Synthesis and optical properties of covalently bound Nile Red in mesoporous silica hybrids – comparison of dye distribution of materials prepared by facile grafting and by co-condensation routes

Institut für Organische Chemie Heinrich-Heine-Universität Düsseldorf, Un Germany. E-mail: ThomasJJ.Mueller@unidu Institut für Anorganische Chemie und Str Düsseldorf, Universitätsstraße 1, D-40225 D Institut für Organische Chemie, Universitä Stuttgart, Germany † Electronic supplementary informa characterization of hybrid materials 6, TEM), spectroscopic characterization of p 7 (UV/Vis and uorescence), red-edge exc 7e, uorescence quenching of hybrid 10.1039/c5ra22736d Cite this: RSC Adv., 2016, 6, 6209


Introduction
Aer the discovery of pure inorganic mesoporous silica materials in 1992, research on mesoporous composite materials modied by organic molecules quickly evolved. [1][2][3] By chemical and physical manipulation of their pore systems, the properties of rigid silica and various functional molecules can be combined and lead to unique features of the hybrid materials in comparison to the individual components. As a consequence these novel composite materials open novel applications in various topical elds of research, such as catalysis, 4 optical sensing, 5,6 solid state lasers, 7 and drug delivery. [8][9][10] For sensing applications mesoporous silica hybrids are particularly advantageous due to their pore structure (e.g. MCM-41) enabling facile mass transport at high rates, which is essential for quick response times to environmental changes. Furthermore, the shape and size of silica particles can be controlled by varying the synthetic conditions, thus rendering them also favorable for medicinal applications, such as drug delivery or biolabeling. 11 Special sensitivity can be additionally introduced by modication of the silica surface with biochemical functionalities, such as antibodies. 9 The simplest route of doping organic dyes into sol-gel matrices is the adsorption and entrapping of the chromophores during their synthesis. However, plain adsorption inevitably causes leakage and migration of the dyes, although this problem can be circumvented by covalent ligation of the dye to free silanol groups in the silica framework. In this manner, a dye containing a reactive functionality can be graed onto the silanol groups of the silica matrix in the sense of a postsynthetic functionalization. Alternatively and quite elegantly in a highly convergent fashion, a dye with pending trialkoxysilyl functionality can be covalently anchored in the mesoporous structure in statu nascendi (co-condensation). 12 Yet, the determination of the homogeneity of the dye distribution, especially at low dye loading proved to be difficult and only little research has been dedicated to the comparison of dye distribution of hybrid materials synthesized by post graing methods and one-pot synthesis. Especially there is barely any comparison between the two most feasible synthesis methods of hybrid materials with similar structural properties, i.e. graing onto commercially available mesoporous silica and co-condensation. [13][14][15] Taking advantage of the inherent chemical, thermal and dimensional stability of silica hybrid materials various uorescent dyes have already been incorporated into mesoporous silica by adsorption or covalent ligation, thus enhancing the photostability of the organic components. 16 advantageously a co-condensation strategy could give rise to a homogenous distribution, thereby avoiding aggregationinduced self-quenching of the dye molecules. 18,19 A typical uorescence dye displaying emission selfquenching in the solid state is Nile Red (NR). NR possesses high uorescence quantum yields in lipids and unpolar solvents and is widely used in biological applications as a lipophilic stain or a laser dye. [20][21][22] Moreover, the pronounced emission solvatochromicity allows its use as polarity sensor in cellular environments. 23,24 Unfortunately, NR's water insolubility restricts its application to lipids and highly hydrophobic micro-environments. But still since NR's uorescence does not interfere with the cellular autouorescence (typically below 550 nm), NR could be an ideal probe for biolabeling and thus recent work is dedicated to water-soluble, but still luminescent NR derivatives. [25][26][27] As a consequence a new class of luminescent stains for intracellular imaging could evolve.
