Plant leaves as templates for soft lithography

We report a simple fast, practical and effective method for the replication of the complex venation patterns of natural leaves into PDMSwith accuracy down to a lateral size of 500 nm. Optimising the amount of crosslinker enabled the replication and sealing of the microvascular structures to yield enclosed microfluidic networks. The use of plant leaves as templates for soft lithography was demonstrated across over ten species and included reticulate, arcuate, pinnate, parallel and palmate venation patterns. SEM imaging revealed replication of the plants microscopic and submicroscopic topography into the PDMS structures, making this method especially attractive for mimicking biological structures for in vitro assays. Flow analysis revealed that the autonomous liquid transport velocity in 1-order microchannel was 1.5–2.2 times faster than that in the 2-order microchannels across three leaf types, with the sorptivity rule surprisingly preserved during self-powered flow through leaf-inspired vascularity from Carpinus betulus.

tissue, 40 which was then used to transfer the microvascular network onto a silicon wafer.
Here we introduce a simpler alternative approach where microuidic structures are directly replicated from plant leaves.In contrast with other microfabrication techniques, this highly economical approach only utilizes PDMS elastomer for microchip fabrication and eliminates the reliance for costly reagents, e.g., fugitive ink, 36 photoresist, 38 developer [38][39][40] and corrosive chemicals. 39,40Furthermore, clean room facilities and microfabrication equipment including inkjet printer, 36 mask aligners, [38][39][40] CNC milling, 47 or laser ablation are also waived here.2][43] We anticipate this nature-inspired manufacturing methodology may open new opportunities by leading microuidic based microvascular engineering towards more bio-realistic assays.

Microchip fabrication
A schematic overview of the main steps in the use of plant leaves as templates for so lithography is schematically depicted in Fig. 1.Leaves were removed from plants and rinsed under running water for one minute, and gently wiped using laboratory paper and blow dried using a N 2 .On the same day, the leaf was attached to single-sided tape (Tesa 57176-00 Tesapack ultra-strong tr.66 m: 50 mm wide) and then to a disposable Petri dish (Mit 3 Nocken Ø 90 mm, 391-0247) using doublesided tape (Tesa double side tape 10 m: 15 mm wide).PDMS (SYLGARD 184; Dow Corning, USA) was prepared by carefully mixing the PDMS prepolymer and cross linker in a 20 : 1 ratio, and degassed for 1 h in a vacuum desiccator to remove air bubbles.When pouring the PDMS into the Petri dish containing the leaf, any possible bubbles formed could automatically disappear aer few minutes.The PDMS was cured in an oven (Binder, US PATS 4585923) at 45 C for 24 h (Fig. 1f).
Next, the PDMS replica was cut using a box cutter and peeled off the template.The template was again covered with PDMS to protect it for further structure replication.An oxygen plasma system (Diener electronic: 0010915) was used to activate the surfaces of both PDMS replica and glass or PDMS substrates before bonding.Finally, the assembled devices were placed in the oven at 80 C for two hours to increase bonding strength.Images of leaves templates were taken using a consumer CCD camera (Canon ED560D with macro-lens).

Pressure-driven ow
A 5 mL disposable syringe was utilized to ll the microuidic network.The syringe was lled with 2.5 mL of red ink (WATERMAN Fountain Pen Ink, Audacious Red S0110730) before the piston was withdrawn to the 4.3 mL mark.A 2 mm diameter inlet was punched in the PDMS replica of a leaf to connect with the central vein and connected with silicone tubing, sealed in place using uncured PDMS.A pipette tip (1-200 mL, UltraFine™, graduiert, VWR International GmBH) was used to connect the lled syringe with the microuidic network.The system was pressurized by pushing the piston from the 4.3 mL mark to the 3.2 mL mark on the syringe, and xed in this position using a metal wire (Fig. S6, ESI 1 †).

Scanning electron microscopy (SEM)
For correlative SEM analysis of the positive leaf and the negative PDMS replica, a FEI (Hilsboro, OR, USA) Quanta 400 FEG was used.Samples were investigated in low vacuum mode (p ¼ 100 Pa water vapor) at an accelerating voltage of 10 kV using the secondary electron (large eld) detector (30 mm nal lens aperture size; spot size 3; pixel dwell time 30 ms, image size 1024 Â 884 pixels).

