High-Precision Micropatterning of Polydopamine by Multiphoton Lithography

PDA deposition based on multiphoton lithography (MPL) is demonstrated, which enables full spatial and temporal control with nearly total freedom of patterning design. Using MPL, 2D microstructures of complex design are achieved with pattern precision of 0.8 µ m without the need of a photomask or stamp. Moreover, this approach permits adjusting the morphology and thickness of the fabricated microstructure within one deposition step, resulting in a unique tunability of material properties. The chemical composition of PDA is confirmed and its ability for protein enzyme immobilization is demonstrated. This work presents a new methodology for high-precision and complete control of PDA deposition, enabling PDA incorporation in applications where fine and precise local surface functionalization is required. Possible applications include multicomponent functional elements and devices in microfluidics or lab-on-a-chip systems.


Introduction
Inspired by nature, polydopamine (PDA) confers unique chemical and physical properties to the surface of nearly any material irrespective of its chemical composition. By strongly adhering it enhances the surface hydrophilicity, chemical reactivity, and permits facile post-modification by a broad range of molecules advanced PDA applications. Employment of innovative microfabrication techniques such as multiphoton lithography (MPL) is one of the solutions to overcome aforementioned challenges.
MPL is a state-of-the-art microfabrication technique [26] utilized to produce 2D and 3D objects with a high spatial resolution (0.1-1 µm [27] ) by material solidification (or ablation) under the exposure of a tightly-focused femtosecond NIR laser beam. With this technique, the material transformation occurs only in the volume of the focal spot (voxel) where the photon density of the fs laser pulses exceeds TW cm −3 , and therefore, is sufficiently high to induce two-photon absorption (TPA) processes. In this fashion, by moving the focal spot through the unconverted material in three dimensions, 2D or 3D structures can be created with high precision. Compared to Vat polymerization additive manufacturing methods, [28] MPL stands out by enabling high spatial resolution and an almost unrestricted 3D structuring capability. Additionally, neither layer-by-layer approaches nor photomasking/stamping is required. Moreover, intricate features can be made in regions of structures that are impossible to access using other techniques. (e.g., within a microfluidic channel).
Herein, we report a new MPL-based approach for surface modification that offers high-precision, maskless or stampless, localized 2D PDA deposition in any desired shape of micropatterns. We use a benzylidene cyclopentanone-based photo initiator (BAE) in the mixture with dopamine to trigger the PDA formation. The microstructure, therefore, appears as a result of controllable scanning of the focal spot of the MPL laser at the substrate interface. Via TPA processes, the photoinitiator (PI) generates an exited electron transfer complex and thereupon active radical species promoting PDA formation. The chemical composition of PDA is verified from the recorded local vibrational spectra of microstructures. Neither strong oxidants, metal ions nor adjusting pH to alkaline is required for this approach. PDA pattering can be performed in alkaline, neutral, and slightly acidic solutions. The morphology and thickness of polydopamine microstructure (PDA-MS) can be tailored by adjusting the beam scanning velocity and the laser power. With inherent benefits, this technique empowers PDA application in complex microdevices where a high level of temporal and spatial control is essential for device functionality and performance.

PDA Fabrication with MPL Technique
We achieved 2D PDA micropatterning from aqueous solutions containing DA and BAE PI by applying MPL technique. The setup uses either an air or oil-immersion objective, with the laser beam focusing on the interphase between the substrate and the DA solution (Figure 1a). Determined by the optical properties and thickness range of the applied substrate, we suggest two different DA solution-substrate configurations ( Figure S1, Supporting Information). PDA build-up was observed following the scanning path of the light focal spot (Videos S1 and S2, Supporting Information) and resulted in PDA-MS fabricated at unprecedentedly high precision ( Figure 1b). Adjustment of the beam scanning velocity and laser exposure power during MPL fabrication allows tailoring of the morphology of the PDA surface ( Figure 1c) and influences the surface roughness ( Figures S2 and S3, Supporting Information). A smooth, bulky-like (Figure 1c-1) topography is achieved at low velocities and beam power intensities (e.g., 20-50 µm s −1 ; 10 mW, respectively), whereas an increase of these parameters leads to the formation of rougher (Figure 1c-2), void- (Figure 1c-3) and finally grain-like surfaces (Figure 1c-4). The density of the PDA grains is inversely proportional to the fabrication speed and can therefore be adapted to application requirements. Thus, with the flexibility of MPL, PDA-MS featuring a combination of different surface morphologies and/or their gradients within one fabrication procedure are achieved.