Upon incorporation of NR into mesoporous silica, aggregation-induced self-quenching in aqueous media should be suppressed, leading to hybrid materials suitable for biotechnological applications. 28,29 In addition, these hybrids could be ligated to biomolecules by decorative functionalization of the outer surface of silica particles. 9 Finally, these NR-silica hybrids should be still luminescent in the solid sol state. Potential applications can be envisioned in solid state dye lasers where the luminescent properties of the dye are concatenated with the inherent stability of the silica host structure, concomitantly avoiding toxic solvents and ensuring easier handling with potentially adjustable shape, e.g. as bres. 7,30 Moreover, solid-state uorescent NR hybrids could be employed in security technology as near-infrared solid state dyes. 31 Here we report the synthesis of NR-functionalized hybrid silica by the two most feasible synthetic methods i.e. postsynthetic graing of commercial available MCM-41 and in situ co-condensation. Materials prepared by both routes were studied with respect to homogeneity as well as the effect of applied synthesis route and dye loading of the hybrid. Furthermore, the structural and optical properties of these novel hybrid materials are thoroughly studied and discussed.

Synthesis
For the covalent ligation of NR to the silica materials a side chain with a terminal triethoxysilyl group had to be introduced. Starting from 3-diethylaminophenol (1), via the formation of the nitroso derivative 2, the 2-hydroxy substituted NR derivative 3 was obtained according to a literature procedure (Scheme 1). 32 By Williamson ether synthesis the phenol 3 was transformed into the propargyl ether 4 in 24% yield. Finally, by CuAAC (Cucatalyzed alkyne-azide cycloaddition) 33 of alkyne 4 with (3-azidopropyl)triethoxysilane the required terminal triethoxysilyl group was introduced 34 to give the triethoxysilyl functionalized NR precursor 5 in 45% yield.
The NR silica hybrid materials 6 were synthesized via postsynthetic functionalization of commercially available MCM-41 with the precursor molecule 5 (postsynthetic graing) and NR silica hybrid materials 7 were prepared via in situ cocondensation upon simultaneous formation of the mesoporous structure (in situ synthesis). To exclude dye leakage from the hybrid materials, Soxhlet extraction was performed until no more dye could be detected in the supernatant.
During the postsynthetic graing, the triethoxysilane terminus of precursor 5 reacts with the free silanol groups exposed on the surfaces in the pores. The major advantage of this strategy is retaining the initial pore structure of the silica material. However, the major drawback is the potential nonuniform distribution of the organic molecules and pore clogging at elevated substrate concentrations as a consequence of preferential functionalization at the pore openings.
In contrast, a homogenous distribution can be achieved by the in situ co-condensation approach where tetraethoxysilane (TEOS), the precursor dye molecule, and the templating agent are present in the same pot. Especially in cases where selfquenching of the emission of uorophores can be expected at higher concentrations a homogeneous distribution and dilution is indispensable. Furthermore, it is still possible to obtain materials at higher degrees of loading since pore clogging is not an issue. However, since the dye molecules are immediate components of the silica material, an increase in organic functionalization might cause a decrease in mesoporous order, inevitably causing a total collapse of the structural order at high doping levels (Scheme 2).

