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Abstract
Two novel photoresists were developed for the fabrication of sub-diffractionally sized polymeric nanostructures with chemically reactive surfaces. Using multiphoton polymerization as well as stimulated emission depletion (STED) lithography, chemically functional monomers were copolymerized with highly crosslinking triacrylate monomers to yield stable nanostructures. The polymer structure was thereby supplemented with chemical functionalities for further covalent modification reactions. The reactivity of mercapto- and carboxylate groups on the surface of the nanostructures was proved by orthogonally labeling them with reactive fluorophores. The photoresists can be used for stimulated emission depletion lithography and lateral line widths down to 55 nm were achieved. A three-dimensional structure is shown that is made up with three compounds: a frame out of a un-functionalized acrylate photoresist, and two intermediate networks made up with thiol- and carboxyl functional photoresists.
© 2017 Optical Society of America
1 Introduction
The growing importance of three dimensional (3D) micro- and nano-structures in research areas such as photonics [1–3], microelectronics [4,5] or biomedicine [6–9] led to increased research efforts to improve fabrication techniques as well as to develop new materials [10]. Multiphoton polymerization (MPP) is one of the most powerful techniques and allows for fabrication of complex 3D shaped structures [11–16]. Careful composition of the photoresist used for MPP allows for tailoring of material properties: structures with defined mechanical stiffness [17–19], surface energy [20], degree of hydrophobicity [21] or improved cell adhesion [22–24] are reported in literature. However, the achievable minimal structure size and resolution in MPP is limited by diffraction. Inspired by stimulated emission depletion (STED) microscopy [25], STED lithography was developed and allows for diffraction unlimited structuring [26–30]. Similar to STED microscopy, a second laser is used to deplete previously excited photoinitiators in the outer rim of the point spread function (PSF). Thereby, the capability of the photoinitiator to start the polymerization is locally inhibited, effectively shrinking the polymerization volume and allowing to break the diffraction barrier. In MPP, feature size decreases with the excitation wavelength, which was demonstrated for 520 nm and 405 nm [31,32]. However, using STED allows fabrication of ~55 nm features while using an NIR laser for excitation [29], with an improved stability against laser power fluctuations [33].
To extend the range of applications of 3D MPP microstructures, functional groups were introduced, allowing for chemical modification. For example, microstructures were demonstrated which can be selectively coated with metal [34], which are based on hydrogels [8] or organic-inorganic sol-gel materials [35], or thiol-ene reactive structures [36], or which are capable of binding proteins [7]. Further, microstructures possessing one or more reactive surfaces are employed nowadays, and a variety of different functional monomers, which can be structured using MPP, have been developed [34,36–42].
In the meantime, functional structures which are fabricated by MPP found widespread application in the biomedical field [43], like 3D scaffolds for neural tissue engineering [6] or biomimetic hydrogel scaffolds for guidance of cellular organization [44]. Currently, STED lithography, when compared to MPP, stays behind in the development of functional structures because it imposes stricter requirements on the monomers than MPP. This so far resulted in a rather limited choice of photoresist composition. These limitations raise a demand to widen the library of STED photoresists in order to extend the application of sub-diffraction nanostructures. Lately, our group reported a new metal-oxo cluster based photoresist which allows structuring with 70 nm feature sizes, and successful post-modification was shown by labeling experiments [45]. However, moisture sensitivity of the metal-oxo clusters makes handling of this photoresist cumbersome and the minimal lateral feature sizes were limited to 70 nm, not reaching the current state of the art 55 nm in STED lithography [29,46].
In this work, we present two new photoresists which allow for MPP and STED structuring of surface reactive nanostructures. The activity of the reactive groups is proven by covalent binding of fluorophores. Sub-diffraction structuring down to the 50 nm range is enabled using STED lithography. In addition, the purely organic photoresist is stable against moisture. Compared to the metal-oxo clusters that are functionalized with methacrylate groups [45], the reactivity ratio and the miscibility are improved by using acrylate groups. Furthermore, the photoresists can be used for the fabrication of compound structures which allow for chemically orthogonal functionalization.
2. Methods
The photoresists are composed of the commercially available monomer 2-carboxyethylacrylate (CEA) or the non-commercial monomer 3-mercaptopropanoyl-oxyethyl acrylate (MPOEA) in combination with pentaerythritol triacrylate (PETA). In the past, PETA was successfully used in STED lithography [29,27] but has the drawback that it limits the choice for post-modification to thiol-ene reactions. Photoresists for STED and MPP experiments were prepared by mixing 10% wt. of CEA or 5% wt. of MPOEA with PETA.
