Fluorescent Nanoclays: Covalent Functionalization with Amine

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Fluorescent Nanoclays - Covalent Functionalization with Amine Reactive Dyes from Different Fluorophore Classes and Surface Group Quantification Tom Felbeck, Katrin Hoffmann, Ute Resch-Genger, Marina Lezhnina, and Ulrich H. Kynast J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b01482 • Publication Date (Web): 11 May 2015 Downloaded from http://pubs.acs.org on May 12, 2015

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The Journal of Physical Chemistry

Fluorescent Nanoclays – Covalent Functionalization with Amine Reactive Dyes from Different Fluorophore Classes and Surface Group Quantification

Tom Felbeck,†,‡ Katrin Hoffmann,† Ute Resch-Genger*,† † BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Str. 11, 12489 Berlin, Germany, Email: [email protected] Marina M. Lezhnina,‡ Ulrich H. Kynast‡ ‡ Münster University of Applied Sciences, Stegerwald-Str. 39, 48565 Steinfurt, Germany Received date xx.xx.xxxx

ABSTRACT

The ever increasing applications of fluorescence techniques in conjunction with the interest in enhanced detection sensitivities in bioanalysis, biosensing, and bioimaging are closely linked to the rational design of novel non-toxic fluorescent nanomaterials with improved brightness and stability that can be reproducibly synthesized from inexpensive starting materials in simple one-pot reactions and easily surface functionalized. This encouraged us to investigate the potential of the commercially available water-dispersible nanoclay Laponite® RD with the empirical formula Na0.7(H2O)n{(Li0.3Mg5.5)[Si8O20(OH)4]}, forming 25 nm-sized disk-shaped particles, as nanocarriers for different fluorophores. The Si-OH functions at the rims of these 1 ACS Paragon Plus Environment


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disks can be selectively grafted with 3-aminopropyldimethylethoxysilane (APES), thereby enabling subsequent coupling to amine-reactive molecules ranging from target-specific organic ligands and biomolecules to amine-reactive fluorescent labels. Here, we present different strategies for the surface functionalization of nanoclays and the subsequent quantification of the density of synthetically introduced surface amino groups exploiting analytical methods which rely on different detection schemes including, elemental analysis, colorimetric assays and fluorophore labeling strategies. In this respect, we systematically assess the potential of negatively and positively charged, neutral, and zwitterionic dyes to act as fluorescent labels for amino functionalities at the surface of negatively charged nanoclays. Our studies underline the strong influence of dye charge and aggregation tendency on the brightness of the bound dyes and on surface group quantification. Best results regarding surface group analysis and coupling yield were obtained for a neutral dansyl derivative and fluorescamine.

INTRODUCTION Commercial

nanoclays

like

the

disk-shaped

laponite

Na0.7(H2O)n{(Li0.3Mg5.5)[Si8O20(OH)4]} present an inexpensive and very versatile platform for tailor-made water-dispersible fluorescent hybrid materials. Loading of nanoclays with positively charged compounds can be easily achieved via cation exchange, thereby substituting interlayer Na+ by quaternary amines or cationic dyes.1, 2 This adsorption-driven loading circumvents tedious dye functionalization as required for the preparation of many other fluorescent nanomaterials like the majority of fluorophore-doped silica nanoparticles.3, 4, 5, 6, 7

The potential of laponite as carrier for water insoluble neutral organic dyes like indigo,

nile red, coumarin 153, and the perylene Lumogen F Red 305 was demonstrated only recently.8, 9 The prevention of dye dimerization and aggregation in the interlayer galleries of the clay sheets constitutes a bottleneck for this approach, as this can favors self-quenching for 2 ACS Paragon Plus Environment

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the vast majority of dyes except for fluorophores like pyrene that form emissive excimers.10, 11 Except for fluorophores that form emissive excimers or J-aggregates, dye-dye interactions typically result either in a loss in brightness due to a decrease in fluorescence quantum yield as well as in a reduction in the quantum yield for singlet oxygen generation which is disadvantageous for signaling and bioimaging applications and photodynamic therapy.12,

