 80 eV for survey spectra and 20 eV for the core-level and valence band spectra. The electron emission angle was 0° and the source-to-analyzer angle was 60°. The binding energy scale of the instrument was calibrated following a Kratos Analytical procedure which uses ISO 15472 binding energy data.20 Spectra were taken by setting the instrument to the hybrid lens mode and the slot mode providing approximately a 300 × 700 μm2 analysis area. In case of insulating samples, the charge neutralizer was used. The binding energy scale was furthermore corrected for charging,21 using an electron binding energy of 285.0 eV22 for the C 1s level of aliphatic hydrocarbon. Spectra are decomposed with the CasaXPS peak fit program, version 2.3.15 from Casa Software Ltd. (United Kingdom). A Gaussian/ Lorentzian product function peak shape model GL(30) (70%

Scheme 1. (a) Chemical Derivatization Reaction of Poly(acrylic acid) (PAA, left) Grafted Microparticles Using EDC-Mediated Amidation with 2,2,2-Trifluoroethylamine (CF3CH2NH2, TFEA) Yielding Poly(N-(2,2,2trifluoroethyl)acrylamide) (PTFEAA, right) and (b) Chemical Structure of Poly(methyl methacrylate) (PMMA) and Poly(N-vinylpyrrolidone) (PNVP)

The measurand is the fraction of carboxylic acid carbon atoms before TFEA labeling expressed as a percentage, xCO2H, of the total carbon amount described by eq 1. nCO2H xCO2H = × 100 nCtotal (1) with nCtotal = nCO2H + nCR

(2)

where nCO2H is the amount of carboxylic acid carbon atoms, nCtotal the total amount of carbon, and nCR the amount of all remaining carbon atoms that are not located in CO2H groups. The quantitative elemental analysis (QEA) approach for quantification of xCOOH was applied by quantification of XPS survey scans, using the intensities of C 1s and F 1s photoelectron peaks (measured in atomic percent, atom %).24 The QEA data set has to be corrected for additional TFEA related elements (C, N, and F) that are introduced during the derivatization reaction. According to the mass balance law, a more generalized equation follows from Scheme 1. Cx − (CO2 H)y + yCF3 CH 2NH 2 → Cx − (CONHCH 2CF3)y + H 2O

(3)

with Cx − (CONHCH 2CF3)y = Cx + 2yH 2yOy (NH)y F3y

(4)

where y=

1 IF1s [F] = 3 RSFF1s 3

(5)

and 20394



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Figure 1. SEM micrographs at 30 000× magnification of PMMA microparticles (diameter ca. 6 μm) with the following amounts of grafted PAA (according to conductometry): 35 (P35, a), 99 (P99, b), and 946 (P946, c) μmol/g. (SEM micrographs of PMMA cores are shown in Figure S1, Supporting Information (SI).)

x=

IC1s RSFC1s

− 2y = [C] −

2[F] 3

Cleveland, USA) at 285.4 eV.26 Normalized spectra are shown in units of the absorption edge jump after subtraction of the pre-edge count rate.25 Time-of-Flight Secondary Ion Mass Spectrometry. All sample measurements were performed on a ToF.SIMS IV instrument (ION-TOF GmbH, Münster, Germany) of the reflectron type, equipped with a 25 keV bismuth liquid metal ion gun (LMIG) as primary ion source mounted at 45° with respect to the sample surface. The LMIG was operated at 0.5 μA emission current in the so-called “high current bunched” mode (high mass resolution, low lateral resolution). Bi3+ was selected as primary ion by appropriate mass filter settings. To improve the focus of the primary ion beam, the pulse width of the Bi3+ (25 keV) ion pulse was reduced to 15 ns and the lens target was adjusted to obtain a sharp image on a structured sample (e.g., silver cross) in the secondary electron mode. The primary ion current was directly determined by using a Faraday cup located on a grounded sample holder. Operation conditions with these settings comprised a target current of 0.3 pA for the selected primary ion. The total primary ion dose density was set to 5 × 1011 ions/cm2 ensuring static conditions. Scanning area for analysis was 100 × 100 μm2 with a pixel resolution of 64 × 64. The vacuum in the analysis chamber was in the range of 10−9 mbar during all measurements. ToF-SIMS spectra were acquired in negative ion mode with 5 spots per sample analyzed. The mass scale was internally calibrated by using a number of well-defined and easily assignable secondary ions. C−, C2−, and C4− were used for the mass calibration keeping the error in calibration below 10 ppm. Principal Component Analysis (PCA). The peak list creation strategy to perform PCA relied on selecting solely secondary ions significant for PMMA and PAA in the mass range of m/z 0−150. The corresponding reference spectra for peak selection were available from the built-in database of the SIMS software SurfaceLab6 (ION-TOF GmbH, Münster, Germany). As mentioned above, the uncertainty in the calibration of the ToF-SIMS mass scale was kept below 10 ppm to ensure minimum scattering in the peak position and to minimize uncertainties related to the choice of the integration limits which are relevant for a reliable determination of secondary ion yields. This ensures that the variance in the given data set is due to real sample differences. Integration limits in m/z regions with overlapping peaks were placed tightly around each peak to ensure consistent and accurate measurements of all peak areas. PCA was performed by using the software R version 2.15.2. (R is an open access program for statistical computing downloadable from http://www.r-project.org.) Each peak intensity was normalized to the sum of the selected peak intensities to