1% wt. IRGACURE 819 was used as initiator for MPP lithography and 0.25% wt. 7-diethylamino-3-thenoylcoumarin (DETC) was used as initiator for STED lithography [27,29]. The chemical structures of PETA, CEA, MPOEA, DETC and IRGACURE 819 are shown in Fig. 1. MPOEA was synthesized as outlined in Appendix D and Fig. 9. Briefly, the mercapto group of 3-mercaptopropionic acid was protected with tritylchloride to yield 3-(tritylthio)propionic acid (trtSPrAc). The protected acid was then reacted with 2-hydroxyethylacrylate to obtain 2-((3-(tritylthio)propanoyl)oxy)ethyl acrylate (trtSPrOAcr) in good yield. The mercapto-functional acrylate monomer MPOEA could then be obtained by removing the trityl protection group as colorless oil. The ATR-IR spectrum clearly shows the weak thiol stretching band at 2367 cm−1, as well as the C = O band at 1734 cm−1 and the olefinic band at 1636 cm−1, which stem from the acrylate group. Additionally, the monomer was characterized by NMR, IR and HR-ESIMS spectroscopy and data are given in Figs. 10–21 in Appendix D.
The STED lithography setup is essentially identical to the one described before in Ref [29]. and a scheme is presented in Fig. 6(a) in Appendix A1. In brief, an ultra-short pulsed NIR laser (780 nm) is used for multiphoton excitation of the polymerization starters. In STED lithography, a depletion beam (532 nm) is additionally applied to deactivate the photoinitiators in the outer rim of the excitation volume. For two dimensional (2D) STED lithography, a ring shaped, donut like point spread function (PSF) is formed by passing the STED beam through a 2π spiral phase mask. In case of 3D structuring, the STED PSF is shaped into a bottle beam using an annular phase retardation by π.
3. Results and discussion
To determine optimal structuring parameters for the new photoresists, lines were written with varying depletion powers, as shown in Figs. 2(a) and 3(a). All powers are given as powers entering the objective lens and a 2D donut shaped STED PSF was used. At both ends of each line, the excitation intensity was increased by 10% in order to improve adhesion and visibility. We will first discuss results obtained with the CEA/PETA photoresist. Figure 2(a) shows a series of lines, written with constant excitation power (3.2 mW). The depletion power increased from 0 mW from left to right in 2 mW steps. Hence, the most left line was written by MPP without depletion beam. Figure 2(b) shows a zoomed image of this line, exhibiting a width of 97 nm. The chemical nonlinearity of polymerization thresholds allows writing continuous lines with NIR lasers with widths just below 100 nm [33].
Fig. 2 Testing the CEA/PETA photoresist. (a) SEM images of lines with thickened ends for improved attachment, written with 3.2 mW excitation power and different depletion powers (increasing from left to right from 0 mW to 30 mW in 2mW steps). A 2D donut was used for the STED PSF. (b) Zoom in on the line written without STED and (c) with 12 mW STED power (c.f. white dashed boxes in (a)). (d) Evaluation of line widths as a function of the applied STED power.
Fig. 3 Testing the MPOEA/PETA photoresist. (a) SEM images of lines with thickened ends for improved attachment, written with 3.0 mW excitation power and different depletion powers (increasing from left to right from 0 mW to 7.5 mW in 0.5 mW steps). A 2D donut was used for the STED PSF. (b) Zoom in on the line written without STED and (c) with 5 mW STED power (c.f. white dashed boxes in (a)). (d) Evaluation of line widths as a function of the applied STED power.
Applying increasing depletion powers first decreases the linewidths until a minimum line width is reached. A minimal feature size can be obtained by 12 mW depletion power as depicted in Figs. 2(a) and 2(c). From 14 mW depletion power onwards, the lines broaden due to residual absorption of the depletion laser beam [47]. With 12 mW depletion power, smallest line widths of 53 nm were obtained, corresponding to λ/14 of the excitation wavelength as shown in Fig. 2(c). Figure 2(d) shows the achieved line widths as a function of STED power. We note that the minimum achievable feature size using only MPP was 89 nm for the CEA photoresist. The feature height, which should theoretically be independent of a donut-shaped depletion PSF [33], can be reduced in fact by a lateral confinement due to diffusive effects [45,48]. However, to characterize the axial resolution, we use the bottle-beam depletion PSF for 3D fabrication (see Fig. 5).
Similar experiments were performed using the MPOEA/PETA resist. MPOEA/PETA shows an enhanced polymerization at higher depletion powers due to residual absorption of the depletion beam as shown in Fig. 7 in Appendix B. Hence, the concentration of MPOEA in the photoresist was adjusted to 5% wt. only, compared to 10% wt. CEA. A series of lines is depicted in Fig. 3(a), written with 3.0 mW excitation power and with depletion powers ranging from 0 mW to 7.5 mW in 0.5 mW steps. Similarly as observed for the CEA/PETA resist, line widths first decrease with increasing depletion power until a minimum is reached at 5 mW STED power. Residual absorption results in a broadening of the lines for depletion powers exceeding 5 mW, see Fig. 3(a) and Fig. 7. Smallest line widths of 59 nm could be obtained at 3.0 mW excitation power and 5.0 mW depletion power, corresponding to < λ/13 of the excitation wavelength, see Figs. 3(a) and 3(c). The minimum MPP feature size achievable with the MPOEA photoresist was 86 nm.
