Stimuli-responsive protein-based micro/nano-waveguides

Abstract

In this letter, customized protein-based micro/nano-wires with diameters of ≥150 nm have been facilely fabricated from bovine serum albumin (BSA) aqueous ink by maskless and noncontact femtosecond laser direct writing (FsLDW) technology. With the reproducible optimized morphology, the protein-based micro/nano-wires here can be applied as optical micro/nano-waveguides with transmission loss of ∼0.059 dB μm⁻¹ for 633 nm light. Based on the nature of as-formed protein micro/nano-hydrogels (stimuli-responsive shrinking or swelling), their “smart” environmental stimuli (e.g., pH) responsive features are proved experimentally (∼50% output light decrease corresponding to pH value tunning range of ∼0.4).

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Recently, biopolymeric hydrogel based optical waveguide devices are drawing increasing attention and efforts for biosensing and biomedical applications motivated by their excellent biocompatibility, biodegradability, customizable flexible mechanical characteristics, and facility of functionalization.^1–4 In particular, the micro/nano-scale biopolymeric hydrogel based waveguide devices may produce significant improvements of response speed,^3,5 integration,^5,6 and portability.^1,3 Compared to the hot topic nowadays of graphene-based materials,^7–10 polymeric hydrogels have distinct advantages including biocompatibility, abundance, renewability, environment-friendship,^11–17 protein-based biopolymers (e.g., silk,^2,11 gelatin,³ albumins,^12–15 …) are important and irreplaceable building materials in some situations for promising novel hydrogel optics among numerous artificially synthetic and natural product alternatives.^18,19 More importantly, it is of great help for the facile functionalization of micro/nano-waveguide devices without complex subsequent modification to use proteins as building materials for their intrinsic biological, chemical and physical properties, for instance, stimuli-responsiveness^13,14,20 and enzymatic activity.²¹ Up to now, numerous fabrication methods have been developed to produce optical element, such as transferring-and-printing processes, self-assembly, nano-imprinting, and hot-embossing technique.^2,22–27

However, either expensive masks or cumbersome operations were needed, or nanoscale processing precision, high designability or reproducibility was difficult to be achieved in previous related work. Therefore, in this letter, we applied promising femtosecond laser direct writing (FsLDW) to fabricate protein-hydrogel-based stimuli-responsive single micro/nano-wire optical waveguides from bovine serum albumin (BSA) aqueous ink. The noncontact and maskless FsLDW implementation scheme here comprehensively and simply realized relative facility,^28,29 good designability,^30–32 extensive applicability,^28–35 high spatial resolution down to sub-20 nm resolution.^36,37And high-quality customization of functionalized protein-based optical micro/nano-waveguides were ensured with nanoscale spatial resolution (diameters ≥ 150 nm for BSA nanowires), excellent morphology (∼5 nm average roughness),¹⁴ acceptable optical performances for sensing applications (transmission loss of ∼0.059 dB μm⁻¹ for 633 nm light), and environmental stimuli responsiveness (pH value here), which might be useful for integrated micro/nano-biophotonics and biosensing.

In the experiment, as-designed protein-based micro/nano-wire waveguides were directly and facilely “written” out on the MgF₂ slice from BSA aqueous ink (BSA, 500 mg mL⁻¹; photosensitizer methylene blue, 0.6 mg mL⁻¹) by FsLDW as shown in schematic of Fig. 1(a). Two-photon absorption of methylene blue occurred in the core region of laser beam focal spot (femtosecond laser, 80 MHz repetition rate, 120 fs pulse width, 790 nm central wavelength; oil-immersion objective lens (OL), 60× , numerical aperture, 1.35),which produced singlet oxygen (1O₂). 1O₂ further catalyzed crosslinking of BSA molecules with photosensitive groups (Tyr, Trp, His, Met, Cys, etc.).^8–10 Combined with computer-programmed 3D scanning of the focused laser spot (vertical scanning by a piezo stage (PZT), horizontal scanning by a two-galvano-mirror set),³⁸ arbitrarily designed micro/nano-wire waveguide devices could be constructed after necessary water rinsing to remove residual BSA ink. Here, MgF₂ substrate was applied for its lower refractive index (1.39) than that of as-formed protein hydrogel (∼1.55 in air and ∼1.45 in water),^13–15 which met the requirements of total reflection and light propagation along the waveguides (see Fig. 1(b)).⁴ With a silica fiber nano-taper, incident light was launched into the protein single micro/nano-wire waveguide by evanescent field coupling as illustrated schematically in Fig. 1(c). And the output light could be collected and measured at the end of the waveguides with a CCD camera or a spectrum detector.