(6)

where IF1s and IC1s are the XPS intensities of F 1s and C 1s determined from the survey scan after TFEA derivatization, and RSFF1s and RSFC1s are the relative sensitivity factor of F 1s and C 1s, respectively, taken from the Kratos element library. Finally xCOOH can be calculated from eqs 1−6 according to eq 7. xCO2H =

y [F] × 100 = × 100 x 3[C] − 2[F]

(7)

The derivatization reaction yield (X) can then be calculated by taking into account that 1/3 of all carbon atoms in PAA are located in carboxylic acid (CO2H) moieties. X = 3xCO2H

(8)

Alternatively the derivatization reaction yield (X) can be calculated by using relative CF3 component peak areas from the C 1s core level spectra according to eq 9.

X=

CF3,exp. CF3,stoich.

× 100 (9)

Here CF3,exp. refers to the CF3 relative peak area as obtained from the fit of measured C 1s core-level spectra, while CF3,stoich. is the fraction derived from the theoretical stoichiometry of a completely derivatized PAA shell (cf. Scheme 1). And from the chemical structure of PTFEAA (the quantitatively derivatized PAA) shown in Scheme 1 it follows CF3,stoich. = 20%. Near-Edge X-ray Absorption Fine Structure (NEXAFS). NEXAFS spectroscopy was carried out at the HE-SGM monochromator dipole magnet beamline at HelmholtzZentrum Berlin (synchrotron radiation source BESSY II, Germany). Spectra were acquired at C, N, and O K-edges in the PEY (partial energy electron yield) mode, using an electron detector based on a channel plate and a retarding field of −150 eV.25 The resolution E/ΔE of grid 1 at the carbonyl π* resonance of CO (hν = 287.4 eV) was found to be on the order of 2500. The slit width used was 200 μm. Raw spectra were divided by the monochromator transmission function, which was obtained with a freshly sputtered Au sample.25 C and O Kedges were recorded at an angle of 55° measured between the surface plane of the sample and the direction vector of the incident linearly polarized light beam. Energy alignment of the energy scale was achieved by using an I0 feature referenced to a C 1s → π* resonance measured with a fresh surface of HOPG (Highly Ordered Pyrolytic Graphite, Advanced Ceramic Corp., 20395



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Figure 2. High-resolution C 1s (left column) and O 1s (right column) core-level XP spectra of a spin-coated PAA film (top) and PMMA microparticles P0 (bottom). Additional components occurring in the P0 particles due to PNVP are shown in gray.

entiation between the PMMA core and a grafted surface layer of PAA is quite challenging as to be expected owing to the similar XPS signatures of both polymers. The C 1s XPS spectrum of a reference PAA film shows two clearly separated peaks around 285 and 289 eV, which can be assigned to aliphatic carbon atoms (CC/CH, CH−CO2H) and the CO2H group. The most characteristic difference in the C 1s XP spectrum of bare PMMA microparticles (P0) is an additional component peak (3) at 286.7 eV, which is clearly absent in PAA and can be assigned to the O−CH3 carbon of the methyl ester group of PMMA.22,23 Both O 1s core-level spectra consist of two intense peaks around 532−533 and 533− 534 eV, which can be attributed to OC and OC oxygen species in accordance with reported values for PAA and PMMA.22 In addition to the expected carbon and oxygen, significant amounts of nitrogen were detected in unmodified PMMA microparticles P0, which originate most probably from poly(N-vinylpyrrolidone) (PNVP) which is used as aggregation inhibitor during PMMA microparticle synthesis. This assumption is supported by additional components in the C 1s and O 1s XP spectra (CN, NCO, and NCO) of the P0 particles which had to be included to obtain reasonable fits of these spectra and can be assigned to PNVP (cf. Figure 2, Table S1 (SI), and ref 22 for more details). XPS survey spectra of the microparticles, which have been functionalized with varying amounts of surface-grafted PAA, were recorded next. The surface composition of the analyzed microparticles was obtained from these survey spectra by using an implemented quantification routine of CasaXPS software (which is valid for homogeneous and isotropic samples) based on peak intensities and relative sensitivity factors. Here, we have to consider the “XPS 95% information depth” z95.29 For polymers (e.g., PMMA), z95 values for C 1s, N 1s, O 1s, and F 1s of approximately 10, 9, 8, and 7 nm result from calculated