Knowing the optimal writing parameters for STED lithography, composite structures were fabricated. Orthogonal, covalent fluorescence labeling verified the activity and selectivity of the reactive groups on the surface. Figure 4 shows MPP and STED fabricated grids made of both photoresists. The compound structures were written sequentially, combining carboxylate- and mercapto-functionality in one structure. First, a grid was written into the MPOEA/PETA photoresist using MPP, and unreacted photoresist was gradually replaced with excess CEA/PETA photoresist, followed by writing of the second grid. The vice versa fabrication (writing the CEA/PETA grid before the MPOEA/PETA grid) is also possible (vide infra: STED lithography).
Fig. 4 Orthogonally functionalized PETA grids. (a-c) Prepared with MPP, (d-f) prepared with STED lithography. (a, d) Red channel of the fluorescence microscope (excitation with (a) 642 nm and (d) 660 nm): Cy5-maleimide selectively binds to mercapto-groups of MPOEA/PETA grids. (b) 488 nm excitation channel of the fluorescence microscope: CF-488A-amine binds preferentially to carboxy-groups of the MPP structured CEA/PETA grid. (e) 532 nm excitation channel of the fluorescence microscope: Cyanine3-amine binds preferentially to carboxy-groups of the STED structured CEA/PETA grid. (c) and (f): Overlays of (a, b) or (d, e), respectively. The insets show zoom-ins on the white dashed boxes. The linear color scales are shown in the insets of (a, b, d, e). (g) SEM image of the grids shown in (f), after metallization with 12 nm platinum. The inset shows line widths of 55 nm for the MPOEA/PETA grid and 61 nm for the CEA/PETA grid.
Wet chemical labeling experiments on the bifunctional CEA/MPOEA compound structures were carried out by first labeling the mercapto functional part of the structure with Cy5-maleimide as depicted in Fig. 4(a). Then, the carboxy groups were converted in NHS-ester and reacted with amine functionalized CF-488A as shown in Fig. 4(b). The overlay of both fluorescence images shows highly selective labeling as depicted in Fig. 4(c). The carboxy conversion and labeling procedure are described in more detail in Appendix D. Further, Fig. 8 in Appendix C shows a two-compound structure consisting of a plain PETA grid and two MPOEA/PETA grids. Functionalization with Cy5-maleimide labels only the MPOEA/PETA, but not the plain PETA structures. Plain PETA structures do also not react with amine functionalized fluorophores, see the 3D structure discussed below.
Next, composite grids were fabricated with STED lithography. First, a droplet of CEA/PETA photoresist was placed on a piranha cleaned glass slide. The excitation power was set to 2.7 mW and a depletion power of 12 mW was used to write the first grid. Afterwards, the unreacted CEA/PETA photoresist is diluted and gradually replaced with excess of MPOEA/PETA resist. The MPOEA/PETA grid was then written using 2.4 mW excitation power and 5 mW depletion power, offset by half a lateral period of the first grid. After removal of the unreacted MPOEA/PETA resist with acetone and subsequent drying, the composite grids were first labeled with Cy5-maleimide which binds selectively on the MPOEA/PETA grid, see Fig. 4(d). Next, carboxy groups were converted to NHS-esters and reacted with the fluorophore Cyanine3-amine.
Cyanine3-amine was used to label the STED lithography grids rather than CF488A-amine, because the fluorescence of CF488A-amine overlaps with the auto-fluorescence of the initiator DETC. The result is shown in Fig. 4(e). A slight tilt of the slide during the production process leads to an incomplete CEA/PETA grid. The overlay of the fluorescence images shown in Fig. 4(f) demonstrates orthogonal covalent coupling to the two PETA grids with different chemical functionality. The line widths of the STED fabricated grids were determined from SEM images and are 55 nm for the MPOEA/PETA grid and 61 nm for the CEA/PETA grid as depicted in Fig. 4(g).
