FIELD OF THE INVENTION

The present invention is in the field of nanotechnology and particularly relates to the nanoprocessing and hetero structuring of natural fibers like silk etc. The present invention involves ultrafast laser based modification and seamless micro-welding of natural fibers including spider silks.

BACKGROUND AND PRIOR ART

Spider silk is an ancient biomaterial that is tougher than steel, yet elastic and light weight. The superior physical properties of spider silk make it an attractive biocompatible functional material. Various applications of silk in water collection, biomedical devices and optics have been demonstrated. Silk fibers with enhanced toughness and possessing electrical and magnetic properties have been created by infiltrating them with various (non-) metallic nanoparticles. A great deal of experimental and theoretical efforts has been made to understand relation between structure and its functional properties. However, to realize true potential of silk for diverse nanoengineering, technological and other applications two fundamental difficulties exist. First, it is difficult to manipulate the silk with nanoscale precision due to high toughness and finesse. One needs to devise a noninvasive nano- processing approach for silk while preserving its key structural building blocks and also retaining its excellent physical properties. Second, it is not yet fully explored, how to integrate silk with other modern materials such as metals, dielectrics, synthetic polymers thereby combining their best properties. Thus, it is vital to devise a new approach to remove these two bottlenecks.

Previously, chemical processing techniques have been proposed to make silk films and bulk structures for various applications. Ablation of spider dragline by nanosecond (ns) pulses of deep ultraviolet (VUV) radiation has also been studied. Moore et al., in 2006 made a first attempt to process the dragline silk of black widow spider (Latrodectus hesperus) using nanosecond UV laser (λ=157 nm) under vacuum controlled conditions. Besides requiring high vacuum conditions, the VUV light degraded the silk structure leading to loss of function and strength. Thus, it is apparent that it is difficult to nano-machine silk fibers in controlled conditions. Moreover, it is difficult to micro-weld silk fibers such as spider silk with themselves or other materials.

Recent advent of amplified ultrafast lasers has drawn considerable interest for material processing. These lasers could provide higher precision and less thermal damage than conventional lasers with rather long pulse durations of several tens of nanoseconds. Ultrafast lasers are very efficient for direct micromachining of materials because of their non-contact nature, which allows micro-processing and surface patterning of materials with minimized mechanical and thermal deformations by rapid pulse energy deposition. Since last decades, short femtosecond (fs) pulses have evolved into a preferred tool over picosecond (ps) and nanosecond (ns) pulses in precise material processing with low collateral damage. Femtosecond (fs) lasers have made a mark to be used in corneal refractive surgery and many more surgical procedures. The effects of different fs-laser parameters such as pulse energy, wavelength, pulse width, and tissue depth on the operation performance have been widely investigated. But, fs-lasers processing technology has not been used yet to fuse/weld threads/fibers/sutures that holds the donor tissues in place during healing. Fs-lasers could be proposed as a future surgical tool for seamless micro-welding for joining biological tissues including cornea. Therefore, it is a vital need to establish an optical approach to modify threads/fibers/sutures and micro-weld on surfaces using such lasers.

The present invention is an attempt to overcome problems in the prior art and for the first time shows that the interaction of spider silk with few-cycle fs pulses generated by fs lasers offers a unique opportunity for its controlled nanoprocessing and hetero structuring with diverse materials in air. Precisely, in order to overcome limitations in the prior art, the inventors have developed femtosecond laser based framework to modify and weld (seamlessly) the thin silk fibers with a high precision. This is a significant step to realize spider silk fibers and other natural fibers in utilization as threads/sutures for fabricating nano- devices, allografting, designing pressure, force and vibration sensors. OBJECTIVES OF THE PRESENT INVENTION

The main objective of the present invention is to provide a novel approach for nanoprocessing of silk in air, including spider silk by application of few-cycle fs pulses. Yet another objective of the present invention is to modify optical as well as physico- chemical properties of natural spider silk fibers and seamless welding of such ultrathin fibers using super high speed lasers. Yet another objective of the present invention is to provide non-invasive localized nanoshaping of silk while retaining its key molecular structure.