Fig. 1 (a) Schematic diagram of FsLDW of protein single micro/nano-wire waveguides. (b) Optical microscopic (OM) image of as-prepared protein single micro/nano-wire waveguides on MgF₂ substrate. Scale bar, 10 μm. (c) Schematic of the test of a protein single micro/nano-wire waveguide.

During FsLDW processing, parameters such as laser power (∼20 mW, measured before OL), exposure time (1000 μs at every point) and scanning step (100 nm in three dimensions (3D)) were sufficiently optimized to achieve outstanding quality of morphology including device geometry as-designed and high surface smoothness (∼5 nm Ra)¹⁴ [Fig. 2]. Importantly, the highly reproducible excellent morphology was guaranteed by table FsLDW system equipped with an output feedback system and proper enclosure. Additionally, the local temperature (22 ± 0.2 °C) and humidity (relative humidity, ∼20%) around the laser was maintained constant in the super clean lab. Therefore, protein-based single micro/nano-wires with high morphology quality, different diameters faithfully as designed and satisfactory repeatability could be customized by the optimized FsLDW [Fig. 2]. Protein nanowires with diameters from 150 nm to 650 nm were written out exactly as designed by a computer. The uniformity of every set of micro/nano-wires in Fig. 2(a) well proved the good repeatability and controllability of positioning and geometry of FsLDW preparation. Fig.2(b) is the partially enlarged view of Fig. 2(a), which exhibits the high surface and structure quality. By atomic force microscopy (AFM) characterization [Fig. 2(c)], surface Ra was measured to be ∼5 nm which is smooth enough for waveguiding applications.⁴ In addition, 3D geometry of the protein micro/nano-wires was proved to be like partial cylinder [Fig. 2(d) and (e)]. This might be caused by so-called self-smoothing effect that was also helpful for achieving lower surface roughness.^13–15,39 In our previous work, the transmission loss of the protein-based single microwire waveguide was estimated to be ∼0.0592 dB μm⁻¹.⁴⁰ Although the 59 dB mm⁻¹ transmission loss does be high compared to existing polymer micro/nano-waveguides (usually less than 0.1 dB mm⁻¹),⁵ the prototyping protein-based optical waveguide micro/nano-devices here show great potential to be used as ideal platforms for flexible functionalizations (especially for biorelated applications).^15,17 Meanwhile, protein-hydrogel micro/nano-devices have been proved to exhibit long-term stability (with appropriate working environment or proper packaging) and therefore good practicability.¹⁷

Fig. 2 (a) SEM image of a set of the same three protein nanowires (100 μm long and 600 nm width). Scale bar, 10 μm. (b) The enlarged image of the marked area in (a). Scale bar, 500 nm. (c) AFM characterization of a protein microwire in air. Scale bar, 1 μm. (d) Tilted 3D view of (c). Unit of coordinate: μm. (e) The curve of section contour at the marked position of the protein microwire in (d).