13

An alternative route for laponite modification and the design of fluorescent nanoclays presents the covalent attachment of functional fluorophores, bearing reactive groups to derivatizable surface groups of the laponite, utilizing either naturally occurring OHfunctionalities or chemical linker groups previously grafted onto the laponite surface. This broadens the choice of applicable fluorophores and may enable a better control of chromophore location. For example, Wheeler et al. showed that laponite modification with ethoxysilanes occurred only at the rim of the nanoclay.14 This was confirmed by Bujdak et al. who bound fluorescent ethoxysilanes to laponite surfaces.15, 16 This functionalization strategy should enable to selectively modify two different sites, i.e., the rim and the layer surface of the laponite platelets, similar to the controlled functionalization of external and internal surfaces of zeolite L.13,

17

Only recently, Wu et al. demonstrated a modification of both

laponite sites with interlayer-loaded doxorubicin and rim functionalization with folic acid via an amine linker.18 The amine functionality must not necessarily be attached to the laponite rim, but can become even part of the tetrahedral silica layer of the clay backbone as shown by in situ aminoclay synthesis approaches of Datta et al.19 For the straightforward surface modification of laponite with different types of functional molecules like fluorophores and target-specific ligands such as biomolecules, we reacted the laponite rim with 3-aminopropyldimethylethoxysilane (APES). For the analysis of the amino groups, we chose elemental analysis and different optical assays. For this purpose, we developed different dye labeling schemes using fluorophores of varying charge, size, and aggregation tendency in order to systematically assess the number of truly derivatizable 3 ACS Paragon Plus Environment


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amino surface groups. Thereby, we also addressed the eventually desired rational design of fluorescent laponite using amino-functionalized laponite (L–NH2) as nanocarriers for fluorophores. Fluorescent reporters employed included a small, neutral, and solvatochromic dansyl fluorophore20, 21, 22, 23, 24 widely used as fluorescent probe for environment polarity, and neutral fluorescamine (also termed Fluram), which becomes emissive only upon coupling to primary amines.25, 26 In addition, three red emissive amine-reactive dyes of different charge, commonly applied as labels for proteins and the design of dye-bioconjugates were studied: the NHS esters of the negatively charged hemicyanine DY681,27 the zwitterionic BODIPY 581/591 (BODIPY)28 and the positively charged pyrylium reporter Chromeo P503 (Py-1).29, 30, 31

The latter transforms into a spectroscopically clearly distinguishable pyridinium dye

upon binding to primary amines (see Figure 1). These dyes differ also in their aggregation tendency, with planar dyes like cyanines and BODIPYs, which mostly reveal an emission from a locally excited (LE) state, showing an enhanced tendency to the formation of nonemissive H-type dimers compared to charge transfer (CT)-operated fluorophores like dansyl derivatives that are barely prone to such affects.32

Figure 1. Schematic presentation of the different fluorescent labels covalently linked to the rim of laponite disks via 3-aminopropyldimethylsilane.

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EXPERIMENTAL SECTION Chemicals Laponite® RD clay Na0.7(H2O)n{(Li0.3Mg5.5)[Si8O20(OH)4]} supplied from former Rockwood Additives (now belonging to BYK Additives & Instruments) was used as received.33 The solvents dimethyl sulfoxide (DMSO; purity 99.9%), dimethylformamide (DMF; purity > 99.8%), propylamine (purity > 99%), and ethanol (purity > 99.9%) purchased from Sigma Aldrich and Merck, respectively, were applied as received. Toluene p.a. supplied by Sigma Aldrich was dried with a 4 Å molecular sieve for one week before use. The linker 3–aminopropyldimethylethoxysilane (APES) made by ABCR with a purity of 97 % was used as received. The reactive dyes fluorescamine (Sigma Aldrich), Chromeo P503 (Active Motif), and the NHS esters of dansyl (dansyl-X SE; AnaSpec Inc.), DY681 (Dyomics), and BODIPY 581/591 (Life Technologies, former Invitrogen) were used without further purification. Ninhydrin (for analysis) was purchased from Merck and anhydrous sodium acetate (pure) from AppliChem, respectively.