Atomic force microscopy (AFM)
For AFM measurements 20 nm gold was sputtered on the PDMS replica to eliminate charging during investigation.A Nanowizard 3 (JPK Instruments, Berlin, Germany) operating in tapping mode was used to reveal the 3D structure of the replicated leaf surface.AFM tips from NANOSENSORS (Neuchatel, Switzerland), type PPP-NCLR were used for height measurements.The PDMS pre-polymer is poured over the leaf and cured (e) before the PDMS replica is removed (f).An enclosed microchannel is formed by sealing the PDMS replica with a glass or PDMS substrate after oxygen plasma treatment (g).The biomimetic chip is placed in an oven at 80 C for 2 hours in order to stabilize bonding strength (g).

Elastic modulus (E-modulus) of PDMS templates
Varying the degree of crosslinking in the polymer network allows tuning its mechanical properties in a wide range.To estimate mechanical properties of PDMS produced with different amount of crosslinking from the each of at templates a cylinder with a 3 mm diameter was cut for the subsequent compression test performed under 25 C with a TA DMA Q 800 instrument (TAINSTRUMENT, Eschborn, Germany).
Head-space gas chromatography mass spectrometry (HS-GC-MS).To verify the chemical stability of the produced PDMS templates HS-GC-MS analysis was performed.A gas sample of 500 mL at 50, 70 and 100 C from the headspace above the sample (0.15 g) (5 min incubation time; agitator speed, 500 rpm; ll speed, 100 mL s À1 ; injection speed, 500 mL s

Leaf-inspired microuidic devices
There is a large variation in the size, geometry and venation between leaves from different plant species, and in this work, leaves from Tilia platyphyllos, Prunus cerasifera, Viburnum davidii, Prunus avium, Plantago lanceolate, Carpinus betulus, Fraxinus excelsior, Glechoma hederacea, Acer pseudoplatanus and Aegopodium podagraia were all successfully replicated in PDMS.
Examples of leaves and their replicates of a reticulate, arcuate, parallel, pinnate and palmate venation are given in Fig. 2a-j, with close-up images of the microvascular channels in Fig. 2k-o.More information about microchip fabrication from other leaves is also added to Fig. S1 (ESI 1 †).The widths of the rst ordered channels were measured to be 0.54 mm, 0.81 mm, 1.20 mm, 0.82 mm and 0.43 mm for Tilia platyphyllos, Aegopodium podagraia, Plantago lanceolate, Frangula alnus and Acer pseudoplatanus leaves, respectively.Remarkably that, the 1 st ordered channel in Plantago lanceolate was 2.8 times wider than in Acer pseudoplatanus.Across all species, the smallest veins were around 8 mm wide (Fig. S2, ESI 1 †), much smaller than previous reports. 39,40As the replicated vascular structures were copied from the outside of the leaf, internal structures like the phloem and xylem cannot be replicated by means of this method.The vascular geometry and size distribution across the bifurcating channels, however, was maintained as demonstrated by the reduction in channel width moving down from the rst order or main vein as shown in Fig. 2k-o.