Yet another objective of the present invention is to provide an optical approach (using fs pulses) to fabricate diverse topological microstructures.

Yet another objective of the present invention is to provide robust heterostructuring of silk with Cu, glass, Kevlar, Polyvinyl carbonate(PVC), Poly (methyl methacrylate) (PMMA) and Poly-di-methyl-siloxane (PDMS) by its seamless micro-welding. Yet another objective of the present invention is to provide an apparatus for micro structuring and heterostructuring of silk, which comprises of a laser light source generating a femtosecond laser beam so that the same can be applied for modification and seamless welding of silk fibers. SUMMARY OF THE INVENTION

The present invention offers a novel approach for nanoprocessing and heterostructuring of silk by application of few-cycle fs pulses. An optical approach for modifying optical, physico-chemical properties of natural spider silk fibers and for seamless welding of such ultrathin fibers with artificial and biological materials using a super high speed laser has been presented. The technique of the present invention quantified the silk response, i.e., initially bulging and later ablating as a function of applied laser fluence. These regimes were exploited for non-invasive localized nanoshaping (nanocut, grooves, rods, nanotip) while retaining its key molecular structure. The present invention successfully utilizes the bulging regime for seamless microwelding of two independent silk fibers. So far, there is no known route to merge two silk fibers in air, after they have been spun by the spider. This is essential to develop capability to fabricate all- silk based designer structures. The present invention demonstrates merging of the contact region of two different silk fibers, when these were exposed to fs pulses for 10-60 s. The optical microscopy, confocal microscopy, and SEM images of welded region confirmed that welded regions had no cracks and weld-joints retained good surface smoothness. To demonstrate robustness and repeatability of techniques we welded silk fibers of various diameters in different configurations. Most remarkably, robust heterostructuring of silk with Cu, glass, Kevlar, Polyvinyl carbonate (PVC), Poly (methyl methacrylate) (PMMA) and Poly-di-methyl-siloxane (PDMS) by its seamless micro-welding was demonstrated. The optical approach allowed the inventors to fabricate diverse topological microstructures, such as the Mobius strip, chiral helices, and knots which were shown to be propelled by light in air or on water with good control.

Moreover, analysis of Raman bands of micro-welded joints reveal that the polypeptide backbone remains intact while perturbing its weak hydrogen bonds. The approach was used for fabricating a new class of silk based functional topological microstructures, such as Mobius strip, chiral helices, and cantilever or trampoline-like sensors, which were optically propelled in air as well as on fluids remotely with good control.

In one aspect, the present disclosure relates to a non-invasive method for nanoprocessing of silk comprising: quantifying optical non-linear multiphoton response of a silk material as a function of laser fluence induced by a pulsed femtosecond laser beam of predetermined laser characteristics; and micro structuring or heterostructuring of silk material upon exposure of the femtosecond laser beam.

In another aspect, the present disclosure provides a system for quantifying optical response of a silk material, the system comprising: a focusing unit for focusing a pulsed femtosecond laser beam of predetermined laser characteristics at a focal point; a three dimensionally movable translation stage support of holding the silk material for exposing a portion of said silk material at said focal point; displaying unit for displaying real time image of focused portion of the silk material and thereby determining change in physical dimension of said portion of the silk material by varying the laser fluence at the portion of the silk material. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematics of the experimental set up. The fs pulses (7-10 fs, 800 nm, 2.2 nJ at 85 MHz rep. rate) were focused on to 5 μιη spot diameter through a chirp-mirror based dispersion compensated objective. Silk sample was mounted on a computer controlled 3 -axis nano-positioner stage. Insets: SEM image of silk sample, and its typical optical diffraction pattern. S: shutter, ND: neutral density filter, DM: Dielectric broadband dichroic mirror, CM: chirp mirror, M: Ag-mirror, Obj: objective, Stage: Automated X-Y-Z translation stage, CCD: two high resolution charged couple devices (CCD), sp: spectrophotometer.