The thickness of the deposited structures was influenced by the MPL fabrication protocol (Figure 1d). An increase of the fabrication speed to 200 µm s −1 allows thinner structures in the range of 30-150 nm to be obtained, whereas slowing down the scanning speed results in more than 150 nm thicker structures. This correlation was observed for both objectives applied in this study.
To demonstrate the universal adhesion behavior of PDA, PDA-MS were fabricated on substrates of different nature ( Figure S4, Supporting Information). PDA was successfully microstructured on polychlorotrifluoroethylene and polyethylene terephthalate films, silicon wafers, and fluorinated glass slips. Moreover, PDA-MS were selectively produced within the channel of polydimethylsiloxane (PDMS)-based microfluidic devices ( Figure S4, Supporting Information).

Polydopamine Micropatterns: Formation Conditions, Topography, and Chemical Composition
In contrast to reported conventional PDA coatings, PDA-MS fabricated with the MPL technique is, to the best of our knowledge, a novel approach. Herein, we focus on the understanding of PDA-MS build-up, its chemical structure, and the influence of the solution composition (e.g., buffer, pH, reactive oxygen scavengers (ROS)) on the PDA-MS topography.

PDA-MS Thickness
The average thickness values of PDA-MS fabricated from the range of dopamine solutions were obtained by scanning the microstructures applying atomic force microscopy (AFM) in tapping mode (illustrated in Figure 2a). The results are plotted versus the applied beam power at the scanning velocities of 50 (Figure 2b,c) and 200 µm s −1 ( Figure S5a,b, Supporting Information). We find that the PDA-MS morphology and thickness can be significantly affected by the pH and the solution medium leading to the change in the average thickness from 40 nm up to 900 nm (Figure 2b,c). This effect is less pronounced at 200 µm s −1 ( Figure S5a,b, Supporting Information) where structures with thicknesses between 40 and 400 nm are obtained. The PDA-MS topography and surface morphology differ from rough and grain-like to relatively smooth and compact, therefore, effecting the average thickness values and fabrication throughput estimation. In order to better correlate the thickness to PDA-MS topography, Scanning electron microscopy (SEM) images of the samples were recorded (Figures S6-S17, Supporting Information).
In most of the studied solutions with Tris buffer and no additives, we observe that lower beam energies are in favor to obtain thicker structures (Figure 2b,c). This tendency indicates that the thermal effects and avalanche ionization leading to the build-up of heat in the material at high beam intensities [29] have no significant contribution to the PDA formation. Moreover, it was reported that elevated temperatures speed-up auto-oxidation of dopamine. [30] This phenomenon would be expected at higher MPL laser intensities, nevertheless, it was not observed for Tris buffer solutions. This suggests a different nature of the processes underlying the MPL-initiated PDA build-up in the presence of Tris buffer at pH 8.5.
On the other hand, the solutions with phosphate buffer or only DI water as well as most of the formulations in ROS inhibiting environment (e.g., with ascorbic acid, ectoine, or argon-enriched solutions) revealed an increase of thickness with increasing beam intensity (Figure 2b,c). This difference in behavior suggests that the Tris buffer is chemically involved in the PDA formation, whereas the phosphate buffer is not. [31][32][33] Moreover, we observed the formation of high-quality PDA-MS at neutral pH in 0.1 m Tris buffer solution, in contrast to very poor to no outcome detected when phosphate buffer was used instead (in the range of 0.1 to 0.35 m). PDA-MS could also be deposited by using DI water without buffering option. However, the deposited microstructures were thinner, indicating a decreased efficiency of PDA build-up in DI water solutions. In conclusion, Tris formulations are considered for further development of MPL deposition method.