Optimization of crosslinker : monomer ratio
Initially, the so lithography experiment was carried out using the standard 1 : 10 crosslinker : monomer ratio, but the obtained structures could not be sealed in a leakage-tight manner to glass or PDMS substrates.This was hypothesized to be due to the stiffness of the PDMS preventing the textured leaf replica from sealing with the at substrate.It is known that the stiffness of PDMS can be reduced when the amount of initiator reaches 1 : 20 ratio. 44,45The so lithography process was repeated using PDMS mixed in 1 : 15, 1 : 20 and 1 : 30 crosslinker : monomer ratios.The templates obtained using over 1 : 30 PDMS ratio were sticky to touch and le residues, indicating that PDMS was not be fully cured even when extending the curing time to days.For this reason PDMS template synthesized with 1 : 30 crosslinker : monomer ratio has been eliminated from the further experiments.The 1 : 20 ratio provided a PDMS template that was soer than the PDMS obtained using the 1 : 10 or 1 : 15 ratio (Table 1).
In addition, PDMS template mixed in 1 : 20 crosslinker : monomer ratio was not sticky and did not leave residues.Moreover, it was important for microuidic applications because it enabled leakage-tight sealing to at glass and PDMS substrates.
Whilst a soer PDMS can also be obtained by partially curing the PDMS in a shorter curing time, this is not recommended as it increases the variability.Inspection of incompletely cured  structures by SEM (data are not shown) revealed a highly heterogeneous replication, randomly distributed smooth areas amongst highly structured regions.
Next, PDMS prepared in 1 : 10; 1 : 15 and 1 : 20 ratios were inspected by SEM (Fig. 3).The microscopic images showed that structures obtained under 1 : 20 ratio PDMS have been replicated in greater detail versus templates synthesized in 1 : 15 or 1 : 10 ratios.Sub-micron scale structures of Aegopodium podagraia leaf (Fig. 3d-f) showed the replication of the hair-like appendages also known as trichomes.
Because both vascularit [21][22][23][24][25][26][27][28] and surface topography 3-20 can play important role for downstream applications, it was considered to be important to not only preserve the vascularity, but also the microscopic topography of the leaf.To reveal the accuracy of the replicated templates (1 : 20 ratio), a stoma of the back side of Tilia platyphyllos was chosen.As shown in Fig. 4, the line structure next to the stoma can be replicated with high accuracy down to a lateral size of 500 nm.A penetration depth of 90 mm was also found for Tilia platyphyllos replica (Fig. 4d and h).In contrast to this, the nanostructures inside the stoma unfortunately were not replicated into the PDMS using this technique.
On the next step, AFM measurements revealed 3D structure of the replicated leaf of Tilia platyphyllos on PDMS.The liner structure next to the stoma was transformed into the PDMS up to a height variation of several hundreds of nanometer (see Fig. S3, ESI 1 †).For the depth of a hole of a stoma that was replicated into the PDMS a height of more than 1 mm was found (see Fig. S3, ESI 1 †).
Remarkably that produced PDMS replicates (1 : 20 ratio) showed also an excellent chemical stability estimated by HS-GC-MS versus pure PDMS matrix that maybe important in the future for bio-applications (for more information see Fig. S4, ESI 1; † HS-GC-MS chromatograms showed for pure PDMS matrix, natural Tilia platyphyllos leaf aer contact with PDMS and PDMS template aer contact with a Tilia platyphyllos respectively).

Inuence of the storage conditions of leaves templates on the so lithography efficiency
Having optimized the casting conditions using Aegopodium podagrai and Tilia platyphyllos (see above), the delity of replication and structural diversity between plant species were studied using Plantago lanceolate, Viburnum davidii, and Glechoma hederacea.SEM images of the original leaves (Fig. 5a-c) conrmed the diversity in surface morphology between the species.The SEM images at low magnication revealed a variety of surface features.Fig. 5d-i conrmed this diversity is transferred into the PDMS replica, with biodiversity in both vascularity and surface topology preserved using plant leaves directly as template in so lithography.The 800Â images showed the stoma, the mouth-like features used for gas exchange, was also replicated well into PDMS.It is well known that cells culture in a more biologically realistic manner on structures that are not smooth but structured.Studies using the PDMS plant replicas in our laboratory have conrmed a more in vivo-like migration pattern for human melanoma cells (in submission -LC-TIN-01-2016-000076).  Whist the biodiversity between leaves provides a wide variety of structures, this means the optimal leaf for a certain bioassay may have to be identied experimentally.Additionally, the precise size and geometry between leaves will vary, which may introduce an unwanted variability between studies conducted with PDMS replicates from different leaves.
To avoid this issue, and the seasonal dependence, the longevity of the templates was investigated.Initially, fresh leaves (processed on the same day they were picked) were used as templates for replication in PDMS to avoid the structural changes as the leaf dehydrates.The template was then again covered with PDMS and stored for up to a year.The Tilia platyphyllos template (Fig. 6a) has been replicated into PDMS for at least thirty times over a one year period.The discoloration of the leaves (Fig. 6d) indicates oxidative processes as well as the loss of water through the permeable PDMS.When comparing 'b' and 'e', some shriveling and loss of microscale features can be observed aer a year storage, but a surprisingly high level of detail of the microstructures is preserved, as indicated by the SEM images of the stomata (Fig. 6f).
As aforementioned, low heated temperature of 45 C was required for the curing of PDMS on fresh leaf template to preserve the microscopic structure.In the other case, the template would change color from green to black and microstructures were found to have collapsed.Interestingly, it was found that for aged templates stored over three months, the use of higher temperatures like 70 C or 80 C could be used for curing PDMS without damaging the template.Whilst creating a daughter template from the rst PDMS replica, for example NOA 63 resist, 35 will guarantee uniformity across the replicated structures, the ability to store the templates over an extended period of time adds to the versatility of the approach.Longevity of the Carpinus betulus leaf template was also investigated, indicating similar endurance results as Tilia platyphyllos leaf (Fig. S5, ESI 1 †).

