U.S. patent application number 14/765202 was filed with the patent office on 2015-12-24 for silk-based nanoimprinting.
The applicant listed for this patent is TUFTS UNIVERSITY. Invention is credited to Mark A. Brenckle, David L. Kaplan, Florenzo G. Omenetto.
Application Number | 20150368417 14/765202 |
Document ID | / |
Family ID | 51354599 |
Filed Date | 2015-12-24 |
United States Patent
Application |
20150368417 |
Kind Code |
A1 |
Omenetto; Florenzo G. ; et
al. |
December 24, 2015 |
SILK-BASED NANOIMPRINTING
Abstract
Protein-protein imprinting of silk fibroin is introduced as a
rapid, high-fidelity, and/or high-throughput method for the
fabrication of nanoscale structures in silk films, through
controlled manipulation of heat and/or pressure. High resolution
imprinting on conformal surfaces is also demonstrated.
Inventors: |
Omenetto; Florenzo G.;
(Lexington, MA) ; Kaplan; David L.; (Concord,
MA) ; Brenckle; Mark A.; (Northborough, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TUFTS UNIVERSITY |
Medford |
MA |
US |
|
|
Family ID: |
51354599 |
Appl. No.: |
14/765202 |
Filed: |
February 14, 2014 |
PCT Filed: |
February 14, 2014 |
PCT NO: |
PCT/US2014/016630 |
371 Date: |
July 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61765621 |
Feb 15, 2013 |
|
|
|
61827544 |
May 24, 2013 |
|
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Current U.S.
Class: |
428/156 ;
101/463.1 |
Current CPC
Class: |
Y10T 428/24479 20150115;
C08L 2205/025 20130101; B41C 1/10 20130101; G03F 7/0002 20130101;
C08J 2389/00 20130101; B82Y 40/00 20130101; C08L 89/00 20130101;
C08L 89/00 20130101; C08L 89/00 20130101; C08J 5/18 20130101 |
International
Class: |
C08J 5/18 20060101
C08J005/18; B41C 1/10 20060101 B41C001/10 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
W911NF-11-1-0254 awarded by the United States Army, Army Research
Office. The U.S. government has certain rights in the invention.
Claims
1. A composition comprising: a first silk fibroin material
comprising a predetermined nanostructure fabricated thereon;
wherein the first silk fibroin material has a first beta sheet
content and a first glass transition temperature (T.sub.g1); a
second silk fibroin material, having a second beta sheet content
and a second glass transition temperature (T.sub.g2); wherein the
first silk fibroin material is in close contact with the second
silk fibroin material, such that the predetermined nanostructure on
the first fibroin material is substantially replicated onto the
second silk fibroin material to produce an inverse imprint of the
predetermined nanostructure.
2. The composition of claim 1, wherein the inverse imprint of the
predetermined nanostructure has a resolution of at least 200
nm.
3. The composition of claim 1, wherein the inverse imprint of the
predetermined nanostructure has a resolution of about 100 nm.
4. A composition comprising: a first silk fibroin material
comprising a predetermined nanostructure fabricated thereon;
wherein the first silk fibroin material has a first water content
and a first glass transition temperature (T.sub.g1); a second silk
fibroin material, having a second water content and a second glass
transition temperature (T.sub.g2); wherein the first silk fibroin
material is in close contact with the second silk fibroin material,
such that the predetermined nanostructure on the first fibroin
material is substantially replicated onto the second silk fibroin
material to produce an inverse imprint of the predetermined
nanostructure.
5. The composition of claim 4, wherein the inverse imprint of the
predetermined nanostructure has a resolution of at least 200
nm.
6. The composition of claim 4, wherein the inverse imprint of the
predetermined nanostructure has a resolution of about 100 nm.
