Ultrahigh resolution, serial fabrication of three dimensionally-patterned protein nanostructures by liquid-mediated non-contact scanning probe lithography

Abstract

To mimic the functionality of the complex surface morphology of living systems, the fabrication of artificial 3D-patterned structures has attracted increasing attention and much progress has been made in recent years. Here, a liquid-mediated non-contact scanning probe lithography method was developed for the serial fabrication of patterned structures of protein. Very importantly, protein structures with different heights were successfully fabricated by precisely controlling the slow scan direction, set point value, and scan pixel density. What is more, sub-100 nm dot and sub-50 nm line arrays of protein structures were fabricated. This method opens up a new path for the rational design of surface-tethered 3D-patterned architectures directly from biomolecules.

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The immobilization of biomolecules on a solid surface in well-defined micro- and nanopatterns has attracted increasing attention because of its potential applications in proteomic research, drug discovery, tissue engineering, clinical diagnostics and some fundamental studies of biological processes.^1–7 Various strategies for fabricating small-scale patterned architectures of biomolecules including nanoimprint lithography,^8–10 micro-/nanocontact printing,^11–13 electron-beam lithography,^14–17 microstamping^18,19 and scanning probe lithography (SPL)^20–23 have been developed in recent years. As one of the scanning probe techniques, atomic force microscopy (AFM) has been widely used to characterize the nanometer-scale surface topography of different materials, especially biological samples in solution. Besides imaging, AFM-based lithographic techniques have been developed to fabricate micro- and nanostructures of biomolecules, such as dip-pen nanolithography,^24–27 conductive AFM lithography,²⁸ nanografting and nanoshaving.^29–32 In general, pretreatment of the AFM tip and modification of the solid substrate are needed for those AFM-based lithography techniques. This makes the process of patterning biomolecules more complex and time-consuming. What is more, unlike patterning of small biomolecules, the fabrication of high-resolution patterns of biomacromolecules (e.g. protein) are more challenging, owing to their much larger sizes, flexible conformation, complex chemical/physical property, and fragile bioactivity. Thus, it is important to develop facile and rapid AFM-based lithography methods for patterning biomacromolecules under a physiological environment, where their biological activity still can be well maintained.^33–38

To address the above challenges, herein, “liquid-mediated non-contact scanning probe lithography” (L-NCSPL), a facile and rapid AFM-based lithographic method is successfully developed to fabricate high-resolution three-dimensional (3D)-patterned structures of protein on a solid surface in a physiological environment. Unlike our previously-developed “liquid-mediated scanning probe nanosculpting” (LSPN) for the fabrication of high-resolution 3D-patterned polymer structures with the AFM tip operated in contact mode,³⁹ the present L-NCSPL uses an AFM tip operated in the non-contact mode in a protein solution. During the non-contact scanning process, protein molecules are trapped and pushed onto the scanned area by the AFM tip, forming patterned structures of protein within the scanning area.

This method has two unique features. First, the amount of protein deposition can be controlled by the slow scan direction during the non-contact scanning, i.e. the y scan direction. As the non-contact AFM operated in the top-to-bottom slow scan direction, a larger amount of protein are deposited on the scanned area, while just tiny amount of protein are deposited on the scanned area in the bottom-to-top slow scan direction. Therefore, patterning protein on a solid surface can be performed in top-to-bottom slow scan direction, while the in situ imaging of the as-made patterned structures of protein can be performed in bottom-to-top slow scan direction at the same time. Second, the amount of protein deposited at scanned area strongly depends on the set point value and scan pixel density of non-contact AFM. Therefore, patterned structures of protein with different height, i.e. 3D structures, can be fabricated by precisely controlling the fabrication conditions. We have demonstrated the L-NCSPL is capable for making sub-100 nm dots, sub-50 nm lines, and even grids with controllable height of protein structures.