correct for variations in the total secondary ion yields between different spectra. The data were then mean centered. Detailed information on PCA and especially PCA performed with SIMS data can be found in a current review by Graham.27

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RESULTS AND DISCUSSION Characterization of PAA-Coated Microparticles. Surface Grafting of PAA. For this study, four different PMMA particle populations (diameter = 6 μm) with varying amounts of surface-grafted PAA 0, 35, 99, and 946 μmol/g (determined by conductometry14,28) designated here as P0, P35, P99, and P946 were selected. P0 is the nongrafted PMMA core. The particles’ size, shape, and topography were first investigated by scanning electron microscopy (SEM). SEM images show a rather narrow size distribution with a mean particle diameter of 6.0 ± 0.5 μm (histograms are shown in Figure S2, Supporting Information) and an increasing roughness of the surface with increasing amounts of surface PAA (Figure 1). While the surface of P35 and P99 appeared as smooth as the native PMMA core P0, SEM images of P946 indicated a slight roughness of the particles’ surface. This observation agrees well with our previous presumption of a flexible, “pore-like” structure of the surface PAA chains.14,28 With the goal to verify surface grafting of PAA to PMMA cores in conjunction with the attempt to characterize surface species on PAA-coated microparticles, X-ray photoelectron spectra of unmodified PMMA microparticles and of a spincoated PAA film on silicon were recorded first to obtain reference spectra of the polymers used for particle preparation. XPS survey spectra (see Figures S3 and S4, SI) agree well with published data for these polymers.22 Careful inspection of high-resolution photoelectron C 1s and O 1s core-level spectra (Figure 2) indicate that the differ20396



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Table 1. CO2H Groups in the PAA Shell and Elemental Compositions Derived from XPS of PAA-Coated Microparticles P35, P99, and P946 Together with Data Obtained with Nongrafted PMMA Microparticles P0 and a PAA Reference Filma sample

[CO2H]b [μmol/g]

O [atom %]

C [atom %]

N [atom %]

Oc [atom %]

Cc [atom %]

C/O ratiod

P0 (PMMA) P35 P99 P946 PAA filme

0 35 99 946 n.a.

21.6 23.3 27.5 35.3 35.8

75.1 73.2 70.0 64.0 64.2

3.3 3.5 2.5 0.7 0.0

24.9 27.5 31.3 35.9 35.8

75.1 72.5 68.7 64.1 64.2

3.0 (2.5) 2.6 2.2 1.8 1.8 (1.5)

a

An uncertainty of measurement of 10% is estimated for XPS surface concentrations. bTotal amount of CO2H groups in the PAA shell as determined by conductometry14,28 (uncertainty ca. 9%, see ref 14). cElemental composition excluding PNVP contributions. dFor comparison stoichiometric values for PMMA and PAA are given in parentheses. eSpin-coated from a 3% (w/w) solution of PAA in H2O on silicon.

Figure 3. Comparison of nitrogen surface concentrations on nongrafted PMMA microparticles P0, a PAA reference film, and PAA-coated microparticles P35, P99, and P946 measured by XPS and ToF-SIMS. The nitrogen surface concentration is expressed in atom % for XPS (from the N 1s peak intensity, left) and as the secondary ion intensity ratio ICN−/IO− taken from normalized negative static ToF-SIMS spectra (right). [An uncertainty of 20% is estimated for the XPS nitrogen surface concentration. The given statistical uncertainties of the ICN−/IO− intensity ratio result from standard deviations derived from five repeated SIMS measurements.]