7. A composition comprising: a plurality of negative silk fibroin
imprints and a plurality of positive silk fibroin imprints; wherein
a positive silk fibroin imprint comprises a nanopattern, and
wherein a negative silk fibroin imprint comprises an inverse of the
nanopattern present on the positive silk fibroin imprint; and,
wherein the negative silk fibroin imprints are substantially
replicas of one another, and wherein the positive silk fibroin
imprints are substantially replicas of one another.
8. A method comprising the steps of: (i) providing a crystallized
silk fibroin master comprising a predetermined nanostructure
thereon; (ii) layering the crystallized silk fibroin master with a
silk fibroin material having a high water content; (iii) applying
heat, pressure, or combination thereof, under a condition
sufficient to generate an imprinted silk fibroin material having an
inverse of the predetermined nanostructure.
9. The method of claim 8, further comprising (iv) annealing the
imprinted silk fibroin material from step (iii) to induce
crystallization.
10. The method of claim 8, further comprising using the imprinted
silk fibroin material from step (iii) as a template for repeating
steps (i) and (ii).
11. A method for high throughput imprinting comprising repeating
the method of claim 8.
12. The method of claim 10, wherein the repeating is performed
about 2-30 rounds.
13. The method of any one of claims 8-12, wherein the high water
content is at least 95%.
14. The method of claim 13, wherein the high water content is at
least 99%.
15. The method of any one of claims 8-14, wherein the condition
comprises heating for a duration of about 2 seconds to 120
seconds.
16. The method of any one of claims 8-15, wherein the condition
comprises heating at about 75.degree. C. to 130.degree. C.
17. The method of any one of claims 8-16, wherein the condition
comprises heating for a duration of about 5 seconds at about
120.degree. C.
18. The method of any one of claims 8-17, wherein the condition
comprises heating for a duration of about 30 seconds at about
100.degree. C.
19. The method of any one of claims 8-18, wherein the condition
comprises heating for a duration of about 60 seconds at about
80.degree. C.
20. The method of any one of claims 8-19, wherein the condition
comprises applying pressure at about 10-100 PSI.
21. The method of any one of claims 8-20, used for conformal
imprinting.
22. The method of any one of claims 8-21, wherein the step (iii) is
performed with the use of an embosser.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Provisional Applications 61/765,621, filed Feb. 15, 2013, entitled
"IMPROVED SILK-BASED NANOIMPRINTING" and 61/827,544, filed May 24,
2013, entitled "SILK-BASED NANOIMPRINTING," the contents of each of
which are incorporated herein by reference in their entirety.
BACKGROUND
[0003] Nanoimprinting is a lithography technique for fabricating
micro-, submicro- and nano-scale patterns. In such methods, a mold
or template made of such materials as metal, is pressed onto a
thermoplastic material, such as silk-based materials, heated above
its glass transition temperature, and the softened material
conforms to the mold due to applied pressure. Such techniques have
been previously described, in for example, WO 2010/126640, entitled
"NANOIMPRINTING OF SILK FIBROIN STRUCTURES FOR BIOMEDICAL AND
BIOPHOTONIC APPLICATIONS," the entire contents of which are
incorporated herein by reference. Such methods already described in
the pertinent art typically utilize materials such as metals and
silicon as a mold or template (i.e., hard master) to carry out
nanoimprinting.
SUMMARY OF THE INVENTION
[0004] Among other things, the present application provides
improved imprinting methods using silk-based materials, including
nano- and micro-scale imprinting. The invention includes novel
protein-protein imprinting "PiP" technique for high throughput
nanoscale imprinting of silk fibroin films.
[0005] The invention is based at least on the recognition that
crystallized silk material itself can be effectively used as a
template or "master" for carrying out the process of imprinting
involving the use of silk materials for imprinting, by
differentially controlling material properties of silk fibroin.
Accordingly, the methods described herein leverage glass transition
temperature (Tg) of silk materials to carry out rapid, high
fidelity imprinting capable of achieving high resolution. In some
embodiments, resolution of about 100 nm may be achieved.