Further, the nature of the as-prepared protein micro/nano-hydrogels endowed the protein-based single micro/nano-wire waveguides with environmental stimuli responsive capability [Fig. 3].^13,14 In Fig. 3(a), the output intensity of the 633 nm-laser coupled protein micro-waveguide in aqueous solution decreased during the process of tuning surrounding pH value from 9.4 to 10.4 by NaOH titration. In detail, 10 μL mg mL⁻¹ NaOH aqueous solution every time was titrated into 50 mL mg mL⁻¹ NaOH solution where the evanescently coupled protein microwaveguide was immersed. Then, 10 μL solution was drawn off after 2 minute standing for sufficient diffusion of NaOH, and the output images were taken after another 2 minutes' standing to avoid interference of solution-level sloshing. The decrement of output intensity was up to ∼50% of original value. And it was noted that the major and linear reduction occurred correspondingly to the small range from pH ∼9.6 to ∼10.0. Similar linear decrease (∼40%) of output intensity could be observed by tuning pH value from ∼2.4 to ∼2.05 by HCl titration [Fig. 3(b)] (10 μL mg mL⁻¹ HCl aqueous solution every time into 50 mL mg mL⁻¹ HCl solution with similar procedures of NaOH titration). As the control experiment, the 633 nm coupled protein micro-waveguide was immersed in pure water and the same procedures of titration but with pure water was operated, which didn't induce obvious change of output intensity [Fig. 3(c)]. The OM images in insets of Fig. 3(a)–(c) visually showed the variation of output light before and after respective tests. This pH-sensitive phenomenon was probably caused by pH-induced swelling and shrinking of protein-hydrogel-based micro/nano-waveguides. While the pH value increased or decreased from 7.0, refractive index of protein waveguides was decreased owing to swelling of protein hydrogel [Fig. 4(a)].^13,14 It might induce less light launched in by evanescent field, lower restriction and more leakage of light during propagation, which probably increased the transmission loss as a result. Since all the tests in Fig. 3(a)–(c) were carried out using the same protein micro-waveguide, the stimuli responsiveness of protein-based micro/nano-waveguides here was proved to be reversible, dynamical and repeatable. Further, the protein-based micro/nano-waveguides might be used as pH sensors after facile modification of pH-sensitive fluorescein isothiocyanate (FITC, bright 515–555 nm yellow-green fluorescence under 465–490 nm light in basic environment, modified by simple immersion in 0.02% (wt) FITC aqueous solution).⁴¹ Modified FITC helped to determine whether it was acidic or basic [Fig. 4(b)] and pH-responsive output variation was utilized for precise detection after proper calibration.

Fig. 3 Points of normalized output intensities and exponentially fitted curve with (a) surrounding pH value changed from 9.4 to 10.4 by NaOH titration, (b) from ∼2.4 to ∼2.05, and (c) water titration for control. Insets: the OM images the variation of output light before and after respective tests. Scale bar, 10 μm.

Fig. 4 (a) The OM image of focal lengths of the PHDM in buffers vs. pH value. Scale bar, 20 μm. (b) The OM images of the FITC modified protein-based micro/nano-waveguides by pH changing from 3.0 to 7.0 to 11.5. Scale bar, 20 μm.

Conclusion

In summary, we have reported protein-based single micro/nano-wire waveguides constructed by FsLDW using BSA aqueous ink. The excellent nanoscale quality control of device morphology and highly reproducible customization of the protein micro/nano-waveguides were guaranteed by carefully optimized and stabilized FsLDW. Therefore, as-formed protein-based micro/nano-wires operated well as optical waveguides. Transmission loss was ∼0.0592 dB μm⁻¹ for 633 nm incident light launched in by evanescent field coupling here, which might be applicable for uses as sensing probes. Owing to the nature of protein molecules as “building blocks”, protein-based micro/nano-waveguides exhibited “smart” dynamically stimuli-responsive feature (up to ∼50% linear decrease output intensity for pH variation from ∼9.6 to ∼10.0 and ∼40% linear decrease for pH variation from ∼2.4 to ∼2.05). Moreover, the protein-based micro/nano-waveguides might be used as pH sensors after facile modification of FITC. Therefore, the protein-based micro/nano-waveguides in this letter shows great promise for novel biophotonic and biomedical applications, and may be used in environment-friendly polymer and hydrogel optics, optical sensing, and “smart” integrated photonic micro/nano-biosystems.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (61107024) and State Key Laboratory of Luminescence and Applications.

Notes and references

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