Structural characterization
Nitrogen sorption isotherms. Analysis of the hybrid materials by N 2 sorption gives isotherms which are of type IV in the IUPAC classication. These isotherms represent the particular situation of mesoporous materials possessing adsorption inside micropores at low relative pressures followed by multilayer adsorption and by capillary condensation in the pressure region p/p 0 between 0.25 and 0.40 ( Fig. 1 and S1 and S2 in ESI †). 35 Pure MCM-41 exhibits a H4 hysteresis loop in the higher pressure region (p/p 0 ¼ 0.45 to 1) which can be attributed to slit-shaped pores or internal voids of irregular shape. [35][36][37] This behavior is also found for the co-condensed and graed materials but with a less pronounced hysteresis loop. In addition, these materials show a H1 hysteresis loop in the region of the capillary condensation step which can be rationalized with a high degree of pore-size uniformity. 38,39 A further distinction between the co-condensed (7) and graed (6) materials can be made by means of their specic surface areas of $1000 and $800 m 2 g À1 , respectively (Table 1). These differences can be rationalized by the modied synthesis conditions of the co-condensed material relative to the synthesis of pure MCM-41 material. Within the series of cocondensed materials, hybrids 7f-h exhibit slightly lower surface areas and pore volumes than the other samples, as well as higher structural disorder, as can be seen in the hysteresis loop of the capillary condensation step. This could be due to a higher dye content inside the pores as this would cause a decrease in surface area. But it is more likely that this decrease is dependent on the modied synthesis conditions as it is known that the structure of MCM materials can be controlled by the addition of alcohols. [40][41][42] Thus, as in the synthesis of 7f-h a higher amount of methanol was applied due to solubility issues of the precursor molecule, it is very likely that this caused a decrease in the surface area rather than a higher dye content in the micromolar range. For the graed hybrids 6a-i the surface areas range between 748 and 878 m 2 g À1 , whereas neat MCM-41 has a surface area of 806 m 2 g À1 . As the experimental error in BET surface area determination can be AE50 m 2 g À1 the values overlap with their error margins. So functionalized materials 6a-i do not differ much from neat silica, indicating that dye doping with 0.6-23 mmol g À1 does not lead to a significant variation of the surface area and pore volume.
The pore size distributions of the hybrid materials were obtained by DFT calculations from the N 2 isotherms (Fig. 2, S3 and S4 in ESI †). These calculated distributions are bimodal and relatively narrow around diameters of ca. 1.5 and 2.8 nm for MCM-41 and its co-condensed and graed hybrids. Noteworthy, the volume fraction of the smaller pores around 1.5 nm and of pores larger than 3 nm has decreased in 6 and especially 7 compared to neat MCM-41. As there is no decrease of the volume fraction for low and high loaded materials, this change is attributed to the dye extraction conditions leading to slightly modied pores.
These results imply that the surface area as well as the pore size distribution is an inherent feature of the employed silica material or rather its synthesis conditions. They are not affected by the loading with varying amounts of dye. The shape of the sorption isotherms and their capillary condensation steps show no distinct change or shi at higher loadings of dye as was the case for organic molecules at concentrations up to 0.77 mmol g À1 incorporated in the MCM-41 type material. 43 Small angle X-ray scattering (SAXS) In order to gain deeper insight into the structure of the hybrid materials the samples were studied by small angle X-ray scattering (SAXS) ( Table 2  1 : O3 : O4 : O7 which is typical for two-dimensional ordered hexagonal mesostructured material possessing P6mm symmetry. 38,44 Thus, no change in the mesostructure can be observed in comparison to neat MCM-41. Furthermore, the post-graed hybrids as well as the neat MCM-41 exhibit lattice planes around 4.0 nm which is almost unchanged compared to the co-condensed hybrid materials. Like in the gas sorption analysis, the co-condensed materials 7g-h show slightly smaller lattice planes as a consequence of the adjusted synthesis conditions.
In a detailed investigation of the lattice parameters for the differently dye loaded materials, no signicant deviation can be observed. All co-condensed materials possess d 100 spacing in a range from 3.88 to 4.03 nm corresponding to lattice parameters ranging from 4.47 to 4.65 nm. In addition, no decrease of the reection intensity is observed which also suggests that the hexagonal structure is not inuenced by the addition of dye. In summary it can be concluded that the applied dye concentrations in a micromolar range do not interfere with the formation of the mesoporous structure as long as the synthesis conditions remain constant.