[0006] In some embodiments, a method for nanoscale imprinting of
silk fibroin materials (such as films) is provided, in which a
nanopatterned silk fibroin film ("master") is used to pattern a
second, unpatterned silk fibroin film ("blank"). The imprinting
process thus involves the transfer of a pattern (e.g., nanopattern)
from the master to the blank, such that a replica of the pattern
present on the master can be generated. Pattern transfer from the
master to the blank is accomplished by the application of pressure
(such as, for example, .about.50 Psi) along with heating the blank
above the glass transition temperature of the film. In some
embodiments, the master is treated prior to imprinting to increase
content of the beta-sheet secondary structure motif in the protein
by any suitable method. In this way, crystallized and
non-crystallized silk materials may be processed differentially by
manipulating conditions such as temperature and pressure. This
method may also be used to functionalize silk fibroin films for
high technology applications in a high-throughput manner.
[0007] As described in more detail herein, in some embodiments, the
nanoimprinting techniques described herein are suitable for the
fabrication of a wide variety of micro- and nanosructures,
including, without limitation, photonic structures on silk
materials, such as silk films. It was previously shown that is
possible to make nanoimprinting on silk films with very high
resolution by means of a hard master, such as metal molds used as
templates. In some embodiments of the invention, provided methods
may employ a hard master for the first imprint, and then an
imprinted silk may be used as master for subsequent step(s) of
generating positive/negative replicas of nanostructures, such as
photonic structure. The possibility of imprinting nonplanar
surfaces (e.g., curved or uneven surfaces) with good resolution is
also demonstrated, using this technique.
[0008] The present application encompasses various embodiments, in
which multiple replicas may be generated via serial imprinting. In
any of the embodiments, the invention also encompasses the silk
based protein-protein imprinting technique that allows for rapid,
multi-generation, high-throughput, bench top imprinting
applications.
[0009] In some embodiments, provided methods are applicable to
generating patterning on nonplanar surfaces, including biological
substrates, which allows for conformal imprinting.
BRIEF DESCRIPTION OF THE DRAWING
[0010] FIG. 1 provides schematics of the PiP process, and
replication mechanism. (a) Each imprint was made by layering an
untreated (blue) film on top of a crystallized silk fibroin master
(yellow). These samples were placed on a heated substrate at
120.degree. C. for 60 seconds, with .about.50 Psi pressure. Reflow
of the untreated films (SEM micrographs, inset) complete the
pattern transfer. Duplication can be carried out by using each
generation to imprint the next, making the technique truly
high-throughput. (b) Lap shear bond strength for overlapped,
PiP-pressed films of untreated and 120.degree. C. 60 seconds
pre-treatment. A 6 fold drop of in bond strength was seen with the
crystallized films. (c) Crystallinity kinetics and residual water
content for films throughout the PiP process. A crystallized
plateau was reached after 15 seconds of pressing. Water loss
follows the inverse pattern of crystallization.
[0011] FIG. 2 provides characterization of multi-generation PiP
imprint, showing mean AFM cross section, AFM topology, and
darkfield optical response of 200 nm diameter nanopillar lattice.
Scale bars 1 .mu.m. (a) Third generation imprint. (b) First
generation PiP imprint. (c) Silk master generated through
nanoimprint lithography. (d) Silicon master fabricated through
electron beam lithography.
[0012] FIG. 3 provides characterization of PiP parameter space. (a)
AFM topography of PiP imprint process for parameter space
characterization. (b) Darkfield optical response and AFM
cross-section of silk master. (c) Characteristic optical response
and AFM section of PiP imprints. Imprinted at 110.degree. C. for 30
seconds. (d) Darkfield optical response and AFM cross-section of
silk master after 18 uses. (e) Interpolated contour plot of imprint
quality as measured by hole depth over PiP parameter space.
Differences in kinetics with temperature are clearly seen. No
response seen below 80.degree. C.