We then examined the effects of Tris buffer concentration on the deposition efficiency of PDA-MS. In good agreement with literature, 0.1 m Tris concentration provided an optimum environment for PDA deposition at pH 7 and 8.5. We observe the formation of PDA patterns with an average thickness ranging between 50 and 315 nm ( Figure 2b, Figure S5a  750 nm (Figure 2b), which is an outstanding deposition amount compared to previous studies. [12,31,34,35] Nevertheless, further increase of Tris concentration up to 0.2 m revealed an inhibiting effect leading to thinner structures. That seems to explain thicker PDA-MS structures obtained for argon-enriched solutions (Figure 2c) where the gas purging possibly led to water evaporation and, therefore, to an increase of Tris concentration comparable to 0.15 m. MPL fabrication of PDA was possible at pH 8.5, standard conditions used for auto-oxidation of dopamine, as well as from the neutral (pH 7.0) and acidic (pH 6.0) solutions (Figures S6, S7, and S16, Supporting Information). We were able to achieve tightly packed PDA structures for both, alkaline and neutral solutions, with a pattern average thickness of up to 315 and 250 nm, respectively. However, the structures formed under acidic conditions (DI, pH 6.0) exhibited a low density of coverage ( Figure S16, Supporting Information), showing limited efficiency of PDA formation when compared to basic solutions (DI, pH 8.5, Figure S17, Supporting Information). Moreover, the average thickness of the PDA at pH 6.0 did not exceed 100 nm. This effect of acidic pH can be explained by the hindrance of amino group deprotonation required for the intramolecular Michael addition during PDA build-up. [36] Summarizing, we suggest pH 7.0 ( Figures S4 and S6, Supporting Information) to be a good alternative with minimized side effects of auto-oxidation processes, therefore, leading to minimized PDA contaminants on the substrate.

PDA Chemical Composition
AFM-based infrared spectroscopy (AFM-IR) is a hybrid technique where chemical characterization provided by infrared (IR) spectroscopy can be obtained with a spatial resolution of  AFM. [37,38] This is realized by using gold-coated tips to locally detect the thermal expansion of a sample resulting from local absorption of IR radiation and leading to AFM cantilever deflection (Figure 2d). Consequently, in this method, the AFM cantilever acts as the IR detector. Therefore, AFM-IR technique overcomes the spatial resolution limits of conventional IR microscopy and permits to collect local information (the surface area around 30 nm) from MPL produced PDA-MS structures.
The spectra obtained by AFM-IR confirm the PDA structure of the MPL-formed micropatterns. The IR absorption spectrum of PDA-MS fabricated from Tris buffer (Figure 2e) indicates the presence of two IR bands centered at 1610 and 1518 cm −1 that corresponds to the ring structure (benzene and nitrogen heterocycle) of PDA. The results are consistent with indole and/or indoline structures proposed earlier elsewhere. [39,40] The peaks located in 1453-1426 cm −1 range are typical for C-H stretching vibrations. The CO vibrations of PDA-MS present in the range of 1730-1722 cm −1 are more pronounced than that of conventionally auto-oxidized PDA films reported before. [34] For conventional PDA oxidation, the typically observed peak at 1722 cm −1 can be assigned to the quinone structure formed by autooxidation of dopamine or to oxidation products (e.g., CO or COOH) of residual OH groups. [34,41] On the other hand, CO peak in the range of 1722-1730 cm −1 for PDA films formed by auto-oxidation in Tris buffer was not observed ( Figure S18a, Supporting Information). Consistently, the origin of the CO band in MPL-fabricated PDA-MS could be a contribution from the BAE PI, which also exhibits a peak at 1737cm −1 ( Figure S18c, Supporting Information). Since PDA build-up is supposed to be initiated via interaction with excited BAE states, the formation of PDA-BAE structures cannot be excluded.