electron attenuation lengths (ALs) using Seah’s general formula for organic materials.30,31 It has to be noted that for very thin PAA shells, where the PAA film thickness is smaller than z95, quantitative information from XPS will be biased because of the sample’s chemical in-depth inhomogeneity within the probed volume (thin overlayer on a sphere). To avoid this bias, alternative XPS quantification models have to be used which address the layered structure and curvature of the surface as well (cf. refs 32−37). Unfortunately, in the case of the studied PAA-coated microparticles these improved quantification models could not be used due to the very similar chemical nature of PAA and PMMA (vide supra) that does not allow unambiguous discrimination between these two polymers by XPS. The results of the XPS compositional analysis for PAAcoated microparticles with varying amounts of grafted PAA are summarized in Table 1. Reminiscent of the nongrafted PMMA microparticles, PNVP-related nitrogen was also found in all PAA-coated microparticles with surface concentrations decreasing vs increasing amounts of PAA in the shell. The PNVP contributions to the overall elemental composition, i.e., in P0 particles with 3.3 atom % nitrogen, 3.3 atom % oxygen, and 19.8 atom % carbon, could not be neglected. Thus, recalculated elemental compositions excluding PNVP contributions were used for obtaining the C/O ratios. Otherwise, the carbon

concentration and hence the C/O ratio would be overestimated due to the PNVP fraction, which accounts for about 1/5 of all carbon atoms in P0. As follows from Table 1, the carbon-to-oxygen atomic ratio was largest for nongrafted PMMA microparticles P0 and decreased with increasing amounts of PAA until the carbon-tooxygen ratio became identical with that of the spin-coated PAA reference film (cf. Figure S3, SI). The theoretical carbon-tooxygen atomic ratio of PMMA and PAA resulting from stoichiometry (cf. Scheme 1) is somewhat increased owing to additional contributions from adventitious hydrocarbons usually found in XPS. Similar to XPS observations a decreasing nitrogen content due to an increasing PAA shell thickness was observed in ToFSIMS, when using the CN− fragment secondary ion intensity normalized to the O− secondary ion intensity as a measure of N surface concentration, see Figure 3. Since the oxygen content of the individual polymers, PAA [C3H4O2]n, and PMMA [C5H8O2]n is constant, i.e., the same number of O atoms per repetition unit (cf. Scheme 1), this seems to be a valid method to compare the nitrogen content within different microparticle populations. Interestingly, the intensity ratio ICN−/IO− corresponds very well with the nitrogen surface concentration obtained by quantitative XPS (cf. Figure 3). For the PAAgrafted microparticles, the highest ICN−/IO− ratio was found for P35 followed by P99 and P946 microparticles. As to be 20397



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Figure 4. High-resolution C 1s (left column) and O 1s (right column) XP spectra of PAA-coated microparticles P99 before (top) and after derivatization with 2,2,2-trifluoroethylamine (TFEA, bottom). Components of fitted spectra are numbered according to the chemical structures shown in the insets. C 1s and O 1s components originating from PMMA (CH3O, CO) and PNVP (C−N, N−CO, N−CO), respectively, were also included and are shown in black and gray, respectively (for a detailed assignment of the components see Table S2, SI, and Table 3).

expected a significantly higher ICN−/IO− ratio was measured for the bare PMMA microparticles P0 when compared to P35 data. It is somewhat striking that XPS data for P0 are too low compared to P35. This might be due to the problem that we compare data from a surface most probably homogeneous within z95 (P0) with data obtained from layered samples (P35− P946) which are definitely inhomogeneous within z95 resulting in an unknown bias of the quantification routine implemented in standard XPS software. Nevertheless, the correlation of XPS and ToF-SIMS data is obviously good for the studied PMMA microparticles coated with PAAs P35−P946 (cf. Figure 3). This exceptional correlation can only be explained if either the shell is very patchy, or the “information depth” of XPS is similar to that of SIMS (i.e., with a characteristic length of ∼3 nm for organic materials38). The latter is not unreasonable when using a 25 keV Bi3+ analysis ion source and CN− as the secondary ion because with that combination light ions like CN− can originate from at least 2 nm deeper than molecular secondary ions as shown recently in a VAMAS study on organic depth profiling.39 Moreover, a homogeneous PAA coating on the microparticles is assumed based on previously reported results14 and the data presented here. High-resolution photoelectron C 1s and O 1s core-level spectra of PAA-coated microparticles P35, P99, and P946 were also recorded (cf. Figure 4 and Figures S5−S7, SI). Highresolution C 1s and O 1s XP spectra of P35 and the PMMA core P0 look very similar to each other (cf. Figures S4 and S5, SI) and those for P99 reveal a mixture of spectral signatures from PAA and PMMA (for comparison see the top panel of Figures 2 and 4).