Transmission electron microscopy (TEM)
In contrast to the gas sorption and SAXS analysis of the hybrid materials where a hexagonal ordering of the silica is deduced from the experimental data, the characterization by transmission electron microscopy (TEM) gives unambiguous evidence for the structural ordering as a direct image of the sample. As shown in Fig. 4, the hexagonal, honeycomb-like ordering of the pores as well as the two-dimensional organization of channels is evident when the sample is viewed in direction of the pores and perpendicular to the pore channels, respectively. The quality of the obtained silica structures are comparable to commercial MCM-41 and show no dependence on the amount of incorporated dye. The assumption of voids inside the hybrid material, as indicated by the hysteresis in the sorption isotherms, can be conrmed in the TEM images. Thus all structural analysis are mutually consistent with each other and conrm the assumed hexagonal mesoporous ordering as well as the absence of an inuence of the dye incorporation (in the micromolar range) on the structural ordering.

Excitation and emission properties
Determination of dye incorporation. As a consequence of low amounts of dye employed in the hybrid synthesis to avoid self-quenching of uorescence, only UV/Vis spectroscopy proved useful for the determination of the effective dye concentration. Therefore, suspensions of the hybrid materials in DMSO were analyzed. But as the loading of dye in the hybrid materials had to be calculated from the UV/Vis spectra via the Lambert-Beer's law, the molar extinction coefficient for the silica matrix-embedded dye had to be determined. However, this cannot be done precisely as there is no reference substance and thus the molar extinction coefficient had to be estimated. For a reasonable estimation the environment of the dye inside the hybrid material was modelled and the molar extinction coefficient of the precursor molecule was determined. Although it cannot completely be ruled out that these calculations are error-prone due to the estimation of the molar extinction coef-cient, the determination of 3 in DMSO should give reasonable estimate as the absorption spectrum of the free precursor molecule is essentially identical to the absorption spectra of the hybrid materials suspended in DMSO. This suggests that the environment of the silica matrix-embedded dye is comparable to pure DMSO. Otherwise the absorption would be signicantly shied caused by changes of the environment polarity. The regression analysis of determined concentrations vs. applied concentrations served for gaining higher accuracy by recalculating the concentrations (Fig. 5, Table 3).
The incorporation of dye into the silica material proceeds more effectively by graing than by co-condensation. The slopes of the regression lines indicate an incorporation of approximately half of the applied dye concentration in the hybrid material obtained by graing, whereas for the co-condensed material only one third of the applied concentration is incorporated. Although a lower concentration of reactive silanol groups can be expected due to calcination of the commercial MCM-41 source a higher degree of incorporation of dye by graing was proven.
As a consequence the lower applied dye concentration is still sufficient for the graed materials, in comparison with the equivalent loaded co-condensed hybrids.

Fluorescence quantum yields
The total uorescence of nine graed (6) and eight cocondensed (7) hybrid materials in the solid state was determined using an integrating sphere (Ulbricht setup). The quantum yields in dependence on the matrix-embedded dye concentration are shown in Fig. 6 and Table 4. Both types of materials, co-condensed as well as graed, reveal a similar dependence of the quantum yield on the concentration.
These materials display uorescence even at very low dye doping levels, such as in the nanomolar range. With increasing dye concentration a steep increase in quantum yield is detected up to a maximum concentration around 2 mmol g À1 . Quantum yields of 18 and 23% for the co-condensed and graed material, respectively, were determined. At higher dye loading the quantum yields decrease even below the quantum yields for the nanomolar dye doping levels. This asymptotic convergence to a completely quenched uorescence very likely arises from the uorescence quenching of NR at higher concentrations, as reported for free NR in the solid state.
Although a similar dependence of the quantum yield on the dye loading can be observed, the maximum quantum yield of the co-condensed material 7 is about 4% less than for the graed material 6. Moreover, the graed hybrids reach a quantum yield of 7% even at high concentrations, whereas the co-condensed materials are nearly quenched (2%). A possible explanation for this behavior could be the weak acidity of the silanol groups (pK a z 7.1), 45 which can protonate the NR molecules, and thereby cause uorescence quenching.