In Figure 2f, the CO region with a broad peak in the range of 1730-1694 cm −1 for MPL PDA-MS structures from different buffer solutions is compared. A shift of the CO band towards lower wavenumbers with a shoulder at 1694 cm −1 observed for PDA from Tris buffer can be attributed to the interactions of Tris buffer with the PDA via H-bonds. A similar shift was detected for auto-oxidized PDA. [42] This confirms the impact of Tris buffer on PDA build-up, as discussed above. In contrast, almost no shift of CO vibrations was observed for PDA from a phosphate-based medium or DI water solution.
In addition to AFM-IR, we performed X-ray photoelectron spectroscopy (XPS) analysis to evaluate the chemical groups of the formed PDA-MS. Moreover, its surface ability for postmodification with trypsin was tested. The XPS survey revealed signatures originating from C, N, and O atoms, identifying the PDA nature of the microstructures (Figure 3a-d). The results are in good agreement with PDA obtained by UV-exposure [19] or by conventional oxidation in alkaline medium. [17] The atomic concentrations of the main elements are given in Table S1, Supporting Information. The functional groups were further analyzed quantitatively by deconvolution of C 1s, N 1s, and O 1s peaks ( Figure S19, Supporting Information, and Table 1  and CN bonds, suggesting both catechol and quinone groups. The presence of CO observed by AFM-IR can be confirmed in the C 1s peak with its maximum at 287.7 eV. These functional groups were also identified by XPS in conventional PDA being assigned to quinone and carboxyl oxygen in OCO groups. [17] Further, the cyclization of dopamine is confirmed by the presence of aromatic N groups (399.7 eV). However, the major contribution to N 1s is originated from CNH groups and not from aromatic N. This identifies the presence of open-chain dopamine units in the structure or/and inclusion of Tris buffer into the PDA structure.

PDA Post-Functionalization with Trypsin
The immobilization of enzymes is of great importance in numerous biomedical applications including protein detection. Herein, we demonstrate the ability of our PDA-MS to act as a primer for covalent immobilization of protein enzymes based on a facile one-step surface post-modification of the PDA-MS with trypsin. The difference in chemical composition before (PDA-MS) and after trypsin immobilization (PDA-MS-Try) is inferred from differences in the respective XPS spectra. Figure 3 shows an alternation in C 1s, N 1s, and O 1s intensities ( Figure 3a) and peak shapes (Figure 3b-d) caused by the presence of trypsin. We observe an elevation of the N 1s signal (Figure 3a) and an increase of the N/C ratio by a factor of two (Table S1, Supporting Information) in PDA-MS-Try compared to PDA-MS. This is due to the raise of aliphatic N-contained groups (CNH 3 + , CNH) present in trypsin in contrast to the aromatic N, which is inherent to PDA (Table 1). Moreover, the C 1s peak of PDA-MS-Try shows increased amounts of COH, CN, and CO groups (285.8, 287.7 eV) that are typical for polypeptides [43] and reveals the presence of trypsin. Similar to our observations, an increase of N 1s and CO was previously reported as an indication of trypsin immobilization for conventional PDA. [43] Those changes in the surface composition of our PDA-MS confirm their successful surface post-functionalization with trypsin.

MPL Performance
DA microstructures were fabricated in this work by using highprecision oil-immersion ( Figure S20, Supporting Information) or long-working-distance air objectives (Figures S6 and S17, Supporting Information). The first one achieves a higher resolution of the structures, whereas the air objective is more convenient due to large working distances allowing fabrication onto complex surfaces with a different axial profile.