[0013] FIG. 4 illustrates conformal PiP and surface transfer. (a)
Modification to the PiP process for conformal imprint, showing the
addition of aluminum tool for normal pressure application. (b) Lens
characteristics, 9.8 mm radius of curvature, 15 mm diameter. Inset:
Imprinted lens. (c) Transfer of film imprinted on lens to curved
biological surface (scale bar 4 mm). Inset: change in colorimetric
response of the imprinted film with the addition of ethanol to the
surface (scale bars 1 mm). (d) AFM topology. Scale bar 5 .mu.m. (e)
AFM cross section of conformal imprint, showing good pattern
replication across all dimensions.
[0014] FIG. 5 illustrates certain embodiments of an overall scheme
of silk fibroin processing.
[0015] FIG. 6 provides schematics of protein micro- and
nano-fabrication. Left: the "shadow mask deposition" technique;
center: the "casting and lift-off" technique using a mold; and
right: "rapid nanoimprinting" technique.
[0016] FIG. 7 depicts general relationship between crystallinity,
water content and glass transition temperature, as applied to the
rapid nanoimprint lithography technique.
[0017] FIG. 8 provides a graph showing structural relationship of
random coil and beta-sheet silk fibroin as applied to improved
nanoimprinting lithography.
[0018] FIG. 9 provides schematics of protein-protein imprinting
("PiP").
[0019] FIG. 10 shows temperature- and time-dependent
nano-imprinting.
[0020] FIG. 11 shows PiP replication of imprints using a silk
fibroin master film.
[0021] FIG. 12 shows non-limiting applications of the PiP
technique.
[0022] FIG. 13 shows a non-limiting embodiment of a desktop
embossing system.
[0023] FIG. 14 shows an example of nickel master.
[0024] FIG. 15 shows an imprinted silk fibroin film.
[0025] FIG. 16 provides an illustration of a non-limiting example
of an embossing system, drawn in 3D.
[0026] FIG. 17 provides an illustration of an example of a bottom
plate that forms the embossing system of FIG. 16 above.
[0027] FIG. 18 provides an illustration of an example of a center
plate that forms the embossing system of FIG. 16 above.
[0028] FIG. 19 provides an illustration of an example of a top
plate that forms the embossing system of FIG. 16 above.
[0029] FIG. 20 provides an illustration of a front view of the
embossing system of FIG. 16 above.
[0030] FIG. 21 provides an image of Allen-Bradley Family of
PLCs.
[0031] FIG. 22 shows an embodiment of a heated-plate design.
[0032] FIG. 23 provides a back view of an exemplary heated
plate.
[0033] FIG. 24 provides a schematic of an embosser control
software.
[0034] FIG. 25 provides exemplary temperature control rungs.
[0035] FIG. 26 shows an exemplary TC2 module selection.
[0036] FIG. 27 shows an exemplary configuration of TC2 module.
[0037] FIG. 28 shows exemplary OF2 module selection.
[0038] FIG. 29 shows exemplary configuration of 2080-OF2
module.
[0039] FIG. 30 illustrates an exemplary embossing ladder rung.
[0040] FIG. 31 depicts an exemplary configuration of onboard serial
port.
[0041] FIG. 32 provides an exemplary parse_incoming_MSG function
block.
[0042] FIG. 33 shows an exemplary report_temperature function
block.
[0043] FIG. 34 shows an exemplary embosser user interface.
[0044] FIG. 35 provides an image of a non-limiting example of a
completed embosser (front view).
[0045] FIG. 36 provides an image of a non-limiting example of an
embosser and controls during software development applications.
[0046] FIG. 37 provides an image of a silk battery.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0047] Control of the interface between biological tissue and high
technology materials is paramount for the development of future
applications in biomedicine, especially in the case of implantable
integrated devices for signal transduction (D.-H. Kim et al.,
Science 2011, 333, 838-43; D. Kim et al., Nature Materials 2010, 9,
511-517; J. Viventi et al., Nature Neuroscience 2011, 14,
1599-605). Such work requires careful materials design to develop
devices that can efficiently perform technological functions while
retaining biocompatibility and biological integration.