Figure 2. Mechanisms of laser-silk interaction, a, Absorption spectra of dragline silk in UV- VIS-IR range, b, Absorption of silk as a function of peak intensity (I) of fs pulses. Red line is a mixed fit corresponding to non-linear multiphoton absorption. Dotted lines represent pure 2-, 3-, 4-photon absorption, c The emission spectra of silk at three different intensities. The cut-off in emission below 380 nm coincides with the maximum of silk absorbance.

Figure 3. Bulging and ablation response of the silk as a function of laser fluence. a-c: show SEM images of natural, bulged and ablated silk, respectively, d: shows that bulging regime is characterized by the relative change in silk diameter versus the laser fluence. The ablation regime was quantified by plotting the cut width (W). Error bars indicate average over three measurements.

Figure 4. Non-invasive nano-machining of silk fibers, a-c: Silk rods of 8-26μιη in length, d: nano-grove of width 300 nm to 15 μιη. The exposure time of fs pulses is labeled on the picture, e-f: Optical and electron microscope images of a nano-tip fabricated at the end of the fiber, g: Raman spectra of the silk at three different locations near the micro-cut.

Figure 5. Confined micro-bending of silk fibers, a-d: Different bend angles are approximately 10°, 30°, 102°, and 180°, respectively. The exposure time of fs pulses is labeled on images, e: Raman micro spectra of silk taken at the bending site along with the native silk. The spectra are normalized to 1234 cm ^"1 peak. Figure 6. Fabrication and characterization of micro-welded silk hetero- structures, a-d: Images of welded silk with silk, Kevlar, Cu, and glass, e: The stress-strain curves of silk-silk, silk- Kevlar, silk-Cu and silk-glass joint along with the native silk. The stress is computed by assuming a constant silk diameter, f: Raman spectra accumulated at the welding cite g. Schematic and real image of a contact lens (0:6 gm) suspended from a point welded silk fiber h. SEM micrograph of welded silk on PDMS surface.

Figure 7. Silk-based functional topological micro- structures, a-c: a micro-spring, coiled- solenoid, and a knotted L-shaped beam, d-e: chiral structures, f: a hemispherical silk particle, g-i: Mobius strips like structures with topological charges. Triangular arrow indicate exposure cite for fs pulses, j: Optical propulsion of silk-rods by a periodic driving using low power green laser in air and in water, h: indicates the displacement of silk structure after the laser irradiation.

DETAILED DESCRIPTION OF THE INVENTION

While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention.

Accordingly, the present invention provides a novel approach for nanoprocessing and heterostructuring of silk by application of few-cycle fs pulses. The process of the present invention involves non-invasive localized nanoshaping of silk while retaining its key molecular structure. Importantly, the optical approach (using fs pulses) of the present invention is utilizable to fabricate diverse topological microstructures, such as the Mobius strip, chiral helices, and knots which were shown to be propelled by light in air or on water with good control. Robust heterostructuring of silk with Cu, glass, Kevlar, Polyvinyl carbonate (PVC), Poly (methyl methacrylate) (PMMA) and Poly-di-methyl-siloxane (PDMS) by its seamless micro-welding has been achieved. Accordingly, a system according to the present invention for modification and seamless welding of fibers comprises: a fs-laser light source; a neutral density attenuator controlling the power of the fs-laser beam; a fixing unit (rectangle frame) to hold the spider silk sample in air; a dispersion compensated chirped mirror based optical setup for optimal pulse width (7 - 10 fs) to process spider silk placed on fixing unit; and a focus control part controlling the focus of the focused laser beam.

The approach of the present invention involved exploitation of the non-linear multiphoton absorption response of fs- pulse towards silk material to bulge or ablate the silk material.