If the functionalization of the co-condensed hybrids 7 can be assumed to take place preferentially inside the pores, the Table 2 Lattice parameters and lattice planes of grafted (6) and co-condensed (7)    uorophore molecules will face an environment with a higher effective concentration of silanol groups in comparison to predominantly outside bound dyes as expected for the graed case 6. Thus, the dye molecules in co-condensed hybrids 7 will experience a higher degree of protonation and quenching of the NR luminophore compared to the graed hybrids 6. 46

Solvatochromism
The solvatochromic properties of the 2-hydroxy-substituted NR derivative 3 are not signicantly affected by functionalization to give the precursor molecule 5. The excitation as well as emission maxima remain almost unchanged, except for the measurement in diethyl ether where the maxima of the precursor molecule 5 are slightly hypsochromically shied relative to the 2-hydroxy substituted NR 3 (Fig. 7). Upon incorporation of the NR derivative 5 into mesoporous silica materials (as studied for hybrid material 7e) the solvatochromic behavior is mostly retained (Fig. 8). However, it was found that the hybrid materials show a red-edge excitation shi (REES) known from polar dyes in viscous solvents. [47][48][49][50] Thus, since it was not possible to obtain absorption spectra in the solid state and, except for DMSO, in suspension, excitation spectra were recorded for the determination of the absorption maxima. But due to the previous discussion it was not possible to correct these excitation spectra for the inner lter effect. It cannot be ruled out that the measured spectroscopic data are slightly error-prone. However, these systematic errors become negligible as emission spectra were always recorded at excitation in the short wavelength part of the excitation spectra where no shi of the emission spectra could be observed (vide infra Fig. 16).
Upon changing the environment of the dye the spectroscopic properties are also inuenced. The excitation as well as emission maxima of hybrid material 7e are bathochromically shied in comparison to precursor 5 ( Fig. 9 and 10).
Most strikingly, both spectra reveal a much smaller range of emission and excitation maxima for the hybrid material than for the precursor molecule 5. Whereas the emission of precursor 5 shis by 4000 cm À1 from hexane to water, the shi decreases to about 2000 cm À1 for the hybrid material 7e. This can be explained by the dominance of the siloxy environment in   6 Quantum yields of grafted (6) and co-condensed (7) hybrids. the pores, which is only affected to a minor extend upon changing the solvent polarity in the pores. This assumption is additionally supported by the emission maxima of the hybrid material in the solid state as indicated by the grey line at 643 nm in Fig. 9. Only the emission of hybrid material 7e suspended in ether or ester causes a hypsochromic shi of the emission maximum and the suspension in water leads to a signicant bathochromic shi relative to the emission of 7e in solid state. The redshi for the hybrid material in water relative to the emission in the solid state can be rationalized by extensive hydrogen bonding, leading to a better stabilization of the excited state. The reverse is true for hydrogen-bond accepting solvents, such as ethers and esters which induce a blue shi of the emission. The effect of the solvent polarity on the excitation spectra is not strongly pronounced, possibly due to the lower sensitivity of the electronic ground state to polarity changes. However, slight deviations due to an inner lter effect have to be taken into account.
Furthermore, the excitation as well as emission spectra of precursor 5 and hybrid material 7e do not change for measurements in methanol, DMSO and water. In all three cases the environment created by the silica matrix is comparable to the environment of the dye in the respective solvent. This is also reected in the differences in the Stokes shis of the precursor molecule 5 and the hybrid material 7e (Fig. 11).