The C 1s core-level of P946 resembles very much the XP spectrum of pure PAA except for three minor components (relative peak area of only 2% each at enhanced measurement uncertainty) at the binding energy positions that have been assigned to the OCH3 carbon of PMMA’s methyl ester group or PNVP’s CN and NCO moieties, respectively (cf. Figures S3, S4, S7, and Table S2, SI).22 Principal component analysis (PCA) of negative ion mode ToF-SIMS spectra supports these XPS findings. Similar to XPS analysis, P946 and pure PAA appear to be very similar as illustrated in the scores plot of PC1 vs PC 2, see Figure 5 (top). The complete set of microparticles analyzed in this study can be separated on PC1. P0 and P35 are well separated from each other as well as from all other particle populations. Although P99 is already close to P946 and PAA, it is still significantly separated from them. This suggests that the secondary ion mass spectrum of P99 represents again (see the discussion of XPS results above) a kind of a mixed situation of contributions from PAA and PMMA. This becomes more obvious in the loadings plot where positive loadings on PC1 for P0 and P35 can be linked to PMMA-related secondary fragment ions, e.g. OCH3− (cf. Figure 5). P99, P946, and pure PAA in contrast are consistently characterized by negative loadings on PC 1. P946 and PAA cannot be separated on PC 1 or PC 2, indicating identical spectra and no spectral contribution from the PMMA core. Hence, we conclude that for P946 microparticles, the PAA layer is thicker than the information depth of ToF-SIMS when using Bi3+ primary ions. This depth has been estimated to be approximately 3 nm for organic materials (Bi3+ at 25 keV primary energy).38 20398



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Figure 6. Enlarged π* region of O K-edge NEXAFS spectra of PAAcoated microparticles P0, P99, and P946 and a reference PAA film.

show that the P35 particles carry indeed a grafted PAA shell which must be rather thin. The P946 particles carry a substantial amount of PAA and the spectral signatures resemble very much pure PAA. The P99 particles carry a significantly lower amount of grafted PAA, but most probably in a closed shell. More details of shell thickness will be addressed in the next paragraph. PAA Shell Thickness Considerations. ToF-SIMS and XPS in combination with specific signals (fragments and elements) originating exclusively from the core and their attenuation due to the grafted PAA layer provide a handle for estimating the PAA shell thicknesses. To estimate the layer thickness of the PAA shell, we examined the attenuation of a characteristic secondary ion signal from the PMMA core. This can be readily done by comparing the fwhm intensity ratio of suitable characteristic secondary ion signals for PAA and PMMA, respectively. Based on the secondary ion signals highlighted by PCA in Figure 5 and a comparison of the spectra of the pure polymers the intensity of CH3O− for PMMA and C3H3O2− for PAA was chosen. The results are shown in Figure 7. Obviously, the attenuation of the PMMA related CH3O− fragment intensity increases exponentially (i.e., linearly in a log10 diagram) with an increasing amount of surface-grafted PAA. This trend is corroborated by an increase of the secondary ion yield ratio ICN−/IO− with a decreasing thickness of the surface-grafted PAA layer (cf. Figure 3). Based on these findings, the information depth of Bi3+ primary ions,38 and the ToF-SIMS PCA analysis (cf. Figure 5), we conclude a PAA shell thickness of 3 nm for P946 microparticles. The ToF-SIMS shell thickness estimates were supported by our XPS results using a similar approach. According to Shard et al., the attenuation of core-specific XPS signals can be used to calculate shell thicknesses of core−shell particles.33,45 Taking advantage of the nitrogen content from PNVP that is characteristic of the PMMA core only (vide supra), we could use here the attenuation of N 1s photoelectrons (cf. Table 1 and Figure 3). By using that method and if the PAA layer is assumed to be homogeneous in thickness on all particles, then the PAA shell is approximately 0.7 and 4 nm thicker for P99 and P946, respectively, than the PAA shell of P35 microparticles.46

Figure 5. PCA evaluation of negative ToF-SIMS spectra obtained from nongrafted PMMA microparticles P0, a PAA reference film, and PAA-coated microparticles P35, P99, and P946. Top: Scores plot PC1 (94.3%) vs PC2 (3.1%) of the negative ToF-SIMS spectra including the 95% confidence ellipses. Bottom: Loadings plot on PC1 showing the key secondary fragment ions responsible for separation on PC1. The percentage given in the brackets denotes which percentage of the variance is covered by the corresponding principal component PC1 or PC2.