[0048] Silk is a natural protein fiber produced in a specialized
gland of certain organisms. Silk production in organisms is
especially common in the Hymenoptera (bees, wasps, and ants), and
is sometimes used in nest construction. Other types of arthropod
also produce silk, most notably various arachnids such as spiders
(e.g., spider silk). Silk fibers generated by insects and spiders
represent the strongest natural fibers known and rival even
synthetic high performance fibers. Silk is naturally produced by
various species, including, without limitation: Antheraea mylitta;
Antheraea pernyi; Antheraea yamamai; Galleria mellonella; Bombyx
mori; Bombyx mandarina; Galleria mellonella; Nephila clavipes;
Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia;
Araneus diadematus; Latrodectus geometricus; Araneus bicentenarius;
Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus;
Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and
Nephila madagascariensis.
[0049] Silk fibroin proteins offer desirable material
characteristics for a number of applications that take advantage of
the nature of biological materials, such as biocompatibility. Silk
fibroin of the Bombyx mori silkworm has come of considerable
interest in this context, owing to its attractive mechanical (B. D.
Lawrence, et al., Journal of Materials Science 2008, 43, 6967-6985;
S. Sofia et al., Journal of Biomedical Materials Research 2001, 54,
139-48; L. Meinel et al., Bone 2006, 39, 922-31; H.-J. Jin et al.,
Biomacromolecules 2002, 3, 1233-9), biological (M. Santin et al.,
Journal of Biomedical Materials Research 1999, 46, 382-9; E. M.
Pritchard et al., Journal of Controlled Release: Official Journal
of the Controlled Release Society 2010, 144, 159-67), and optical
properties (H. Perry et al., Advanced Materials 2008, 20,
3070-3072; B. D. Lawrence et al., Biomacromolecules 2008, 9,
1214-20) for use in biomedical, optical, electro-optical,
industrial and other applications.
Silk Fibroin Processing
[0050] An exemplary scheme of silk fibroin processing is outlined
in FIG. 5. The process involves silk fibroin protein, known for its
biocompatibilty, biodegradability, and biomedical applicability. In
some embodiments, regenerated silk fibroin proteins are prepared
from natural sources, e.g., cocoons of the mulberry silkworm,
Bombyx mori. Native silk from cocoons contain both silk fibroin, as
well as sericin, which is a glue-like protein that holds the cocoon
together. In some embodiments, silk proteins are processed so as to
remove the sericin protein in a process generally referred to as
"degumming" in order to produce regenerated silk fibroin that is
substantially free of sericin.
[0051] As used herein, "substantially free of sericin" means that
sericin is absent from such a preparation, or present in such a
trace amount that it does not affect the subsequent step or steps
of silk fibroin processing or its downstream application. In some
embodiments, a trace amount of sericin that may be present in a
silk fibroin preparation is present in concentrations less than
about 0.5%, less than about 0.4%, less than about 0.3%, less than
about 0.2%, less than about 0.1%, less than about 0.05%, less than
about 0.04%, less than about 0.03%, less than about 0.02%, less
than about 0.01%, or lower. In some embodiments, a trace amount of
sericin that may be present in a silk fibroin preparation is
present in a concentration that is below a detectable threshold by
conventional assays used in the art.
[0052] The degumming process typically involves boiling of cocoon
samples under an alkaline condition, such as in a sodium carbonate
solution, to remove the sericin. Following that step, the
water-insoluble fibers are first dried, and then dissolved in a
solution, such as a lithium bromide solution. Silk fibroin in the
lithium bromide solution is then typically dialyzed in water.
Dialysis removes the salt, resulting in an aqueous solution of silk
fibroin. In some embodiments, aqueous solution of silk fibroin
prepared this way contain between about 4-10% of pure silk fibroin,
e.g., about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about
6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about
9.5%, about 10% of silk fibroin. Typically, such aqueous solution
contains about 5-7%, for example, about 6% of silk fibroin.