As proof-of-concept, we first demonstrated the fabrication of patterned structures of bovine serum albumin (BSA), a widely-investigated model protein in experiments. Typically, a pre-cleaned Au substrate by piranha solution (Caution: piranha solution reacts violently with organic matter) was placed onto the bottom of the AFM liquid cell filled with a certain amount of a BSA solution (10 mg mL⁻¹ in PBS buffer). The Au surface was first imaged by non-contact AFM with a NCHR cantilever (NanoSensors, spring constant 42 N m⁻¹), as shown in Fig. 1a, there is no obvious deposition of BSA on Au substrate. The AFM tip operated in non-contact mode with set point of 20 nm was then used to scan two 1 μm × 1 μm squares on the Au surface in bottom-to-top and top-to-bottom slow scan direction, respectively, followed by in situ imaging with non-contact mode in bottom-to-top slow scan direction. As shown in Fig. 1b, interestingly, only tiny amount of BSA are deposited at scanned area when scanned by non-contact AFM in bottom-to-top slow scan direction, while in top-to-bottom slow scan direction a large amount of BSA are deposited at scanned area. To demonstrate the reproducibility of this phenomenon, we successfully fabricated arrays of structures of BSA by alternatively switching the slow scan direction both in X and Y scan directions (Fig. S1†). Indeed, a large amount of BSA were deposited and formed patterns in the top to bottom, left to right, and right to left directions (Fig. S2†). This results indicate that the slow scan direction indeed plays an important role in the fabrication of protein structures.

Fig. 1 (a) AFM topographic view of Au substrate obtained under BSA solution. (b) AFM topographic view of two square-shaped microstructures of BSA fabricated by non-contact scanning in bottom-to-top (left) and top-to-bottom (right) slow scan direction, respectively, as indicated in (a). (c–d) Schematic illustrations of the L-NCSPL. (c) Au substrate was scanned by non-contact AFM in bottom-to-top slow scan direction in a protein solution. Deposited protein at the scanned area would be removed by the next line scanning of the AFM tip. (d) Au substrate was scanned by non-contact AFM in top-to-bottom slow scan direction in a protein solution. Protein molecules were deposited at the scanned area and formed patterned structures.

After the process of patterning, the AFM tip was imaged with Scanning Electron Microscope (SEM). As shown in Fig. S3b,† some BSA molecules are adsorbed on the AFM tip after patterning BSA on the Au substrate. Considering that AFM tip oscillates just above the sample surface in non-contact AFM, there should be two possible deposition processes of BSA molecules during the non-contact scanning of the AFM tip. Probably, BSA molecules is first spontaneously adsorbed on the AFM tip and then transferred from the tip to Au surface.³⁴ On the base of this process, direct printing of proteins or small molecules by using cantilever arrays have been reported by Zheng et al.^35–38 Alternatively, BSA molecules in solution can be trapped by AFM tip first and then pushed to the Au surface. To elucidate the deposition mechanism, L-NCSPL experiment was performed by using AFM tip modified with oligo(ethylene glycol) derivative 11-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)undec-1-ene (C₁₁EO₃Me) which has proved to have excellent protein resistance.⁴⁰ Square-shaped microstructure of BSA can also be fabricated by L-NCSPL with this modified tip (Fig. S4†). Because the AFM tip is covered with such protein resistant molecules, the BSA molecules are not adsorbed on the tip (Fig. S3c and d†). Clearly, the deposition process is that the BSA molecules in solution are trapped by AFM tip first and then pushed onto the scanned area.

Based on the deposition process, we can look into the impact of the slow scan direction on the deposition of BSA. As the AFM tip oscillates just above the Au surface in non-contact scanning, local flow of protein solution is induced near the AFM tip indicated by the arrows in Fig. 1c and d, and protein molecules in solution are trapped and pushed by the AFM tip onto the Au surface at the scanned area. When non-contact AFM is operated in bottom-to-top slow scan direction, as shown in Fig. 1c, the XY-piezo scanner moves the Au substrate in the opposite direction of the local flow of protein solution. In this case, the deposited protein induced by the non-contact scanning of AFM tip will be easily removed by the next line scanning. However, as shown in Fig. 1d, when the non-contact AFM is operated in the top-to-bottom slow scan direction, the XY scanner moves the Au substrate in the same direction with local flow of protein solution, which is conducive to the deposition of protein on the Au surface at scanned area during non-contact scanning of the AFM tip and to fabricate corresponding patterned structures of protein. When the non-contact scanning is operated from left to right (or right to left), the moving direction of the substrate is perpendicular to the direction of the local flow, which has little effect on the protein deposition.

Interestingly, the as-made structures of BSA are robust on Au substrate. As for demonstration, square-shaped structures of BSA with an average height of 15.0 ± 0.7 nm were successfully fabricated by L-NCSPL (Fig. S6a†), followed by rinsing sequentially with PBS buffer and water, and drying under a stream of nitrogen. By imaging the structures in air by AFM, significantly, we find that the morphology and height (15.0 ± 0.6 nm) almost did not change (Fig. S6b†). This result indicates that the patterned structures of BSA fabricated by L-NCSPL on Au substrate are very stable.