NEXAFS spectroscopy of the PAA-coated microparticles revealed characteristic spectral signatures of PMMA40−43 and PAA.44 Also for this method the chemical similarity of PAA and PMMA results in rather similar spectral signatures, making a differentiation of P0−P946 quite challenging (cf. Figures S11 and S12, SI). Nevertheless, differences in the O K-edge NEXAFS spectra might be useful as illustrated in Figure 6. O K-edge NEXAFS spectra are dominated by intense O 1s(CO) → π*CO41−44 transitions at ∼532 eV and additional but less intense transitions between 534.3 and 534.8 eV assigned to O 1s(OCH3) → πCO * 41−43 and O 1s(OH) → 43,44 πCO * transitions from PMMA and PAA, respectively. These O 1s → π*CO transitions are located at slightly different photon energies for both polymers. In summary, we could confirm previous results from indirect methods like conductometry, fluorophore labeling, and supramolecular binding and colorimetric assays showing increasing amounts of surface-grafted PAA on a PMMA core by surface analytical techniques such as XPS, ToF-SIMS, and NEXAFS which address the surface chemistry directly. Our results clearly 20399



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derivatization agents are not really known. In the case of XPS, often fluorine labels are used due to its high sensitivity and as fluorine is not present in many application-relevant materials. For a deeper insight into fluorine labeling and the corresponding coupling yields, the initially characterized PAAcoated microparticles were chemically derivatized with 2,2,2trifluoroethylamine (TFEA) to evaluate the amount and accessibility of reactive CO2H groups in the PAA shell by XPS.16−18 Therefore, we labeled all microparticle populations with TFEA following our previously established labeling protocol to covalently bind molecules with amino groups after EDC activation.14,28 Successful TFEA binding to the CO2H groups within the PAA shell on microparticles was confirmed by performing complementary surface chemical analysis tools. Clear evidence for successful TFEA binding, in particular for the formation of covalent amide bonds, was obtained from XPS measurements, see, e.g., the C 1s and O 1s core-level spectra of P99 derivatized with TFEA shown in Figure 4 (bottom). Evidence for covalent binding follows from the O 1s spectrum, where the carboxylic acid related C−O−H peak at 533.4 eV was significantly reduced in intensity, and from the C 1s spectrum, which shows a new peak at 292.7 eV corresponding to CF3 groups (see Figure 4), respectively. The curve-fitting results of C 1s core-level spectra of TFEA-derivatized microparticles are summarized in Table 2. The assignments of the individual components are consistent with the data reported for the trifluoroethyl ester of PAA (PTFEA).22,50 Other clear proofs for successful TFEA binding are the appearance of a new F 1s peak at 688.2 eV and the N 1s peak at 400.2 eV in the survey spectrum (cf. Figure S9, SI), which refer to organic fluorine and an amide moiety, respectively.22,44,51−54 Additional hints of a successful labeling reaction were obtained from carbon and nitrogen K-edge NEXAFS spectroscopy showing amide-specific π*NHCO resonances at photon energies of 288.3 and 401.4 eV as well.40,41,53,55 Further evidence of the derivatization reaction was found in the C Kedge spectrum of TFEA derivatized P946 particles where a new σCF * resonance occurs at hν ≈ 295 eV and in the O K-edge NEXAFS spectrum of P946 microparticles, here the O 1s(OH) →π*CO transition at 534.5 eV diminishes almost completely for TFEA-derivatized P946 due to amide formation. All these changes related to amide formation are illustrated in the C, N, and O K-edge NEXAFS spectra of P946 microparticles before and after TFEA derivatization shown in Figure 8. Additionally NEXAFS difference spectra were included which are helpful to find even small changes of specific transitions caused by chemical reactions. From our XPS, NEXAFS, and ToF-SIMS results it can be concluded that TFEA is a specific reagent for chemical derivatization and semiquantitative estimation of CO2H groups

Figure 7. Secondary ion yield ratio ICH3O−/IC3H3O2− (on a logarithmic scale) taken from normalized negative static ToF-SIMS spectra of microparticles P0, P35, P99, and P946 and a reference PAA film.

