Tailor-made surfaces expressing a homogenous distribution of a certain type of functional group, such as, alcohol, carboxyl, primary amine, thiol, aldehyde or epoxy, are of paramount importance in many areas of the (bio)chemical sciences. For instance, such surfaces are essential in many bioanalytical or diagnostic applications, because these primary functional groups can be used for further attachment of biomolecules, such as, oligonucleotides, peptides, sugars, antibodies or enzymes, rendering them suitable for microarray-based assays or as sensory layers.1–5 The efficiency of the immobilization process and the quality of the final biochemically functionalized surface strongly depend on the density and, even more importantly, on the accessibility of the surface functional groups.6 For the reliable characterization of the chemical nature and surface concentration of functional groups, robust and fast analytical tools are urgently required. Several techniques have for instance been described to determine the density of primary amino groups on solid surfaces—many of the respective publications deal with the characterization of amino groups on polymer surfaces.7 Besides infrared spectroscopic techniques such as Fourier transform IR (FTIR, especially for thin films)8 and time-of-flight secondary ion mass spectrometry (ToF-SIMS),9 X-ray photoelectron spectroscopy (XPS) is one of the most widely used methods. However, XPS often shows inferior performance at low concentrations of surface functionalities and is not suitable for a direct identification and quantification of amino groups on surfaces, when these groups coexist with a manifold of other nitrogen-containing species with similar chemical shifts.10–12 Therefore, protocols have been developed in which the amino groups are labelled with molecular entities containing elements that are originally not present on the surface of the samples, for example, fluorine. After the labelling reaction, the concentration of this new element is assessed by XPS analysis and directly relates to the concentration of the particular functional group on the surface. Up to now, the reagents mostly used for amino-group labelling in XPS analysis are pentafluorobenzaldehyde (PFB),13, 14 (4-trifluoromethyl)benzaldehyde (TFBA),15, 16 3,5-bis(trifluoromethyl)phenylisocyanate17 and trifluoroacetic anhydride (TFAA).18, 19 Other nitrogen-containing functional groups such as amides, imines and nitriles do not react with these derivatization reagents. However, an intrinsic drawback of XPS is that the limit of detection for a certain element lies typically at 0.1–1.0 atom- % (at- %),20 with all the atoms of label, functional group and bulk within the spot of irradiation counting in. To overcome these quantification difficulties of XPS at low labelling concentrations, optical methods in combination with an adequate labelling technique have been increasingly employed. Here, both colorimetric21–23 and fluorometric24–27 methods have been successfully used for surface-amino-group determination, with the second technique showing distinctly higher sensitivities. Dansyl hydrazine and pyrylium dyes are examples of possible fluorescent markers.21–23, 26 The amino-reactive compound fluram (4-phenylspiro-[furan-2(3 H),10-pthalan]-3,30-dione), which reacts with nucleophiles in general but forms a fluorescent product only with primary amines, was successfully employed for various surfaces.28, 29 However, translating the signal detected to an absolute number of functional groups is not trivial. Nonspecific adsorption and binding can result in enhanced background fluorescence and quenching phenomena. In addition, whereas XPS measurements can provide quantitative results that are traceable to a primary standard,30, 31 absolute fluorescence measurements are an intrinsically difficult task already for ideally dilute solutions,32 and most fluorescence techniques only provide relative results. On the other hand, fluorescence scanning techniques are much faster, non-destructive and technically as well as methodologically much simpler than XPS. For traceable and quantitative yet rapid surface chemical analysis, the quest is thus to find a way to directly link both methods with their unique features.
Boron-dipyrromethene (BODIPY) dyes have up to now only been rarely used for fluorescence labelling of surface species,25 despite their popularity as functional dyes, and the tremendous progress in the BODIPY field during the past ten years.33–39 Moreover, the remarkable versatility of BODIPYs has sparked intense research on new modification strategies to enable their attachment to biological substrates and also to tune their optical properties. Today, BODIPYs are widely used as biomolecule markers, fluorescent switches, chemosensors and laser dyes.33–38 Several new strategies for their functionalization have recently been developed, among which halogenation of the BODIPY core is particularly interesting because the introduction of a halogen atom to the dipyrrin core facilitates further derivatization through aromatic nucleophilic substitution and palladium-catalyzed coupling reactions.40, 41 The most important challenges in recent BODIPY chemistry perhaps include the development of dyes with longer-wavelength absorption and emission profiles, and the preparation of dyes with additional functional groups for covalent attachment.
Based on our experience in the field of BODIPY-dye chemistry42–45 and being increasingly confronted with the request for photo- and chemically stable dyes for reliable labelling purposes as well as high-performance dyes for the near infrared (NIR) region, we embarked on the design, synthesis, characterization and application of BODIPY dyes containing a high amount of fluorine atoms.46–48 Regarding the comparability of fluorescence and XPS measurements, we reasoned that highly fluorinated BODIPY dyes might combine several advantageous features, that is, high stability, prominent spectroscopic properties and considerably low limits of detection for a unique XPS-suitable atom such as fluorine. Here, we describe the synthesis and characterization of a series of novel fluorine-containing BODIPY-type fluorophores and labelling agents (Scheme 1). In addition, some of the BODIPYs were functionalized appropriately via the Knoevenagel condensation to generate styryl-substituted NIR-emitting fluorophores. The performance of selected dyes as dual XPS/fluorescence labels is studied, providing detailed results on the chemical composition and distribution of the immobilized fluorescence labels on aminated SiO2 supports.