Preparation

Attachment of the amine linker molecule (grafting). The nanoclay was dried for half an hour at 100 °C under vacuum. For subsequent grafting with the APES linker, the nanoclay (~10 g) was dispersed in dried toluene (50 ml) and different quantities of APES, i.e., 53 µmol, 79 µmol, 106 µmol, 132 µmol, 159 µmol, and 1590 µmol APES per gram nanoclay, respectively, were added. The reaction mixtures were then heated for 6 hours at 100 °C under a steady flow of nitrogen. The resulting amino-surface functionalized laponite samples (1 L–



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NH2 to 6 L–NH2) were washed three times with toluene and twice with ethanol and dried in vacuum for 18 h at 60°C.

Fluorophore labeling. Labeling of the amino-modified laponite samples with five different reactive dyes (see Figure 1) was performed by addition of an excess of the reactive dye (0.3 µmol) to a dispersion of about 1 mg of L–NH2 in 1 ml of dry DMF or DMSO at room temperature. After a reaction time of two hours with occasional shaking, the powders were washed three times with DMF or DMSO. After each washing step, the dye-labeled laponite was separated by centrifugation (Eppendorf centrifuge 5415D; 10 000 G, 15 min.) from the washing solution containing unreacted dye. The fluorophore-functionalized nanoclay particles were dried, redispersed in water, and subsequently analyzed and spectroscopically studied. For each dye-laponite system, control samples or reference systems of laponite without amine functionalization were prepared and analogously treated as the amino-functionalized nanoclay, thereby addressing possible dye adsorption.

Preparation of reference systems. Two reference systems were studied, first the dye dissolved in water-solvent mixtures and secondly, the same system in the presence of the unmodified laponite particles (see Figure 4) lacking amino surface groups. For these experiments, in the case of fluorescamine, 50 to 450 µl of a 0.355 mmol L-1 stock solution of the dye (in DMF) and propylamine (in ethanol; to transform fluorescamine into its fluorescent derivative) in a 1:1 ratio were used. The different solvent volumes added (50 to 450 µl) were adjusted to the same final volume of 500 µl by addition of the accordingly required amounts of DMF and ethanol (450 to 50 µl). Then, the dye solution was diluted with water or the dispersion of the unmodified laponite (0.05 weight-% (wt-%)) to yield a total volume of 2.5 ml. This gave concentrations of 7 – 64 µmol L-1. Analogously, we prepared, control samples for the BODIPY label in DMF-H2O (3 vol-%) and in DMF-H2O (3 vol-%) and for the dansyl NHS ester in DMSO-H2O (12 vol-%) using unmodified laponite (0.05 wt-%), resulting in BODIPY concentrations of 0.05 – 10 µmol L-1 and dansyl concentrations of 4 – 80 µmol L-1, 6 ACS Paragon Plus Environment

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respectively. In the case of dansyl, additionally, also the interaction of dansyl NHS ester with 1 L–NH2 to 5 L–NH2 (0.05 wt-%) was studied in DMSO-H2O (12 vol-%) using dansyl concentrations of 4 – 80 µmol L-1. The solvent content was varied due to dispersion stability issues. Characterization methods

Spectroscopic measurements. The absorption spectra of the dye solutions and laponite dispersions were recorded with the calibrated photometer Omega 10 from Bruins Instruments. To account for the very slight scattering of the laponite dispersions at shorter wavelengths, a baseline correction was always performed with an aqueous dispersion of nonfunctionalized laponite of matching particle concentration. The emission spectra of the fluorophore-modified laponite were measured with a LS50B spectrofluorometer from Perkin Elmer in a conventional 0°/90° measurement geometry. All fluorescence spectra are blank corrected (correction for absorption, scattering, and autofluorescence of the solvent and dark counts at the photomultiplier), yet not corrected for the instrument-specific wavelength-dependent spectral responsivity of the fluorometer (no spectral correction).34, 35 The absorption-weighted integral emission (Figure 6) was determined from blank corrected, spectrally non corrected emission spectra (integration over the complete emisison band) as measure for fluorescence efficiency.