Importantly, patterned structures of protein with different heights were successfully fabricated by precisely controlling the set point parameter during the non-contact scanning. In non-contact AFM, the set point value is an important parameter which correlates to the amplitude of the cantilever vibration. As the set point decreases, the amplitude of cantilever vibration and the tip-to-sample distance decrease. To investigate the impact of the set point on the amount of protein deposition on Au surface, a series of 1 μm × 1 μm squares on the Au surface were scanned by non-contact AFM with set point from 4 nm to 56 nm in top-to-bottom slow scan direction under BSA solution. As shown in Fig. 2a and b, the height of square-shaped microstructures formed by BSA deposition gradually increases from 0.6 nm to 22.1 nm as the set point increases from 4 nm to 14 nm, and then decreases from 21.1 nm to 0.4 nm when the set point is larger than 14 nm. The change of height is resulted from the different amount of BSA deposition onto the Au surface during non-contact scanning of the AFM tip. At small set point, the AFM tip is too close to the Au surface. Therefore, BSA molecules are too difficult to run into the narrow space between the AFM tip and the Au substrate, and then to be trapped by the AFM tip. So only a little amount of BSA molecules are deposited at the area scanned by non-contact AFM at small set point. As the set point increases, the tip-to-substrate distance increases. An increasing amount of BSA molecules can get into the area between the AFM tip and the Au substrate, and then are trapped by the AFM tip and pushed to the Au substrate. Therefore, the height of patterned structures of BSA increases with the increasing set point. However, when the set point is larger than 14 nm, the amount of BSA deposition at scanned area gradually decreases. In this case, tip-to-substrate distance is so large that the BSA molecules trapped by the AFM tip are too difficult to be pushed to the scanned area. Therefore, by precisely controlling the set point value, we are able to control the amount of protein deposition as well as the height of the patterned protein structures.

Fig. 2 (a) AFM topographic view of an array of 1 μm × 1 μm square-shaped microstructures of BSA fabricated by L-NCSPL with different set point from 4 to 56 nm in top-to-bottom slow scan direction. (b) Change of height of square-shaped microstructures of BSA as function of set point. (c) AFM topographic view of four 1 μm × 1 μm square-shaped microstructures of BSA fabricated by L-NCSPL with set point of 20 nm and scan pixel from 64 × 64 to 512 × 512. (d) Change of height for BSA patterned structures as function of scan pixel. (e) AFM topographic view of the patterned structure of BSA fabricated by using the non-contact mode AFM to continuously scan the same area of the Au surface with scan size of 5 μm × 5 μm, 4 μm × 4 μm, 3 μm × 3 μm, 2 μm × 2 μm and 1 μm × 1 μm, respectively. (f) AFM cross-sectional profile of dotted line indicated in (e).

More importantly, the amount of protein deposition can be fine tuned by the scan pixel density and thus 3D structures of protein can be readily fabricated. Notably, scan pixel density is one of the important parameters to fabricate 3D structures of polymer brushes as reported in our previous studies,^41–45 e.g. the higher the scan pixel density, the taller structures of polymer can be fabricated.⁴³ At the present study, a similar phenomenon for 3D patterning of protein structures was observed. For instance, Fig. 2c and d shows the incremental impact of scan pixel density on the height of patterned structures of BSA. Four 1 μm × 1 μm square-shaped structures of BSA were fabricated by non-contact AFM at set point of 20 nm with scan pixel of 64 × 64, 128 × 128, 256 × 256 and 512 × 512 in top-to-bottom slow scan direction. Since the deposition of BSA molecules onto the Au surface is induced by the non-contact scanning of the AFM tip, the amount of deposition of BSA molecules increases with scan pixel density of scan area increasing. Therefore, scan pixel density can be used to control the height of patterned structures of protein. On the other hand, 3D structures of protein can be fabricated by tuning the scan size with the same scan pixel. Fig. 2e and f show the patterned structures of BSA fabricated by using non-contact AFM to continuously scan the same area with scan size of 5 μm × 5 μm, 4 μm × 4 μm, 3 μm × 3 μm, 2 μm × 2 μm and 1 μm × 1 μm, respectively. With the same scan pixel, the height of patterned structures of protein increases as the scan size decreases.