Flow through vascular network
Water transport is one of the most important function of natural leaves.Noticeably, such complicated process totally depends on self-activated pumping with only one stiped inlet.Inspired by this post, we innovated self-powered autonomous ow inside biomimetic PDMS leaves without the need for access holes other than the inlet, to visually reveal how autonomous ow behaves inside leaves' microvasculature.Similar to real leaves, herein uidic lling into PDMS leaves was totally selfactivated, and thus, requiring very low energy [46][47][48] consumption.
Assuming the air in the syringe behaves as an ideal gas, Boyles law P 1 V 1 ¼ P 2 V 2 can be used to predict the pressure 47,48 applied at the inlet of the microvascular network by compressing the air in the syringe to be approximately 2.6 atm (260 kPa).This pressure can be used to ll the microuidic network with the ink, using the gas-permeability of PDMS to squeeze the air out.Altogether three PDMS leaves (Fig. 7) were directly replicated from natural leaves (Tilia platyphyllos, Aegopodium podagraia and Carpinus betulus) for ow studies, with signicant differences in their microvascular networks.The ow studies were used to compare the hydrodynamic resistance of the 1 st and 2 nd -ordered channels branching off the main vein (Fig. S6, ESI 1 †).Image analysis revealed the average ow rate in the 1 st -ordered microchannels and 2 nd -ordered microchannels were 0.08 mm s À1 (n ¼ 1) and 0.04 mm s À1 (STDV, n ¼ 16) for Carpinus betulus, 0.16 mm s À1 (STDV, n ¼ 5) and 0.11 mm s À1 (STDV, n ¼ 7) for Aegopodium podagraia, and 0.45 cm s À1 (STDV, n ¼ 5) and 0.22 cm s À1 (STDV, n ¼ 10) for Tilia platyphyllos (Fig. S7, ESI 1 †).Based on ow analysis inside PDMS leaves, average ow rate through rstordered microchannels was 1.5-2.2times faster than ow rate in the second order channels (ESI 2, 3 †), ranging from Aegopodium podagraia, Carpinus betulus and Tilia platyphyllos leaves.The higher ow rate in 1 st microchannel than 2 nd ordered microchannels, indicates geometrical microvascular conguration in leaves obtained higher overall hydraulic conductivity in rst-ordered microchannels.The ow hierarchy used in plant leaves to ensure equal distribution of nutrients was preserved in the PDMS leaves with replicated uidic structure.Total lling time of all three PDMS leaves was less than 10 minutes, indicating suitability for loading medium for static cell culture.Accounting that both plant leaves and animal circulatory systems are nature evolved microuidic channels 51 obeying Murray's law, 27 plant leaves may become attractive templates to fabricate biomimetic microvascular devices for further animal cell studies.
Further analysis of the ow rate in both the 1 st microchannel (no.17) and 2 nd ordered microchannels (no.2, 4, 6, 8, 10) of PDMS leaf from Carpinus betulus (Fig. S8, ESI 1 †), also surprisingly showed that the ow in leaf-inspired microvascular channel obeys sorptivity rule, 49,50 which is mostly found in capillary absorption, and can be represented by the following equation, where I is the cumulative ow amount, S is sorptivity constant, and t is time.
It's seen from Fig. 8, the owing distance (amount) in both the 1 st microchannel and the 2 nd ordered microchannels of PDMS leaf from Carpinus betulus, were linearly correlate the square root of time, obeying sorptivity rule.