Elemental carbon analysis. The carbon contents of the dried amino-modified laponite and fluorescamine conjugates were measured in threefold determinations (uncertainty of 9 %) with a carbon sulfur analyzer (CS–800 by ELTRA) determining the infrared absorption of the combustion gas (CO2). For this purpose, about 10 mg of the sample were mixed in a fresh crucible with 0.5 g of iron and 1.5 g of tungsten and heated above 2000 °C in an induction furnace under a pure oxygen atmosphere. An iron reference material with 3.734% carbon was used for the carbon determination. The values were corrected for the carbon content of 7 ACS Paragon Plus Environment


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laponite blank samples treated with the same solvents as the L–NH2 samples, but not silanegrafted.

Determination of amino groups with ninhydrin. A 4 mol L-1 sodium acetate solution, adjusted to pH 5.3 with concentrated hydrochloric acid, was mixed with a 0.1 mol L-1 solution of ninhydrin in DMSO in a ratio of 3:1. A calibration curve was generated with lysine dissolved in an acetate buffer. The lysine concentrations were varied between 0.01 and 0.05 g L-1. 1 mL of the amine containing sample was mixed with 1 mL of ninhydrin solution and heated in a water bath at 95 °C for 15 min. Subsequently, it was cooled in an iced bath and 5 ml of ethanol (50 vol-%) was added. In the case of L–NH2 containing samples, the dispersion was centrifuged (10 000 G, 15 min) and the absorbance of the supernatant was measured. All measurements were performed in duplicate.

RESULTS AND DISCUSSION In the following, we present the quantification of amino groups grafted onto laponite surfaces with different fluorescent reporters and other analytical methods and, subsequently, a study of the spectroscopic properties of the dye labeled amino-laponite, thereby critically assessing the different quantification strategies and dye classes used for labeling. Surface functionalization and quantification

Amino

functionalization

and

quantification

with

elemental

analysis.

Amino

functionalization was achieved by reaction of laponite with ethoxysilane linker molecules like APES.14, 36, 37 This surface modification occurs at the broken edges of the clay and less likely, at the octahedral Mg–OH groups in the sheets.1,

14

Measurements of the carbon content

(uncertainty of 9 %) of the samples (1 L–NH2 to 5 L–NH2) revealed the following amounts of edge-surface grafting: 57 µmol, 81 µmol, 99 µmol, 114 µmol, and 137 µmol aminopropyldimethylsilane per gram nanoclay, respectively, see also Figure 2 (black 8 ACS Paragon Plus Environment

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columns). Assuming that the laponite consists of 5.2x1017 disks g-1 (using a laponite density 2.58 g/cm3, a diameter of 25 nm, and a thickness of hydrated smectites of 1.5 nm) and that the silanization reaction occurs exclusively at the rim of the clay sheets (circumference area of 78.5 nm2 with a laponite diameter of 25 nm and height of 1 nm), this equals 0.84 to 2.02 –NH2 groups nm-2. To determine the maximum number of reactive Si-OH groups, we additionally prepared one sample, i.e., sample 6 L–NH2, with a large excess of APES. This yields a grafting maximum of 363 µmol/g, equaling 420 reactive –OH groups per clay particle (about 5 –NH2 groups per nm-2). This number correlates with the average size of the laponite particles and the predicted local modification at the rim (the modification of the sheet –OH groups assuming about 1000 unit cells per particle would give about 10 times higher grafting rates) and agrees very well with the value of 0.36 mequiv g-1 (360 µmol g-1) published by Herrera et al. for the reaction of (3-methacryloxypropyl)-dimethyl-ethoxysilane with laponite in toluene.37 To distinguish between the two different surface regions, i.e. rim and sheet, we subsequently refer to the negatively charged siloxane layer of laponite as “sheet surface”.