A 3D structure consisting of three components was fabricated, in order to show the full potential of multifunctional 3D nanostructuring. An unmodified scaffold of plain PETA (no CEA nor MPOEA added) was written by MPP, namely a 4 × 4 grid of vertical posts with a coarse grid on top, see Fig. 5(a). Subsequently, unreacted plain PETA was exchanged with CEA/PETA. Using an excitation power of 2.7 mW and 12 mW STED power, this time shaped into a 3D STED PSF, four CEA/PETA grids were written into the scaffold. In turn, unreacted CEA/PETA was exchanged with MPOEA/PETA and four grids were written with 2.2 mW excitation and 5 mW STED power. Figure 5(a) shows an SEM image of the whole structure (taken after the fluorescence images and after evaporation of a thin 12 nm platinum shell). The four green shaded horizontal sub-grids within the SEM image represent the CEA/PETA substructure. The red shaded bars represent the MPOEA/PETA subgrids. Only three are visible in the SEM image, because the top most MPOEA/PETA grid was merged with the coarse MPP structured grid of unfunctionalized plain PETA.
Fig. 5 (a) SEM image of a triple component structure, recorded at 60° sample tilt. A scaffold of a 4 × 4 grid of pillars and a cover grid (top) are written with MPP in non-functional PETA photoresist. Using STED lithography, four CEA/PETA grids (shaded green) were written into the scaffold, followed by four MPOEA/PETA grids (shaded red). The top-most MPOEA/PETA grid is merged with the cover grid and therefore not visible. The structure is constricted due to shrinkage of polymers during development. The yellow dashed line indicates the plane of confocal scanning, prior to metallization with 12 nm platinum and SEM imaging. (b) Confocal fluorescence images of the 3D triple compound structure immersed in glycerol. Cy5-maleimide binds preferentially to MPOEA/PETA layers (red), whereas Cyanine3-amine binds to CEA/PETA layers (green). A minimum axial distance between MPOEA/PETA and CEA/PETA layers of 550 nm axially is measured in the confocal images. Cy5-maleimide and Cyanine3-amine were excited with 660 and 532 nm, respectively.
The measured minimal axial line widths were 130 nm for both, CEA/PETA and MPOEA/PETA grids, with a minimal axial distance of 550 nm between the third (from bottom) MPOEA/PETA and the fourth CEA/PETA subgrids. Due to post deposition shrinkage, this distance cannot simply be assigned as axial resolution. The actual resolution is limited by the size of the larger polymerization volumes (prior to shrinking) of adjacent features. Nevertheless, 130 nm axial line widths are clearly below the axial diffraction limit, even if a post deposition shrinkage of the rigid scaffold on the order of 10% is taken into account.
For a visualization of the orthogonal functionalization, labeling with fluorophores was performed as described before for the two-dimensional grids. Figure 5(b) shows confocal fluorescence images, taken at the vertical plane indicated by a yellow dashed line in Fig. 5(a). The red confocal image shows the fluorescence of the Cy5-maleimide attached to the MPOEA/PETA subgrid and the green confocal image shows the fluorescence from the Cyanine3-amine fluorophores bound to the CEA/PETA subgrid. Both confocal images appear as grids of confocal PSFs because the fluorescing objects are the bars with a cross section of approximately 75 × 130 nm (see also Fig. 22) and hence serve as quasi point like fluorescing test objects for a confocal fluorescence microscope (which itself is already a nice application of such 3D scaffolds).
The horizontal red and green arrows depict the vertical (z) position of the alternating planes. The feature sizes as well as the obtained resolution are limited by the material properties, in particular by post-polymerization shrinkage during development.
4 Conclusion
In conclusion, two chemically functional photoresists were developed for STED lithography, enabling the fabrication of multi-component 3D structures with minimum lateral feature sizes of 50-60 nm and axial line widths of 130 nm. The fabricated structures bear functional groups on the surface and allow for chemical post-processing. To the best of our knowledge, this is the first report on composite-nanostructures based on two functional photoresists, applicable for sub-diffraction STED lithography. Two orthogonal coupling reactions with distinct fluorophores to specific parts of multi component structures are shown. The principle of orthogonal coupling paves the way to covalent attachment of other species than fluorophores to the scaffold such as proteins, antibodies or antigens, factors and co-factors, oligonucleotides, aptamers or even vesicles. While MPP has been used to build 3D scaffolds for whole cells [6,7,24,40,49], 3D scaffolds with nanoscale anchors might play a similar role for 3D nanoscopical arrangement of sub-cellular objects. Hence, we are certain that selectively functionalized nanostructures, fabricated by STED lithography, will find applications in areas such as nanoscale biomedical research. Photonic applications may include metallization of the 3D structures or nanoscale placement of metal nanoparticles, semiconductor nanocrystals or perovskite nanoplatelets, e.g. via the mercapto groups.