Theoretically, the multiphoton absorption is determined by the following equation,

= ί¾Ι 4 a _z I ² 4- H ₃ I ³ 4- « J* 4- a _s I ⁵ (1)

where, I is the intensity of incident light beam propagating along the z-axis. The coefficients ai , a ₂ , a ₃ , o , as are one-, two-, three-, four- and five-photon absorption coefficients for a given medium, respectively. For silk fiber of diameter Do, we define absorption, A=(dl/I) (1/Do), which, using Eq. (1) becomes a polynomial of third-order, A = a _t « ₂ / 4 f¾ # ³ 4 a I 4 ίχ ₄ ί* et _s l ^h . This mixed fit was used to fit the experimental data. A comparison of mixed fit with cases of pure 2-, 3-, 4-, and 5-photon absorption processes are also shown for comparison. The values of coefficients are, α ₂ =1χ10 ^{" 2} cm/GW, a =2xl0 ^"5 cm ³ /GW ² , a ₄ =4xl0 ^"6 cm ⁵ /GW ³ , a ₅ =5xlO ^"7 cm ⁷ /GW ⁴ . Our fit analysis suggests that as the intensity of the pulses increases, the absorption is dominated by progressively higher order multiphoton processes. Bulging of silk material allows it to mould/ weld with different artificial or biological materials. Precise ablations using fs- pulses allows to cut the silk material limited to focal volume. Optical microscopy and SEM imaging of the moulded / welded region was done to visualize and monitor the changes. The ablation regime allowed diverse non-invasive machining capability of the silk fibers with nanometer precision. Silk was diced into micro-rods reproducibly. The ends of the micro-rods showed fine cuts as well as good surface quality. The high precision processing and absence of collateral damage allowed the inventors to fabricate the fine silk-nanotip within nano range. The nano-tips, cuts and grooves remained stable in air as well as in vacuum akin to the pristine silk. In order to prove that the fs pulse ablation did not alter the native molecular structure of the silk significantly, the inventors obtained its Raman spectra in the close vicinity of ablated region. It was observed that in spite of the ablation, most of the prominent Raman bands remain preserved. The preservation of the prominent Raman bands after the ablation offers an unparalleled advantage of short fs pulses in non-destructive nanoshaping of silk in air. The bulging regime, however, offers a qualitatively different and new set of nanoprocessing capabilities. As per the existing literature, no route is known to permanently micro-bend the silk because of its high-elasticity and toughness. The inventors exploited the bulging response to demonstrate the confined bending of fibers at any angle. The bent fibers retained good surface smoothness and remained stable in air and vacuum conditions. The inventors successfully utilized the bulging regime for seamless microwelding of two independent silk fibers. So far, there is no known route to merge two silk fibers, after they have been spun by the spider. This is essential to develop capability to fabricate all-silk based designer structures. The inventors demonstrated merging of the contact region of two different silk fibers, when these were exposed to fs pulses. Both the optical microscopy and SEM images confirmed a seamless fusion/welding of the two fibers while retaining the surface smoothness of welded region. Many silk fibers of different diameters were micro-welded in different configurations that demonstrated robustness and reproducibility of the technique.

Remarkably, the bulging regime facilitates the fabrication of precise heterostructures of spider silk by microwelding it with materials like Cu, glass, Kevlar, Polyvinyl carbonate (PVC), Poly (methyl methacrylate) (PMMA) and Poly-di-methyl-siloxane (PDMS). The employed fs pulses also avoid any collateral damage to the silk as well as the substrate material. Moreover, this preserved the key building blocks of silk that allows to potentially combine their best properties. To achieve fine microwelding, the silk fiber was placed in contact with other materials and exposed to the fs pulses. These composite structures remained stable both in air as well as in high vacuum. The ultimate tensile strength of diverse micro-welded joints was measured and was found to be comparable to the strength of the pristine silk fiber from the same spider. The micro-Raman analysis was used to understand the molecular scale deformation in silk for all microwelded joints. It was further compared with the spectra of the native silk. The Raman spectra of the welded silk joint were similar to the native one. This suggests that the polypeptide backbone (C-C, C-H, C=0 etc) was also intact during microwelding. However, the Raman bands were broader, which was probably due to higher heterogeneity and dis-orientation of the side-chains. The reconfigurable nature of the polypeptide chains in silk perhaps make Vander waal or hydrogen bonds with substrate, leading to an atomic scale affinity between silk and diverse materials. Meanwhile, the key molecular bonds of the silk remain preserved that corroborated well with the observed breaking strength of microwelded joints.