Conclusion
A simple method for the replication the venation pattern and microscopic surface morphologydown to a lateral size of 500 nm of plant leaves into PDMS is presented by directly using the leaves as template.The method was applied to leaves from over ten different species, covering the most common reticulate, arcuate, pinnate, parallel and palmate venation patterns.The so lithography process was optimised by reducing the initiator to monomer ratio from 1 : 10 to 1 : 20 to make a soer PDMS, which allows for a leakage-tight seal with a at substrate despite the surface topography.Using reversibly bound PDMS on glass devices, no leakages were observed when lling the microuidic network with pressures up to 260 kPa.Evaluation of the linear ow velocities in 1 st and 2 nd order microchannels revealed the linear ow rate in the 2 nd order channels was about half of that in the 1 st order structure, facilitating ordered lling of the leaf.It's surprisingly found here that ow in both 1 st and 2 nd ordered microchannels of PDMS leaf from Carpinus betulus, obeys sorptivity rule that's believed mainly applied to capillary absorption in conventional views.We'll make a deeper study on this discovery in our future work.
With a demonstrated ability to replicate from a wide variety of vascular structures, this methodology provides access to a variety of uidic structures, with an even larger variety in functionality provided by presence, location and structure of micro-and nanometer scaled structures caused by structures like stomata and trichomes.The surface of leaf-replicated channels is not smooth and may be more bioequivalent than lithographically fabricated structures, with preliminary studies conrming a more in vivo-like migration pattern for human melanoma cells.The variety in three dimensional vascularity and inhomogeneous surfaces offered by this new, simple and affordable approach for biomimetic microfabrication may benet many in vitro cell-based assays.

Fig. 1
Fig.1Schematic steps of the direct replication of microvascular networks from natural leaves into a microfluidic PDMS device.The natural leaf (a) attached to single-sided tape (b) is mounted onto a Petridish (d) using double sided tape (c).The PDMS pre-polymer is poured over the leaf and cured (e) before the PDMS replica is removed (f).An enclosed microchannel is formed by sealing the PDMS replica with a glass or PDMS substrate after oxygen plasma treatment (g).The biomimetic chip is placed in an oven at 80 C for 2 hours in order to stabilize bonding strength (g).
À1 ) was injected by a PAL auto sampler (CTC Analytics, Zwingen, Switzerland) into the QP5050 (Shimadzu, Japan) GC-MS system.A Phenomenex (Torrance, CA, USA) ZB-WAX-plus column (30 m Â 0.25 mm; lm thickness 0.25 mm) was utilized for separation.An injection temperature of 200 C (split ratio 1 : 35) was used, with the temperature program kept at 50 C for 1 min, then raised to 200 C at 20 K min À1 and held at the nal temperature 250 C for 5 min.The column inlet pressure was adjusted to 100 kPa, giving a ow rate of 1.7 mL min À1 .The transfer line to the mass spectrometer and the source temperatures were 230 C and 200 C, respectively.Electron ionization (70 eV) mass spectra were recorded in the m/z range of 40-920.

Fig. 2
Fig. 2 Diversity in shape and venation in plant leaves.(a-e) Photographs of the fresh leaves; (f-j) photographs of PDMS replicates and (k-o) close-ups of the first order vein of Tilia platyphyllos, Aegopodium podagraia, Plantago lanceolate, Frangula alnus and Acer pseudoplatanus.Scale bar images (k-o) corresponds to 2.5 mm.

Fig. 6
Fig. 6 Tilia platyphyllos leaf stored in PDMS for one year, utilized for microfabrication of over thirty times during this period.(a) Photograph of freshly prepared Tilia platyphyllos leaf.(b) PDMS replica from fresh Tilia platyphyllos leaf showing microvascular structure.(c) Imprint of stomata in PDMS replica from fresh Tilia platyphyllos leaf.(d) Photograph of Tilia platyphyllos leaf after one year.(e) PDMS replica from 1 year old Tilia platyphyllos leaf showing microvascular structure (f) SEM image of PDMS replica of stomata from one year old Tilia platyphyllos leaf.Scale bars (b) and (e) 100 mm, (c) and (f) 10 mm.

Fig. 8
Fig. 8 Quantitative flow analogy inside PDMS leaf containing parallel networks replicated from Carpinus betulus, with flowing distance plotted versus square root of time.

Table 1 E
-Modulus of PDMS templates with different amount of crosslinking This journal is © The Royal Society of Chemistry 2016