We found that air objective B allows for a wider range of fabrication powers (up to 40 mW) and enables thicker structures (Figure 1b) to be deposited. Considering this and the benefits of the air objective with respect to pattering on curved complex surfaces, objective B was chosen for further experiments.
The PDA ornaments shown in Figure 4a-d and Figure S21, Supporting Information, are an example of the accuracy and design freedom of the MPL technique. During the PDA pattern fabrication, no PDA formation was observed in the parts where no laser exposure was applied. Using freshly prepared (Figure 4a) solution of DA (pH 7.0 in 0.1 m Tris) and the same solution stored for 24h at room temperature (Figure 4b), different morphologies of PDA ornaments were achieved for the same printing protocol. A grain-like morphology of the ornaments results from fresh solution, whereas denser and thicker structures were achieved from the medium stored for 24 h. This can be attributed to the more efficient orientation of the DA, Tries, and BAE molecules in the system facilitating molecules interactions during beam exposure. However, the minimum pattern linewidth increased from 1.8 ± 0.2 µm (Figure 4a) to 4.5 ± 0.5 µm (Figure 4b) with storage time. By adjusting the structure design, we achieved the micropattern with linewidth in the range of 0.8 ± 0.1 µm ( Figure S21, Supporting Information).
The choice of the buffer also influences the structure topography and the overall quality as seen for PDA-MS produced from 0.1 m of Tris and phosphate solutions (Figure 4c,d). In  The atomic concentration of each functional group = the percentage of each functional group in the corresponding element × the atomic concentration of the corresponding element in all atoms; b) The uncertainty for the peak position is +/−0.2 eV. The relative uncertainty for the atomic concentration is +/−20%. The uncertainties are applied for a confidence value of 95%. One possibility (path 1) of the BAE complex excited-state species is an electron-transfer complex (I) which was previously suggested as an initial process in the photoreaction of ketones and amines. [44,45] In this case proton transfer leads to the formation of ketyl, aminoalkyl, and biradicals (III-V). The decay of excited species then proceeds via back electron transfer. Another possibility (path 2) is an excimer (II) formed as a result of intramolecular electron transfer. Its decay is followed by the proton transfer giving radicals (III-V).
summary, both fabrication parameters and solution composition and storage time have to be considered while designing the microstructure of interest.

Polymerization Mechanism
The PDA micropatterning with MPL can be achieved only in the presence of the PI active to the MPL fs-laser beam (780 nm) at energies exciding the threshold of photoexcitation. Except for solutions without PI, the PDA structures were formed from all formulations tested in this work. Photopatterning was possible not only in alkaline solutions (pH 8.5) but also in neutral (pH 7.0) and slightly acidic (pH 6.0), in the presence of ROS scavengers, and different buffers like Tris or phosphate as well as in DI water only. The oxygenation of the solution prior to MPL seemed to have no noticeable effect on the thickness and quality of the PDA patterns (Figure 2b 0.2 m Tris with and without O 2 purging, Figures S7 and S8, Supporting Information). In addition, no decrease in performance was detected when purging solutions with argon ( Figure 2c, Figure S13, Supporting Information). Moreover, a decline of PDA formation was detected only at 0.5 m concentration of ectoine radical scavenger ( Figure S11, Supporting Information), whereas no significant effect was observed for 0.2 m ectoine solutions ( Figure S10, Supporting Information). All these observations question the mechanistic route of the MPL in the presence of the PI where auto-oxidation of DA and ROS initiation seems not to be the major pathway for PDA build-up.
The auto-oxidation of dopamine to PDA, usually observed in alkaline media, is a very slow process that generally takes from several hours to several days. [46] In contrast, the formation of a visible layer of PDA during MPL is a matter of minutes, considering that a certain PDA volume should be formed in order to be detectable with the MPL camera (Video S3, Supporting Information). We observe the deposition throughput to form PDA monolayer is between 0.036 and 0.144 mm 2 h −1 depending on the laser scanning velocity (details in Supporting information). Concluding, we observed that MPL-triggered PDA formation exhibits speed more comparable to those observed for accelerating harsh conditions involving radical formation (e.g., oxidant-free microwaving, [47] plasma-activated water [48] ).