These values are in good agreement with typically achieved polymer film thicknesses of 5−10 nm obtained on planar substrates under comparable polymerization conditions as demonstrated with interferometry and ellipsometry. For P946 particles, the 4 nm thick PAA layer corresponds to an equivalent planar film thickness of about 6 nm. The film thicknesses discussed here are only valid for dried microparticles; in solution the swollen PAA layer on the PMMA particles can have a thickness of some microns. Characterization of Derivatized PAA-Coated Microparticles. Chemical Derivatization XPS (CD-XPS). The determination of the amount of CO2H groups on PAA-coated microparticles intended for binding of (bio)molecules with an amine linker is important for the understanding and optimization of such coupling processes that are routinely used in many (bio)medical and diagnostic applications.1,47 A direct identification and quantification of reactive groups by XPS is often hampered by the chemical complexity of the analyzed surfaces. Selective functional group assignments can be achieved by labeling surface moieties with unique chemical groups, easily identified, which contain a specific marker element not present in the studied surface and material. This procedure is called chemical derivatization XPS (CD-XPS).48,49 After the derivatization reaction, the concentration of this new element obtained by XPS analysis is directly related to the concentration of the particular functional group. A high yield of the derivatization reaction and a high specificity are important factors for its application in surface analysis. Often, a quantitative labeling reaction (100% coupling yield) is assumed although the coupling yields of even many common

Table 2. Relative Component Peak Areas (%) from the C 1s Core Level Spectra of PAA-Coated Microparticles P35, P99, and P946 after Derivatization with TFEA

a

sample

CC/CH (1) [285.0 eV]

CH−CO (2) [285.8 eV]

CH2CF3 (3)a [286.7−286.9 eV]

N−CO (4)b [287.8−288.2 eV]

O−COc [288.8−288.9 eV]

CF3 (5) [292.7−292.9 eV]

P35 TFEA P99 TFEA P946 TFEA

40.8 42.1 43.1

19.9 12.6 12.8

14.9 17.9 13.3

8.1 8.8 15.1

11.7 11.6 5.8

4.6 8.1 9.9

Including CH3O from PMMA. bIncluding contributions from PNVP’s amide moiety. cCO group from PMMA core or unreacted PAA. 20400



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Figure 8. Comparison of C (top), O (middle), and N (bottom) Kedge NEXAFS (difference) spectra of P946 and TFEA derivatized P946 microparticles.

and CF3 signals in the respective XP spectra. Obviously, the carboxylic acid related H−O−C component peak at ∼533.4 eV decreases with increasing amounts of surface-grafted PAA due to amide bond formation (for more details see Table S4 and Figures S8−S10, Supporting Information ). This observation leads to the question what the outcome of a quantitative evaluation of the measured XPS data will be. The fraction of TFEA-labeled CO2H groups was quantified by XPS using the quantitative elemental analysis (QEA) approach,24 comparing the integrated peak areas of carbon and fluorine atoms obtained from XPS survey scan spectra (for details about the calculations see the Experimental Section). Based on this approach derivatization yields of 32% for P35, 54% for P99, and 47% for P946 (see Table 3) can be estimated, which means that at maximum, i.e., with P99 or P946 particles, approximately half of all CO2H groups in the surface-grafted PAA shell have been converted into the respective amide. Yields of 25% for P35, 44% for P99, and 52% for P946 were obtained from the ratio of measured peak areas of CF3 component in C 1s core-level spectra to the theoretical CF3 fraction of 20% expected for a quantitative derivatization reaction (calculated from the stoichiometry of poly(N-(2,2,2-trifluoroethyl)acrylamide), cf. Scheme 1, for more details see the Experimental Section). The results of both quantification methods are summarized in Table 3. The increasing derivatization yields from P35 to P99 and P946 microparticles can be understood by a PAA film thickness that is much smaller than the XPS information depth z95 (approximately 7−10 nm), thus, the amount of carbon used for quantification of accessible CO2H groups is overrepresented due to additional contributions from the PMMA core (cf. Scheme 1 and eqs 7 and 9).56 Additionally, an incomplete derivatization reaction within the information depth has to be considered as reported earlier for planar polymer surfaces.24,57−59 Overall, the TFEA coupling yields are significantly higher than those obtained for derivatization with, e.g., a larger FLA.14,28 In the case of the derivatization with TFEA, the coupling yield obviously reaches nearly 50%, a number that also has been suggested as the maximum reaction yield achievable in homogeneously dispersed PAA hydrogels.60 This is due to the formation of an intermediate anhydride between adjacent CO2H groups in EDC-activated coupling reactions which limits the reaction yield.60 The high coupling yield measured for TFEA in the derivatization reaction with PAA-coated microparticles is probably facilitated by the smaller molecule size of TFEA in comparison to the previously used FL-A.14 Another likely contribution originates from the much lower pKa of TFEA’s amino group (pKa = 5.7, see ref 61) compared to aliphatic amines (pKa ≈ 10). Because it is the basic free amine that reacts with the EDC-activated carboxylic acid,47 when a reaction buffer with pH 5.0 is used this leads to an increase in the concentration of reactive species by around 4 orders of magnitude.