Figure 2. Analysis of the carbon content of APES-modified nanoclays (black columns) and the corresponding fluorescamine labeled nanoclays (grey columns) to determine the degree of surface modification and the labeling density of amino groups. In the case of covalent attachment of the reporter fluorescamine, the carbon content of the corresponding L–NH2 sample was always subtracted to provide only the number of laponite-bound dye molecules. 9 ACS Paragon Plus Environment


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Dispersibility and colloidal stability. Amino functionalization of the laponite yielded nanoclay dispersions, which appeared to be transparent for the eye. Absorption measurements, however, revealed a slight increase in optical scattering with increasing surface modification. The sample with the highest amino functionalization density, 6 L–NH2, lost its ability to form a stable transparent aqueous dispersion. This is possibly due to increased interactions of protonated amine functionalities with the negatively charged laponite sheet surface (edge-to-face association) and the loss of hydroxyl groups at the particle rim. Subsequently, for our spectroscopic studies, we exclusively focused on transparent dispersions and studied only low to intermediate amino group densities.

Photometric determination of amino groups with ninhydrin. Quantification of amino groups was performed with the well-established amino reporter ninhydrin by measuring the absorbance of the reaction product (Ruhemann`s purple) in solution after separation of the laponite particles via centrifugation. As this reaction product is negatively charged, undesirable adsorptive interactions with the nanoclay seemed unlikely. Nevertheless, as the reaction of ninhydrin with amino groups to Ruhemann`s purple is complex, involving at least seven intermediates and the corresponding equilibria, possible interactions of one or several of these intermediates with the laponite may still lead to interferences.38 Reaction of the samples 1 L–NH2 to 4 L–NH2 with ninhydrin revealed 29 µmol, 36 µmol, 37 µmol, and 52 µmol aminopropyldimethylsilane per gram nanoclay, respectively. Although these values show a linear trend, the amino labeling densities of the surface-functionalized laponite particles seems to be generally underestimated with this method as follows from a comparison with the functionalization densities derived from previously presented elemental analysis. These systematic deviations are ascribed to complex interactions between the highly charged laponite and intermediates or byproducts38 formed during the reaction of ninhydrin


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with the surface amino groups. Alternatively, some of the amino functionalities may not be as reactive or less accessible as others, which seems to be, however, less likely.

Fluorophore labeling. Subsequently, we studied and compared the differently aminofunctionalized laponite labeled with various fluorophores of different charge and aggregation tendency. Except for the nanoclay modified with the positively charged pyrylium dye Chromeo P503, every fluorophore-labeled laponite system could be redispersed in water, yielding colloidally stable and colored dispersions. For this positively charged dye, even the addition of small amounts of the reactive fluorophore to an aqueous amino-modified laponite dispersion led to immediate particle precipitation. This is attributed to the interaction of positively charged dye-conjugated laponite particles with negatively charged laponite particles resulting in the formation of a non-dispersible 3D network. Increased particle-dye interactions were also reported by Bujdak et al. for a positively charged rhodamine dye covalently bound to laponite.15 In the case of the negatively charged DY681 NHS ester, carrying two sulfonate groups, we did not observe dye conjugation to the nanoclay, even at a high excess of the fluorophore. This is ascribed to electrostatic repulsion between the negatively charged laponite particles and the negatively charged dye molecules. First screening experiments with a fluorescein NHS ester carrying a single carboxylate group, which are not detailed here, however, suggest successful dye binding despite its negative charge. This suggests that it is principally possible to covalently attach a negatively charged fluorophore, i.e., a dye containing only a single negatively charged group, to an aminomodified laponite, also this was not systematically assessed in our study. Fluorescamine as well as the neutral dansyl and the zwitterionic BODIPY reporter could be successfully attached to amino-modified laponite and yielded stable dispersions of fluorescent nanoclays. In the following, we focus on these systems.