In one embodiment of the invention a non-invasive method for nanoprocessing of silk has been provided comprising: quantifying optical response of a silk material as a function of laser fluence induced by femtosecond laser pulses; and micro structuring or hetero structuring of silk material upon exposure of the femtosecond laser pulses.

In one embodiment, the method for micro- structuring of silk material comprises preparation of topological microstructures including but not limited to micro -springs, coiled solenoid, mobius strips, chiral helices or knots.

In another embodiment, the method for hetero structuring of silk material comprises seamless welding of the silk material with an artificial or biological material.

The method for quantifying optical response of a silk material under ambient conditions comprises: focusing a femtosecond laser beam having predetermined laser characteristics at a focal point; exposing a portion of the silk material at said focal point; raster scanning the silk sample in the focus; displaying real time image of said exposed portion of the silk material; and determining change in physical dimension of said image by changing at least one of the laser characteristics.

The proposed optical nanoprocessing framework can be exploited to fabricate silk-based functional microstructures with novel topologies. For example, by controlled twisting and welding of silk fiber, the inventors constructed a "non-orientable" Mobius strip like structure. These were made with different topological charges (1/2, 5/2; 7/2 of turns) in air and also fused with Cu or glass substrates. Chiral silk structures were also made. These were in the form of left or right-handed configuration having double or multiple strands. Moreover, by seamless welding at both ends, these helices became stable in air, glass or water surface. Other topological structures like a micro-spring, a microdroplet, a solenoid-like packing, various knotted bundles etc. were fabricated.

Due to diverse shapes, strength and elasticity in silk material, these microstructures offer various potential functionalities. For example, they can be propelled by external fields such as light with a good control. The inventors made a microscale cantilever of twisted silk bundle and welded it on a glass substrate. The free end of cantilever was a head-like structure which could be propelled in air by green-laser. Analogously, the inventors made a composite Silk-Cu microscale device that remain floated on the water surface. In a similar manner, when such structure was irradiated with cw laser, the heavier Cu-head could be propelled. These optically driven structures were durable and robust that offers a great potential for ultimate applications. It is rather possible, in principle, to articulate any shape that can be miniaturized to nanometer scale.

Thus, this study demonstrates a first optical route for controlled nanoprocessing and hetero structuring of silk by exploiting ablation and bulging regimes induced by short fs laser pulses. A wide variety of nanoshaping capabilities such as dicing, grooving, nano-tips, confined bending without causing any collateral damage. The present approach offered a comparable strength while welding of silk with modern materials including Cu, glass, Kevlar, Polyvinyl carbonate(PVC), Poly (methyl methacrylate) (PMMA) and Poly-di-methyl-siloxane (PDMS).