on particle surfaces used for biomolecule immobilizations utilizing EDC-based coupling chemistry. Quantification of Accessible CO2H Groups in the PAA Shell. Because our TFEA derivatization experiments were done with PMMA microparticles containing different amounts of surface-grafted PAA (cf. Table 1), it is attractive to search for correlations to TFEA derivatization related nitrogen, fluorine,

CONCLUSIONS We investigated microparticles consisting of a poly(methyl methacrylate) (PMMA) core coated with poly(acrylic acid) (PAA) shells of varying thickness and concentration of CO2H groups by SEM, XPS, ToF-SIMS, and NEXAFS spectroscopy that present typical beads used for bead-based assays or as a platform for DNA sequencing. The challenges associated with

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Table 3. TFEA Coupling Yields for PAA-Coated Microparticles Estimated from XPS Dataa sample

[CO2H]b [μmol/g]

Fc [atom %]

xCO2H [%]

yield (QEA) [%]

CF3,exp.c [area %]

yield (CF3) [%]

[CF3]d [μmol/g]

P35 TFEA P99 TFEA P946 TFEA

35 99 946

15.8 20.8 19.9

10.6 17.9 15.6

32 54 47

5.1 8.7 10.4

25 44 52

11 53 443

a

For details concerning the calculations please refer to the Results and Experimental Section. bTotal amount of CO2H groups in the PAA shell as determined by conductometry (uncertainty ca. 9%, see ref 14). cRecalculated values excluding PNVP contributions, assuming a constant amount of PNVP in the particles before and after TFEA derivatization (cf. Table S4, SI). dTotal amount of CF3 groups based on calculated XPS yields (QEA).

Special thanks are due A. Shard for helpful discussions and his invaluable help with shell thickness calculations. We thank HZB for the allocation of synchrotron radiation beamtime at the HESGM beamline. M.H. is grateful for financial support by BAM trough the Adolf-Martens fellowship program and by the Austrian Science Found (FWF) through the Erwin-Schrödinger fellowship program (project no. J 3471-N28). This work is funded by the European Union through the European Metrology Research Program (EMRP). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.

these measurements arise from the rather similar chemistry of core and shell materials. With this combination of analytical characterization methods, information about the topography, morphology, size, elemental, and detailed chemical composition could be obtained. SEM of these microspheres verified a very homogeneous particle size distribution (center at approximately 6 μm) and surface morphology for microparticles grafted with different amounts of PAA (0, 35, 99, and 946 μmol/g for P0, P35, P99, and P946, respectively) as well as different PAA shell thicknesses. High-resolution XPS, C and O K-edge NEXAFS, and ToF-SIMS data demonstrated a rather similar chemical surface composition of P946 microparticles and a neat PAA film, whereas particles grafted with lower amounts of PAA like P35 display spectral signatures more comparable to PMMA. This is related to the method-dependent information depth and different PAA shell thicknesses on the particles. Additionally, chemical derivatization with 2,2,2-trifluoroethylamine (TFEA) demonstrated the accessibility of carboxylic acid groups in the PAA shell for EDC-mediated amide bond formation as revealed by subsequent detection of the CF3 marker moieties by XPS, NEXAFS, and ToF SIMS. In our case, about 50% of the CO2H groups in the nanometer scaled shell of such PAA-coated microparticles could be labeled with TFEA as calculated from XPS data. These results agree well with a proposed reaction yield limit of 50% that is achievable in EDCactivated coupling reactions in homogeneously dispersed PAA hydrogels. This underlines the potential of small probe molecules for surface group analysis and also the need to determine the coupling yield for any quantification method, relying on covalent labeling schemes.

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ASSOCIATED CONTENT

S Supporting Information *

Further SEM micrographs, XPS, and NEXAFS data. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

* Tel: +49-30-8104-3533. Fax: +49-30-8104-1827. E-mail: paul. [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank D. Treu for operation of the XPS instrument, S. Benemann for high-resolution SEM imaging of core−shell samples, and S. Rades for her introduction to ImageJ software. 20402



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Surface Analytical Study of Poly(acrylic acid)-Grafted Microparticles

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