Some solvents hardly show any change in the Stokes shi of the hybrid material 7e compared to precursor molecule 5, although their excitation and emission maxima vary signicantly. A striking example is the identical Stokes shi of precursor 5 (ñ¼ 1600 cm À1 ) and hybrid material 7e (ñ¼ 1600 cm À1 ) in hexane, although the emission and excitation of hybrid 7e is shied bathochromically by 3100 cm À1 in comparison to compound 5 ( Fig. 9 and 10). Thus, it can be concluded that the incorporation of the dye into a silica matrix signicantly changes the spectroscopic properties but the inuence of the solvent polarity on these properties is comparable for both the free precursor 5 and the hybrid material 7e.    Comparing the excitation spectra of the differently loaded hybrid materials the effect of loading levels on the excitation maxima for the graed (6) and co-condensed (7) hybrids can be considered to be only minimal. For instance the excitation maximum of graed material 6a at 589 nm is shied bathochromically for material 6c by approximately 100 cm À1 accompanied by a decrease in intensity. The co-condensed hybrids 7 show a similar trend by redshiing from 572 (7c) to 579 nm (7e) (Fig. 12) by approximately 200 cm À1 concomitantly showing a decrease in excitation intensity with increasing concentration. In addition, the shape of the excitation spectra is altered as at the red-edge a relative sharp signal arises whereas the intensity at 590 nm (graed hybrids 6) and 580 nm (cocondensed hybrids 7) decreases even further. This effect is attributed to the primary inner lter effect leading to an attenuation of the excitation light and resulting in a virtual redshi at high dye concentrations.
The emission spectra indicate a pronounced red shi by 1000 cm À1 upon increase of the dye concentration from 10 À7 over 10 À6 to 10 À5 mol g À1 (Fig. 13). This bathochromic shi is identical for graed (6) and co-condensed (7) materials as indicated by almost superimposable spectra at comparable dye loading.
Thus, this bathochromic shi can be attributed to reabsorption of the hypsochromic part of the emission at higher concentrations by dyes with small Stokes shis. 47 Although NR in general exhibits a large Stokes shi aer incorporation in silica such reabsorption events become possible (Fig. 14). 51 There is no evidence for the formation of aggregates although the excitation spectra display a different shape and a slight red shi at higher dye concentrations. These ndings could imply the forming of aggregates, but considering the spectroscopic data from reports on NR aggregation this scenario seems unlikely in our case. 28,[51][52][53][54] If the alteration of shape in the excitation spectra was caused by aggregation, it would be quite unlikely that for the graed and co-condensed materials, despite their very different synthesis conditions, nearly identical aggregates would be formed. Furthermore, literature reported aggregates of NR adsorbed in MCM-41 materials and suspended in dichloromethane possess full width at half maximum (fwhm) values of their emission band around 1000 cm À1 . 54 Even though these values are quite high, they become possible due to higher disorder in a restricted environment leading to a broadening of the emission band. 55,56 But comparing the fwhm values of the synthesized materials which are around 1500 cm À1 for hybrids suspended in dichloromethane and even 2000 cm À1 in solid state, aggregation seems even more unlikely. In addition there is no reduction of fwhm at high dye loading levels, relative to the low loaded hybrid material.
Moreover, there is no change in the absorption spectra of the suspensions of hybrid materials in DMSO, except for a shoulder at 640 nm ( Fig. S25 and S26, ESI †) for some hybrid materials which can be attributed to protonated NR species. This is additionally supported by uorescence quenching studies. Therefore, reabsorption effects in the solid state causing Fig. 11 Difference between Stokes shifts of precursor molecule 5 and hybrid material 7e. Fig. 12 Excitation spectra of selected grafted (6) and co-condensed (7) hybrid materials with variable dye loading (l emission ¼ 700 nm). Fig. 13 Emission spectra of selected differently loaded grafted (6) and co-condensed (7) hybrid materials (shoulder at 695 nm is a measurement caused artefact). a bathochromic shi are by far more plausible than aggregate formation. 46,57 The effect of REES can be found for the hybrid materials suspended in different solvents as well as in the solid state, although less pronounced. For hybrid materials 6f and 7g the emission shis bathochromically by 133 and 88 cm À1 , respectively, upon excitation at 546 and 664 nm. This effect is much more pronounced for the hybrid materials in suspension as shown in Fig. 15, with maximum shis of about 1000 cm À1 . It is striking that predominantly the graed material shows bigger shis compared to the co-condensed hybrids and that nearly no REES can be observed in water.