Appendix A Experimental setup and instruments
A.1. Lithography setup/Confocal fluorescence imaging:
The setup is depicted in Fig. 6. The two-photon polymerization starters were excited with 780 nm ultra-short laser pulses (FemtoRay780, 50 MHz repetition rate, 100 fs pulse duration, Menlo Systems GmbH, Planegg, Germany) and were locally depleted in the outer rim of the point spread function (PSF) with a depletion beam (532 nm, continuous wave, Verdi-V5, Coherent, Santa Clara, CA, USA). The 532 nm depletion beam was shaped into a donut PSF for 2D structuring using a 2π spiral phase mask (RPC Photonics, Rochester, NY, USA) and a λ/4 wave plate. For 3D structuring, a homemade annular phase mask in combination with a λ/4 wave plate was used to create a bottle beam PSF. Both beams were focused through an oil immersion objective lens (Zeiss α-plan Apochromat, 100x, numerical aperture NA = 1.46). Power adjustment of the excitation beam was provided by an acousto-optic modulator (Q1133, Isomet (UK), Ltd., Torfaen, UK). Excitation and depletion powers are measured in front of the objective lens. An avalanche photo diode (APD-SPCM-AQRH, PerkinElmer Optoelectronic Inc., Waltham, MA, USA) was used for aligning the foci and collection of the fluorescence signal. A three axes piezo stage (P-562.3CD, Physik Instrumente PI, Karlsruhe, Germany) with a bidirectional positioning accuracy of 2 / 2 / 4 nm and a travel range of 200 x 200 x 200 μm was used for sample motion. The high-precision stage was mounted on top of a coarse xy-motor stage (M-686.D64, Physik Instrumente PI) with a travel range of 25 x 25 mm. The stages were driven in closed loop with two controllers (E710.3CD and C-867.260, both from Physik Instrumente PI). For sample positioning, recording of images and controlling the writing process, a LabView (LabView 2011, National Instruments Corporation, USA) program was used. Confocal images were taken using the same setup, now as a stage scanning confocal microscope. The unmodified 532 nm CW beam or a 660 nm CW beam (opus 660, LaserQuantum, Konstanz, Germany) were used for excitation, in combination with a 533/17 nm notch filter (NF533-17, Thorlabs GmbH, Dachau/Munich, Germany) and a 658/25 nm notch filter (NF658-26, Thorlabs GmbH), respectively. Confocal images as shown in Figs. 4(d)-4(f), Fig. 5(b) and Fig. 8 were recorded with 200 nm pixel size. Using ImageJ, images of Figs. 4(d)-4(f) were upscaled by a factor of 4 using bilinear interpolation. Lookup-table minima and maxima were set to average of background and average of brightest line, respectively.
Fig. 6 (a) Scheme of the MPP/STED lithography setup. Red depicts the excitation beam for MPP (780 nm, 100 fs pulse duration) and green depicts the optional depletion beam (532 nm continuous wave CW). The depletion beam is shaped into a donut using a 2π spiral phase plate (PP) or into a 3D STED beam using an annular phase plate retarding the center by π. Abbreviations: acousto-optic modulator (AOM), pinhole (PH), quarter wave plate (λ/4), dichroic mirror (DM), avalanche photo diode (APD). (b) Excitation beam PSF and (c) donut-shaped 2D STED beam PSF, measured by scattering of 50 nm gold nanoparticles. (d) Emission and absorption spectrum of 7-diethylamino-3-thenoylcoumarin (DETC, photoinitiator) in pentaerythritol triacrylate (PETA).
A.2. Widefield fluorescence microscopy:
Images were taken on an inverted Olympus IX81 (Olympus Austria GmbH, Vienna, Austria) equipped with an oil objective lens (100 X, NA = 1.49, Olympus) in combination with two diode lasers with wavelengths of 491 nm (Cobolt Calypso 100, Solna, Sweden) and 642 nm (Omicron Laserage Laserprodukte GmbH—Phoxx 642, Rodgau-Dudenhofen, Germany) for excitation. The fluorescence signal was recorded using an Andor iXonEM + 897 (back-illuminated) EMCCD (16-μm pixel size) (Andor Technology Ltd., Belfast, UK). Samples are moved by a motorized XY-stage (JKP, Berlin, Germany) in combination with a XYZ-piezo stage (Physik Instrumente PI). Illumination was controlled using a custom-made Qt-based (The Qt Company, Espoo, Finland) control software.
A.3. NMR spectroscopy:
Solution NMR spectra were recorded on a Bruker AVANCE 300 (300 MHz {1H}, 75.43 MHz {13C}). CD2Cl2 and DMSO-d6 were purchased from Sigma Aldrich and used as received.
A.4. IR spectroscopy:
Solid state ATR-IR spectra were recorded on a Bruker Equinox 55 FT-IR spectrometer equipped with a KBr window. 128 scans were averaged.
A.5. Mass spectroscopy:
High-resolution ESI mass spectra (HR ESIMS) were obtained using a Thermo Fisher Scientific LTQ Orbitrap XL with an Ion Max API Source.