Quantification via fluorophore labeling. As the absorption and fluorescence properties of each label can be affected by its microenvironment in a dye-class specific manner, for the 11 ACS Paragon Plus Environment


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photometric quantification of amine functionalities via dye labeling, knowledge of the molar absorption coefficient (ε) of the bound reporter in its specific environment is mandatory. For fluorometric quantification, also its fluorescence quantum yield (Φ f) must be known. This approach can be additionally hampered by dye aggregation and closely related selfquenching. Moreover, for all labeling reactions, special care must be taken to discriminate bound reporter molecules from merely adsorbed reporters by stringent purification steps involving several washing/centrifugation cycles for particle systems like our laponite. In this respect, the use of control samples, modeling adsorption, can be very beneficial for the quantification of surface groups at particles as pursued by us. In addition, the coupling yield of the reporter to the surface groups at the respective particle, here the laponite, should be known as this yield directly affects quantification. This coupling yield is often assumed to be 100 % (quantitative labeling). This can be, however, rather erroneous, as recently shown by us for carboxylated polymer beads labeled with a fluorescein derivative.39 The mandatory determination of coupling yields requires the use of an additional analytical method for the determination of the total amount of surface groups. This was done here with elemental analysis, see Figure 2.

Figure 3 Schematic representation of free dyes in solution without laponite (a), dyes (dyead) adsorbed onto laponite (b), and dyes (dyecon) conjugated to laponite (c).

For the calibration of our spectroscopic measurements, we used model dispersions of unmodified laponite containing varying amounts of the respective fluorophores and the same amount of organic solvent as used in the redispersion step of the samples. This enabled a 12 ACS Paragon Plus Environment

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comparison of the absorption and fluorescence spectra of the laponite-dye conjugates (dyecon, schematically illustrated in Figure 3c) with the spectroscopic properties of merely adsorbed dyes (dyead, depicted in Figure 3b). With this procedure, a close match between the absorption and emission spectra of the calibrand dyead essential for a reasonable spectroscopic quantification could be achieved. This provided also the environment-sensitive molar absorption coefficients and fluorescence quantum yields of the covalently conjugated reporters dyecon and allowed consideration of the slight scattering introduced by the laponite particles. Our assumption that dyecon and dyead of each reporter possess relatively similar absorption coefficients and fluorescence quantum yields is supported by the rather close match between the spectral features of both species, see also section Spectroscopic studies of

dye-labeled amino-functionalized laponite and Figure 5. Linear calibration curves obtained photometrically (via absorption (abs.) data) and fluorometrically (via emission (em.) data) for the dyead samples were obtained without further corrections. The results of the spectroscopic quantification of the number of dyes coupled to the L–NH2 samples (dyecon) are summarized in Table 1. Table 1 contains also the data derived from elemental analyses of the respective samples (see also Figure 2, black columns). This allowed us to determine coupling yields for our reporter dyes as well. In addition, exemplarily for fluorescamine, we determined the number of laponite-bound dye molecules with elemental analysis (see Table 1 and Figure 2).

Table 1 Number of amino surface groups at modified laponite samples (1 L–NH2 to 6 L– NH2) as determined by elemental analysis (C-det.) and number of coupled dye molecules per disk (average number) determined by absorption (abs.) and emission (em.) measurements. Adsorbed dyes on unmodified laponite (Figure 3b) were used for the calibration of the optical measurements.



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Number of coupled fluorophores (dyecon) per diska [disk-1]

–NH2 Sample

per diska,b -1

[disk ] 1 L–NH2 2 L–NH2

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Dansyl Abs. Em.

Fluorescamine b

C-Det.

Abs. Em.

BODIPY

DY681

Abs. Em.

Abs. Em.

3 L–NH2 4 L–NH2

66 94 115 132

28 39 57 64

23 48 67 74

20 28 29 33

23 35 42 47

2 7 3 4

5 7 7 10

2 3 3 3

Fluorescent Nanoclays: Covalent Functionalization with Amine

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