A thorough inspection of the REES of the graed (6b) and co-condensed (7e) hybrid materials suspended in acetone clearly shows that at excitation wavelengths lower than 540 nm both materials do not distinctly shi the emission in dependence of the excitation wavelength (Fig. 16). Only upon excitation in the red-edge a shi in the emission maxima can be observed.
As all graed materials show a more pronounced REES it can be assumed that the distribution of dye molecules is more inhomogeneous. While some quite unrestricted molecules are located on the outside surface, some relatively restricted uorophores reside inside the pore channels, and a fraction of molecules which is bound to the pore openings, where high dye concentrations are present, enhancing their restrain. In contrast, the relatively homogenous distribution inside the pores with only slight changes in the restriction of the molecules inside the channels could give rise to a less pronounced REES for the co-condensed materials.

Fluorescence quenching
For further investigation of the different hybrid materials uorescence quenching experiments were conducted. Upon addition of hydrochloric acid to the water suspended hybrid materials 7e and 6b, a decrease in uorescence intensity can be monitored (Fig. 17).    Both materials show an emission maximum at 664 nm with a small shoulder at 720-750 nm in the emissive state. Upon addition of an acid to the samples the emission maximum at 664 nm decreases and slightly shis bathochromically to 670 nm for high acid concentrations. For the two materials with comparable dye loading levels of 1.5 and 1.9 mmol g À1 the emission spectra at low acid concentrations (up to 6.00 mmol L À1 ) look very similar. But upon further increase of the acid concentration, the co-condensed material 7e is more efficiently quenched than the graed material 6b. For the identical relative uorescence intensity for the co-condensed hybrid 7e a smaller acid concentration is need than for the graed hybrid 6b (Fig. 18). This indicates the presence of unequally distributed dye loading inside the different materials, which can also be observed in the plot of uorescence intensities against acid concentrations.
In this Stern-Volmer plot the curves deviate from linearity with increasing acid concentrations towards the x-axis which is indicative for the presence of more than one species of uorophores with variable accessibility to the quencher. These different classes of uorophores can also be deduced from the excitation spectra of the quenched uorophores (Fig. 19).
In the emissive state the different materials show slightly variable excitation maxima. For the co-condensed material 7e the maximum lies at 602 nm, whereas the maximum of the graed hybrid 6b is slightly shied bathochromically to 608 nm. Upon addition of 200 mmol L À1 of acid both materials show the same two excitation maxima at 588 and 640 nm, but in the case of the graed material 6b the signal at 640 nm is signicantly more intense than in the co-condensed case 7e. This newly arising redshied excitation maximum at 640 nm corresponds to the literature known absorption maximum of NR-H + . 46,57 As the graed materials show a more intense signal of the protonated NR species NR-H + than in the co-condensed case it can be assumed that the degree of NR-H + is higher in the graed hybrid 6b than in the co-condensed 7e. This can be rationalized by preferred functionalization with the dye molecules on the outside surface and the pore openings of graed materials enabling an easier protonation. In turn, in the co-condensed material the dye molecules are incorporated within the pores or even within the silica walls to a larger extent. In addition the surrounding silica matrix can act as a proton buffer.

Conclusions
The uorescence dye 2-hydroxy NR was functionalized via propargylation and CuAAC reaction with an azido substituted triethoxysilane derivative to furnish a luminescent precursor for covalent ligation. The formation of mesoporous hybrid materials by incorporation into silica matrices with variable dye loading was accomplished by two synthetic approaches, i.e. by graing onto a mesoporous MCM-41 host or by in situ co-condensation with concomitant formation of the mesoporous silica-dye hybrid. The structural and optical properties of these novel hybrids were thoroughly studied. Analysis of the materials by nitrogen sorption measurements in combination with SAXS and TEM conrmed a two-dimensional hexagonal columnar ordered mesoporous structure which is not affected by functionalization with the NR dye in the applied micromolar concentrations. Investigation of the optical properties revealed a red-edge excitation shi (REES) and uorescence quenching, which were utilized to analyze the dye distribution inside the different materials. Therefore it can be qualitatively assumed that different dye species are present inside the graed and co-condensed materials. While graed materials are predominantly functionalized on the outside surface and in pore openings at high dye concentrations, the co-condensed materials obviously possess a more homogenous dye distribution and predominant functionalization inside the pores or silica walls. The hybrid materials retain, although much less pronounced, some optical characteristics of the NR, such as the solvatochromism. However, in contrast to the native dye, these synthesized hybrids possess quantum yields of about 20% in the solid state and even uorescence in aqueous media which opens new alleys to applications of NR-based hybrid materials. Further studies directed to access novel functional  organic hybrid materials by this approach are currently underway.