Appendix B Depletion patterns written with MPOEA resist
Fig. 7 SEM image of lines with thickened ends (10% additional excitation power), written with 2.9 mW excitation power and depletion powers ranging from 0 mW to 25 mW. Lines were written with the MPOEA/PETA resist, using a 2D donut shaped STED beam. Minimum line widths are observed at 5 mW depletion power. Due to anti-Stokes or multiphoton absorption of depletion light, substantial broadening of the lines is observed for higher depletion powers.
Appendix C MPP fabricated compound structures
To exclude unspecific binding of the fluorophore on plain PETA structures, a labeling experiment with a monofunctional compound structure was performed. The grid was written with the non-functional PETA photoresist and the mercapto-functional MPOEA/PETA photoresist by MPP lithography. The labeling experiment was performed as described before for the MPOEA/PETA grid. Confocal reflection and fluorescence images of the PETA and MPOEA/PETA compound structure are depicted in Fig. 8 in the Appendix C. Cy5-maleimide fluorescence arises only from MPOEA/PETA structures. No fluorescence signal exceeding the background level is observed from plain PETA structures. From this we conclude, that the fluorophore binds exclusively through a maleimide-mercapto coupling reaction and unspecific binding of the fluorophore can be excluded.
Fig. 8 (a) Confocal reflection image (532 nm) of patches and composite grids written with MPP using the functional MPOEA/PETA and a non-functional PETA resist. The upper two patches on the left and the upper most left grid consists of PETA with IRGACURE® 819, the lower patch as well as the middle and lower right grid are written with MPOEA/PETA with IRGACURE 819. (b) Fluorescence image (excitation with 660 nm) of the structures after incubation with Cy5-maleimide. The fluorescence signal emanates selectively from the MPOEA/PETA structures. Linear color scales shown in insets.
Appendix D General chemical and preparation procedures
D. 1. General procedures:
3-mercaptopropionic acid (≥99% Sigma Aldrich), tritylchloride (97% Sigma Aldrich), diethylether (p.a. Sigma Aldrich), 2-hydroxyethylacrylate (96% Sigma Aldrich), dicyclohexylcarbodiimide (DCC, 99% Sigma Aldrich), 4-(dimethylamino)pyridine (DMAP, ≥99% Sigma Aldrich), ethylacetate (p.a., VWR), n-hexane (97%, VWR), acetone (≥99.8%, Merck Chemicals and Life Science GmbH, Vienna, Austria) triethylsilane (99% Sigma Aldrich), trifluoroacetic acid (99% Sigma Aldrich), 3-(trimethoxysilyl) propyl methacrylate (≥98% Sigma Aldrich), pentaerythritol triacrylate (PETA, techn. grade Sigma Aldrich), 2-carboxyethylacrylate (CEA, Sigma Aldrich), IRGACURE® 819 (BASF Schweiz AG, Basel, Switzerland) and 7-diethylamino-3-thenoylcoumarin (DETC, Acros Organics, Geel, Belgium) were used as received. Dichloromethane (p.a. Sigma Aldrich) was freshly distilled over phosphorus pentoxide (P2O5, Sigma Aldrich). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, 99%, Sigma Aldrich) and N-hydroxysuccinimide (NHS-OH, 98%, Sigma Aldrich) and phosphate buffered saline (PBS, 1X solution, sterile, pH 7.4 from VWR International, Vienna, Austria) were used as received. Stock solutions of the fluorophores CFTM488A-amine (1mM, Biotium), Cyanine3-amine (1mM, Lumiprobe GmbH, Hannover, Germany) and Cy5-maleimide (1mM, GE Healthcare) were prepared in DMSO.
D.2. Preparation of 3-(tritylthio)propanoic acid (trtSPrAc):
1 ml 3-mercaptopropionic acid (11.48 mmol, 1 eq.) and 10 ml dichloromethane were placed in a round bottom flask. 3.519 g tritylchloride (12.63 mmol, 1.1 eq.) was dissolved in 5 ml dichloromethane and added slowly at room temperature. After stirring 18 hr. at room temperature, a white precipitate was formed, which was filtered and washed with cold diethylether. After drying, trtSPrAc was obtained as white powder with 98% yield.
1H-NMR (300 MHz, DMSO-d6, 25°C, TMS): 12.22 (broad, COOH); 7.32 – 7.26 (m, 15 H, C6H5); 2.26 (t, 2H, S-CH2); 2.16 (t, 2H, CH2-COOH) ppm. 13C-NMR (75.43 MHz, DMSO-d6, 25°C): 172.7 (COOH); 144.36 (4°C C6H5); 129.09 (meta C C6H5); 128.05 (ortho C C6H5); 126.74 (para C C6H5); 66.17 (S-C-Ph3); 32.89 (CH2-COOH); 26.68 (S-CH2) ppm. IR (ATR): ν = 3060 (w), 2934 (w), 2738 (w), 2665 (w), 2567 (w), 1697 (s), 1593 (m), 1483 (s), 1431 (m), 1407 (m), 1345 (m), 1306 (m), 1249 (s), 1182 (w), 1141 (w), 1083 (m), 1031 (w), 1001 (w), 955 (s), 886 (w), 856 (w), 831 (w) cm−1. HRMS (ESI) (MeOH) positive ion: m/z = 371.1 M-Na+.