Example 1

Nano-processing setup

2 nJ femtosecond-laser system (Femtolaser, Austria) having spectral range of 800 nm, Repetition rate: 85 MHz, and pulse width: 7 fs, S-high speed mechanical shutter (Thorlabs, USA), ND-Neutral density filter (Thor Labs, USA) to control incident intensity, DCM- Dispersion compensated mirrors (Femtolasers, Austria) to maintain the pulse width till focus placed at an angle of 7° to each other. Number of bounces for fs-laser beam was optimized to 4 for maintaining pulse duration of 7-10 fs at focus. DM- Dielectric broadband dichroic mirror ( = 800 nm). Lens-a triplet lens objective having thin glass lens to avoid pulse broadening with Numerical aperture 0.2, The beam spot diameter at focus was about 5 μιη. Sample-Stage- Automated X-Y-Z translation stage with a precision of 1 μιη, CCD- two high resolution charged couple devices (CCD) were installed, one for diffraction imaging and other in line of focusing for real time monitoring before the dichroic mirror. The white light continuum generation at focus detected using a spectrometer (Sp) justifies the non-linear multiphoton interaction fs- pulses with silk material.

Quantifying optical response of silk fiber

In order to achieve targeted delivery of fs pulses on the fine silk fiber (0.8-6 μιη in diameter), it was mounted on a xyz nano -positioner stage. Coarsely, the silk fiber was aligned in the laser focus by CCD imaging through the dichroic mirror. However, for laser fluence below the damage threshold of silk, it produced a stable diffraction pattern on the screen, which was further used to carefully align the silk in focus with 100 nm precision. The pulse energy was controlled by ND filter and the exposure times (laser fluences) was varied using a mechanical shutter. The experiment was performed in the ambient conditions at Temperature 25 + 1°C and 50 + 5 % relative humidity.

The response of dragline silk was further quantified by varying the exposure time for fixed pulse energy. The silk sample (d = 1-5 μιη) was periodically scanned N times in the focus at 2 mm/s (exposure times 10 ms - 100 s) in order to vary the laser fluence on the sample from 0.25-9.0 mJ/μιη ² . We observed that below a threshold fluence of -1.25 mJ/μιη ² , for intensity below 80 GW/cm ² , the silk fiber remained unaffected despite the long exposure times. With increase in the laser fluence, two different regimes, namely multiphoton bulging and plasma- ablation, were observed. Bulging of silk takes place between the fluence range of 1.25 - 3.0 mJ/μιη ² . The diameter of the bulging silk increased within the focal volume as a function of increment in laser fluence. In contrast, in the plasma assisted ablation was observed at 3.0 mJ/ μιη ² or above, while with variation in the fluences, the cutting of silk fiber with sub- 100 nm to micrometer range could be achieved.

Example 2

Micro-welding of silk with artificial materials

The spider silk was micro- welded to a tissue-like material such as poly-dimethoxysiloxane (PDMS) and contact lens of 9 mm diameter and 600 mg mass Poly (methyl methacrylate) (PMMA) by placing a silk fiber on the edge of fresh (wet) contact lens (Figure 6g and 6h). The silk fiber on the lens was exposed to fs pulses such that the damage to contact lens was minimal. This was possible because the contact lens has 500 μιη thickness which is much larger compared to the ~3 μιη for silk. The weld joint was sufficiently strong to hold the contact lens in air even when it was dried. This suggests that the silk can be used to "glue" tissue-like materials under ambient conditions without using any chemicals or post processing steps.

Advantages

The key advantage of fs pulses is that one can process silk in air with nanoscale precision. Moreover, the key structural building blocks and functionality of silk fibers remain preserved. The present study will have a diverse range of applications. For example, the biocompatibility of silk can be exploited for seamless suturing of silk with other biological tissues including cornea. By welding of silk one can fabricate precision scaffolds for a wide variety of biomedical applications. The optical processing might allow protective silk- coatings on tissues and microscopic structures that can be further explored for delivery of materials inside the cellular organisms. One can also construct bio-compatible materials, silk- based nano-engineering and micro-electro-mechanical systems etc. The present invention takes a definite step towards realizing 3D printed micro structures of silk on various substrates. The optical route to infiltrate silk with nanocomposites (Au, nano-tubes, graphene) could lead to responsive polymers that are sensitive to heat, humidity, magnetic fields or light for various applications. Due to advantages offered by fs pulses, analogous approaches on other biomaterials (shells, keratin, collagen fibers) is also worth exploring.