In addition, array of sub-100 nm dots and sub-50 nm lines of protein structures can be fabricated by using L-NCSPL. Taking for demonstration, 6 × 6 BSA nanodots array were successfully obtained, each fabricated by using non-contact AFM with set point of 20 nm and scan pixel density of 10 × 10 to scan squared area at Au substrate with size of 10 nm × 10 nm in top-to-bottom slow scan direction. As shown in Fig. 3a, BSA molecules form uniform nanodots array. The height and the full-width-at-half-maximum (FWHM) of the nanodots are 5.5 ± 1.0 nm and 76 ± 8 nm, respectively (Fig. 3b). Array of BSA nanolines can also be fabricated by L-NCSPL, in which each nanoline is obtained with scan size of 2 μm × 0.01 μm (Fig. 3c). The height and FWHM of the nanolines are 2.7 nm ± 0.5 and 47 ± 5 nm, respectively (Fig. 3d).

Fig. 3 (a) AFM topographic view of a 6 × 6 BSA nanodots array fabricated by L-NCSPL. (b) AFM cross-sectional profile of dotted line indicated in (a). (c) AFM topographic view of array of BSA nanolines fabricated by L-NCSPL. (d) AFM cross-sectional profile of dotted line indicated in (c).

We also tried to use L-NCSPL to fabricate patterned structures of human plasma fibrinogen and lysozyme under physiological condition. When imaging the Au substrate under lysozyme solution (10 mg mL⁻¹ in PBS buffer), as shown in Fig. S7b,† a densely packed layer is quickly formed due to the adsorption of lysozyme from solution. Thus, L-NCSPL is not applicable to pattern lysozyme on Au substrate. In contrast, Fig. S7c† shows that patterned structure of fibrinogen can be successfully fabricated in fibrinogen solution (1 mg mL⁻¹ in PBS buffer). Note that a high concentration of fibrinogen solution is not suitable for the experiment due to its high viscosity. BSA and fibrinogen are negatively charged in PBS buffer due to their isoelectric point lower than 7.4, and they have weakly electrostatic interaction with Au substrate. Yet, lysozyme is positively charged in PBS buffer because of its isoelectric point of 11.1 and it tends to adsorb onto the Au substrate with the strong electrostatic interaction.⁴⁶ Thus, L-NCSPL is suitable to pattern protein on substrate when the protein interacts weakly with the substrate. To further demonstrate this assumption, we tried to fabricate patterned structures of BSA on 1-dodecanethiol modified Au substrate which is very hydrophobic. The strong interaction between BSA and this substrate results in spontaneous adsorption, which causes the failure of patterning BSA on the substrate (Fig. S8†). Fig. S7d† shows the relationship between the height of patterned structures of fibrinogen and set point. The height of patterned structures of fibrinogen first increases and then decreases with increasing set point. Similar phenomenon is observed in the case of BSA. Comparing Fig. S7d† with Fig. 2b, we know that the set point corresponding to maximum height of patterned structures of fibrinogen is larger than that of BSA. Because fibrinogen (M_w ∼ 3.4 × 10⁵ Da) has a larger size than BSA (M_w ∼ 6.8 × 10⁴ Da), larger tip-to-substrate distance is required for fibrinogen to run into the area between the AFM tip and Au substrate and then be trapped by AFM tip. Therefore, larger set point is conducive to fabricate patterned structure of fibrinogen due to its larger size. When we choose set point in patterning protein on Au substrate by using L-NCSPL, protein size should be considered.

Conclusions

In conclusion, we have developed L-NCSPL technique performed with non-contact AFM. L-NCSPL is a simple, rapid and low cost AFM-based lithography for sub-100 nm or even 3D patterning structures of protein under physiological conditions on the basis of the rational control of the lateral spacing between the assembled protein molecules. To the best of our knowledge, this is the first realization of serial fabrication 3D-patterned structures of protein by using the AFM lithography. This finding not only opens a new path for the fabrication of 3D structures with biomaterials, but also enables a better understanding of the assemble behavior of protein molecules in solution. Therefore, we envision that the L-NCSPL method can not only be directly applied to fabricate functional surfaces with precisely control surface chemistry and morphology, but also to be a platform to investigate the behaviour and properties of biomacromolecules.

Acknowledgements

We acknowledge the Ministry of Science and Technology of China (2012CB933802), the National Natural Science Foundation of China (21234003 and 21404072), Shenzhen Science and Technology Foundation (JCYJ20140418182819116) and the Natural Science Foundation of SZU (827000040 and 201447) for financial support of this work, and Prof. Vincent S. J. Craig (The Australian National University) for the helpful discussions.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental section, schemes (S1), SEM images (S1), AFM images (S2, S4, S6–S9), XPS data (S3). See DOI: 10.1039/c6ra07715c

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