D. 3. Preparation of 2-((3-(tritylthio)propanoyl)oxy)ethyl acrylate (trtSPrOAcr):
2g of trtSPrAc (5.74 mmol, 1.1 eq.) was dispersed in freshly distilled dichloromethane (20 ml) and placed on an ice bath. 0.599 ml 2-hydroxyethylacrylate (5.22 mmol, 1 eq.) and then 52.69 mg DMAP (0.469 mmol, 0.09 eq.) was added to the cold solution. 1.20 g DCC (5.84 mmol, 1.12 eq.) was dissolved in 10 ml freshly distilled dichloromethane and added dropwise over a period of 10 min to the cold solution. After addition of the DCC the reaction mixture was stirred for additional 10 min before the ice bath was removed. The reaction mixture was stirred over night at room temperature. The reaction solution was filtered over silica and washed three times with 10 ml 0.5 M HCl and one time with brine. The organic layer was collected and dried over MgSO4. After evaporation of the solvent the residue was purified by column chromatography (Silica 60, hexane/ethylacetate 1:2). The first spot was collected to obtain the product as white powder in 73% yield.
1H-NMR (300 MHz, CDCl3, 25°C, TMS): 7.28 – 7.05 (15H, m, C6H5); 6.26 (1H, d, =CH2, cis); 5.96 (1H, dd, CH=CH2); 5.65 (1H, d, =CH2 trans); 4.14 (4H, m, O-CH2-CH2-O); 2.32 (2H, t, CH2-COO); 2.10 (2H, t, s-CH2) ppm. 13C-NMR (75.43 MHz, CDCl3, 25°C): 171.8 (CH2CO-O); 165.5 (CH-CO-O); 144.6 (4°C-C6H5); 131.6 (HC=CH2); 129.66 (ortho C C6H5); 127.9 (meta-C C6H5); 126.9 (para-C C6H5); 66.8 (COO-CH2-CH2); 62.1 (COO-CH2-CH2); 33.2 (COO-CH2-CH2-S); 26.88 (COO-CH2-CH2-S) ppm. IR (ATR): v = 3060 (w), 2934 (w), 1722 (s), 1633 (w), 1599 (w), 1488 (m), 1439 (m), 1415 (m), 1393 (m), 1368 (m), 1343 (m), 1343 (m), 1297 (m), 1239 (m), 1192 (s), 1169 (s), 1145 (s), 1067 (m), 1051 (m), 1033 (m), 998 (w), 982 (m), 955 (m), 881 (m), 810 (m) cm−1. HRMS (ESI) (MeOH) positive ion: m/z = 469.1 M-Na+.
D. 4. Preparation of 3-mercaptopropanoyl-oxyethyl acrylate (MPOEA):
trtSPrOAcr was dissolved in 20 ml freshly distilled dichloromethane and 2.6 ml Et3SiH was added. Subsequently 2.57 ml TFA was added slowly and the reaction was stirred for 3 hrs. Afterwards the reaction was concentrated, layered with 5ml ethylacetate and neutralized with saturated NaHCO3. The product was extracted three times with ethylacetate. The organic layers were combined, washed one time with brine and dried over MgSO4. After evaporation of the solvent a white precipitate was obtained which was purified by flash column chromatography (hexane/ethylacetate 2:1). The product was isolated as colorless oil with 82% yield.
1H-NMR (300 MHz, CDCl3, 25°C, TMS): 6.43 (1H, d, =CH2, cis); 6.11 (1H, dd, CH=CH2); 5.85 (1H, d, =CH2 trans); 4.36 (4H, m, O-CH2-CH2-O); 2.75 (2H, t, CH2-COO); 2.69 (2H, t, HS-CH2); 2.03 (1H, s, SH) ppm. 13C-NMR (300 MHz, CDCl3, 25°C): 171.8 (CH2CO-O); 166.2 (CH-CO-O); 131.3 (HC=CH2); 128.3 (CH=CH2); 62.2 (CH2-CH2-O); 60.2 (CH2-CH2-O); 38.3 (CH2COO); 19.3 (HS-CH2) ppm. IR (ATR): ν = 2958 (b), 2367 (w), 2322 (w), 1734 (s), 1636 (w), 1510 (w), 1443 (m), 1407 (m), 1371 (m), 1353 (w), 1254 (s), 1191 (s), 1155 (s), 1047 (s), 984 (w), 939 (w), 885 (w) cm−1. HRMS (ESI) (MeOH) positive ion: m/z = 227.03 M-Na+.