General considerations
Reagents, catalyst and solvents were purchased reagent grade and used without further purication. Products were puried with column chromatography on silica gel 60 (0.040-0.063 mm) from M&N using ash technique. 1 H, 13 C and 135-DEPT NMR spectra were recorded on a Bruker AVIII-300 and the resonance of the particular solvent was locked as internal standard ( Excitation and emission spectra were recorded on a Hitachi F-7000 uorescence spectrophotometer at T ¼ 293 K. Excitation of uorescence was always carried out at the excitation maximum. Quantum yield determinations of the hybrid powders were obtained with an integrating sphere. Data analysis and quantum yield calculations were performed with the soware FL Solutions Version 4.0 by Hitachi.

Synthesis of hybrid materials
Graed NR MCM hybrids (6a-f). Six NR-functionalized silica hybrid materials were synthesized by postsynthetic graing.  added to the reaction vessel and stirred at room temp for 20 h, followed by stirring at 80 C for 24 h (for experimental details, see Table 5). The obtained suspensions were centrifuged (10 min, 4000 rpm), decanted and resuspended in ethanol (20 mL) and 2 M aqueous hydrochloric acid solution (1 mL), upon which the red suspensions turned blue. Aer heating to 80 C for 24 h the reaction mixtures were centrifuged, the solids transferred into a Soxhlet extraction thimble and extracted with ethanol over a period of 48 h. The obtained powders were washed with triethylamine (2 mL) and ethanol (20 mL), which led to a color change back to red. The solids were washed with ethanol (3 Â 20 mL) and centrifuged each time as described until the supernatant reached pH 7. The obtained violet powders were dried at 60 C and 10 À3 mbar for 3 days to mass constancy.
Co-condensed NR MCM-hybrids (7a-h). 43 For the synthesis of the co-condensed NR-functionalized silica hybrids a solution of tetraethyl orthosilicate (TEOS), variable amounts of precursor 5, hexadecyl-trimethyl-ammonium bromide (C 16 TMABr), ethylamine, methanol, and deionized water was prepared with molar ratios of 1.00 : x : 0.140 : 2.40 : 2.00 : 100 (molar ratios x of precursor 5 are given in Table 6). First the template C 16 TMABr was dissolved in deionized water and ethylamine, which was employed as a 70 wt% aqueous solution before TEOS and the molar amount x of precursor 5 in methanol were added. For the syntheses of materials 7f-h 10 molar equivalents of methanol were used. The mixtures were stirred at room temperature for 24 h with a speed of 750 rpm before they were heated to 100 C for 24 h. The obtained suspensions were centrifuged (10 min, 4000 rpm), decanted and washed with ethanol before they were centrifuged again. Then the residues were suspended in ethanol (80 mL) and concentrated aqueous hydrochloric acid solution (2 mL) and stirred at 80 C for 24 h. The mixtures were centrifuged, and the solids were transferred into a Soxhlet extraction thimble and extracted with ethanol over a period of 48 h. The obtained powders were washed once with triethylamine (2 mL) and ethanol (80 mL) upon which they turned red again. The solids were washed with ethanol (3 Â 80 mL) and centrifuged as described above till the supernatant reached pH 7. The obtained violet powders were dried at 60 C and 10 À3 mbar for 3 days to mass constancy.