Several DA polymerization pathways and the influence of experimental parameters on the structural properties and the mechanism of PDA growth are suggested in the literature. For instance, DA polymerization via the photochemical route has been shown to be initiated either by generation of ROS under UV, [18,19] in the presence of strong acridinium oxidant [21] active in the visible domain, or by an electron-transfer mechanism involving DA and pyrene photosensitizer with diphenyliodonium (DPI) salt. [20] In the first two cases, UV-and Vis-light triggered PDA deposition, the presence of oxygen was important for oxidation processes and its decline showed a decreased or even no PDA formation. In the third system, the process takes place in oxygenfree conditions where the DPI excited state would lead to the formation of radicals and radical cations and is suggested to lead to DA oxidation via photoinduced electron-transfer between PI and electron-donating hydroxyl units on the aromatic ring of DA. [20] Moreover, the presence of other radicals can also promote PDA formation. For instance, by abstracting a hydrogen atom from the dopamine monomer promotes the generation of DA radical species. [49,50] PDA deposition can also be achieved in acidic conditions either in the absence of oxygen [48] or in the presence of oxidants. [9,51] Here, the polymerization route was suggested to proceed via the formation of dopamine-semiquinone radical [11] and free radical polymerization mechanism. [52] All abovementioned represents the mechanistic versatility of PDA formation and fosters the hypothesis of electron transfer and radical formation mechanisms being present in the MPL driven PDA build-up presented here.
Taking a closer look at the photoinitiation process, the here used BAE is a centrosymmetric D-π-A-π-D chromophore (where D, π and A are donor, π-conjugated bridge, and acceptor, respectively) that proceeds the photolysis via TPA mechanism leading to the excited triplet state. In its structure, tertiary amines are electron-donating groups and carbonyl of cyclopentanone is an electron-withdrawing group. It was shown that the formation of reactive species of PIs with similar structures can include intra-and intermolecular charge transfer, [53,54] hydrogen abstraction; [55] moreover, it can lead to ROS formation. [56] Referring to the research on photoinitiation mechanism of ketone/amine systems [57][58][59] and given similarities between BAE and its functional analog, Michler's ketone, the mechanistic route of BAE photolysis is suggested in Figure 4e resulting in the formation of ketyl, aminoalkyl, and biradicals.
Summarizing, we propose the PDA formation in the presence of BAE being based on electron-transfer mechanisms that can include the following: i) hydrogen abstraction from DA by the electron acceptor groups of BAE electron-transfer complexes resulting in DA-based radicals, ii) generation of singlet oxygen ( 1 O 2 ) by interacting of dissolved oxygen with BAE* that leads to DA oxidation, and iii) attack of DA by BAE radicals (Figure 4e III-V) leading to the formation of DA radicals and their further polymerization, possibly also via a free radical mechanism as suggested elsewhere. [52]

Conclusion
We propose a solution to the limitations of PDA deposition exhibited by existing conventional methods. In this work, for the first time, the deposition of fine 2D PDA microstructures applying the MPL technique was presented. An organic PI was used to promote PDA build-up under NIR fs-pulsed laser beam light. The chemical information collected by AFM-IR and XPS on the surface of patterns confirmed the PDA nature of microstructures. We found that fabrication is feasible under different conditions such as alkaline, neutral, and slightly acidic pH of different buffers, as well as with DI water only. Moreover, the morphology and thickness of the micropatterns can be well controlled by scanning velocity and power of the laser beam, thus, revealing the possibility of creating structures with a topography gradient. High control over pattern design, as well as spatial and temporal deposition with resolution down to 0.8 µm of PDA-MS elements, was achieved without the need for a photomask or stamp. The mechanistic route of PDA build-up is based on the electron and proton