D. 5. Preparation of the photoresists and fabrication of compound structures:
Resists were prepared by mixing 10% wt. CEA or 5% wt. MPOEA with PETA, respectively. 1% wt. IRGACURE 819 as initiator was used for MPP and 0.25% wt. DETC as initiator was used for STED lithography experiments. For the non-functional 3D scaffold frame, PETA (without CEA or MPOEA) with 0.25% wt. DETC was used. Typical writing speeds of 50 µm s−1 were employed. For STED “depletion pattern” structuring, 10 µl of the resist were deposited on untreated high precision cover slips (0.17 mm, 5 µm tolerance, Carl Zeiss GmbH, Germany). For fabrication of 2D compound functional structures, piranha solution cleaned cover slips (Menzel Gläser, Germany) were used to avoid surface contamination. 0.5 µl of the MPOEA resist was deposited on the slide, followed by structuring of the first grid. Subsequently, 20 µl of the CEA resist were used to displace the MPOEA resist. Then, the second grid was written, followed by developing the structures with acetone. 3D structures were fabricated on piranha cleaned cover slips, functionalized with 1 mM 3-(trimethoxysilyl) propyl methacrylate in toluol (Sigma Aldrich) as adhesion promoter [7]. First, a 0.5 µl droplet of the PETA resist was deposited on the slide and the scaffold was written using 6.5 mW excitation power. The resist was exchanged with 5 µl of CEA resist and the rungs were written using 2.6 mW excitation power and 12 mW depletion power (bottle beam phase mask). Next, the resist was displaced using 20 µl of MPOEA resist and the rungs were written using 2.4 mW excitation power and 5 mW depletion power. Finally, the structure was developed with acetone.
D. 6. Preparation of the buffer solutions:
50 mM sodium carbonate (Na2CO3)/sodium bicarbonate (NaHCO3) buffer was prepared in double distilled water and adjusted to pH 9.6. PBS buffer (1x, pH 7.4) was used as received. Buffers were stored at 4°C.
D. 7. Conversion of the carboxylate groups:
The structures were incubated for 30 min. in an aqueous solution of 0.1 M NHS-OH and 0.4 M EDC and washed with PBS buffer subsequently.
D. 8. Labeling experiments:
A homemade reaction chamber (twinsil, picodent GmbH, Wipperfürth, Germany) is glued on the sample, followed by washing with PBS. 100 µl of PBS remain in the chamber. The glass surface was passivated with a lipid bilayer, leaving only the polymeric structures uncovered [50]. 10 µl of DOPC lipid vesicle solution (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids Inc., Alabama, USA) was added for 10 min, followed by washing with PBS. Stock solutions of the fluorophores in DMSO were prepared and stored at −20°C. For the labeling experiments, Cy5-maleimide stock solution was diluted 1:100 with PBS, 1 µl of the diluted solution was added to the reaction chamber and allowed to react for 15 min, followed by washing with PBS before the fluorescence signal was recorded.
Labeling of the carboxy functional structures was done after the carboxylate groups were converted in NHS esters. After washing with PBS, a bilayer is formed as described before. The sample was washed 3x with carbonate buffer (pH 9.6). For labeling of MPP fabricated CEA/PETA-structures, CF488A-amine stock solution was diluted 1:500 with H2O. For labeling of STED lithography structures, Cyanine3-amine stock solution was diluted 1:1000 with H2O. 1 µl of the diluted amine-fluorophore solution was added to the carbonate buffer on the sample. CF488A-amine or Cyanine3-amine readily binds via a NHS-amine coupling reaction, respectively. After 20 min. incubation, the sample was washed 3x with carbonate buffer before recording the fluorescence signal of the grids. For the 3D scaffolds, the carbonate buffer was replaced with glycerol to ensure refractive index matching before the fluorescence readout.
D. 9. NMR, IR and MS spectroscopy data:
Fig. 19 13C-NMR spectrum of MPOEA (CDCl3, 75.43 MHz). Residual solvent peaks stem from ethylacetate.
Appendix E Side view of 3D three component structure
Fig. 22 Magnified side view of Fig. 5(a). The rung-cross-sections are indicated by dashed ellipses. The rungs exhibit an aspect ratio of ~1.8.
Funding
Austrian Science Fund (FWF) (P26039, P26461) and basic funding PolFunk.
Acknowledgments
We would like to thank Heidi Piglmayer-Brezina for taking the SEM images, Markus Himmelsbach for HR-ESIMS measurements and Habed Habibzadeh, Bernhard Fragner and Alfred Nimmervoll for technical support.
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