[0053] In some embodiments, one or more dopants (e.g., active
agents), such as biodopants, may be added to silk fibroin
preparations, which can provide additional functionality, visa vie
sensing capability or structural support. For example, silk fibroin
solution containing at least one dopants can then be processed via
various methods into desirable formats, such as gels, films,
fibers, sponges, meshes, particles, and the like. In some
embodiments, we focused on silk films, which can be further
processed, and micro- or nanofabrication steps can convert them
into useful devices, including optical components, e.g., lens
arrays, gratings, and holograms.
Protein Micro/Nanofabrication with Silk
[0054] A variety of useful applications are contemplated. For
example, within this family of microfabrication tools reported in
the literature (see FIG. 6), we have the means of providing a
number of additional functionalities. For example, by using shadow
mask deposition, we can sputter metals on to films, for
transferable metamaterial sensors. By adding pharmaceutical dopants
and then casting on molds, such as soft PDMS-based molds, we can
fabricate microneedle arrays for transdermal drug delivery with
continuous release profiles. And, by pressing a hard nanopatterned
silicon or glass master into a silk film with the application of
heat, we can generate micro and nanoscale features, such as these
photonic crystal structures as shown in the FIG. 6. Functionally,
these structures may act as optical transducers for biosensing
applications.
[0055] As described herein, the present invention extends and
greatly improves both fabrication processes and end use
applications by improved nanoimprinting methodology.
Rapid Nanoimprint Lithography
[0056] The inventors of the present application have recognized
that, the process of imprinting is intimately affected by, and even
a direct result of, the variable glass transition temperature, or
Tg, of silk materials. Moreover, the inventors have recognized that
the process also depends on their water content. The illustration
provided in the upper left side of FIG. 7 under this subsection is
intended to represent the secondary structure of the silk protein
films, containing random coil, alpha-helix, and beta-sheet motifs
(e.g., secondary structures), along with bound and unbound water
molecules. An air-dried film, as on the left panel, will contain a
mixture, with extensive random coil and beta sheet motifs, as well
as considerable water content. Inducing crystallization of silk
fibroin (e.g., annealing) will cause an increase in the beta sheet
motifs, and a corresponding decrease in water content, as unbound,
and then bound water are pushed out of the film creating
crystalline domains. In some embodiments, annealing involves
contacting a silk fibroin material with an organic solvent or an
alcohol, including but are not limited to: methanol, ethanol,
isopropanol, acetone, or any combination thereof. In some
embodiments, annealing involves water-vapor annealing. In some
embodiments, annealing can be induced by submerging a silk fibroin
material (such as film) in a methanol solution to induce
crystallization of silk fibroin, such that there is an increased
level of beta-sheet content in the fibroin protein.
[0057] This is also exemplified on the graph shown in the lower
left side of FIG. 7, where, in this case, treatment time is time
treated with a 50% methanol solution. As the graph on the right
shows, the silk film Tg can exist anywhere from 50.degree. C. to
150.degree. C., depending on the water content, with a lower Tg
corresponding to a higher water content. In some embodiments, silk
fibroin Tg is, for example, about 50.degree. C., about 55.degree.
C., about 60.degree. C., about 65.degree. C., about 70.degree. C.,
about 75.degree. C., about 80.degree. C., about 85.degree. C.,
about 90.degree. C., about 95.degree. C., about 100.degree. C.,
about 105.degree. C., about 110.degree. C., about 115.degree. C.,
about 120.degree. C., about 125.degree. C., about 130.degree. C.,
about 135.degree. C., about 140.degree. C., about 145.degree. C.,
about 150.degree. C. Below their glass transition temperatures,
silk films form hard glassy structure, and above they will be in a
pseudo-liquid state. It has been recognized that this is the
crucial transition that may be used to leverage to imprint. In some
embodiments, water content of a silk fibroin material (such as silk
fibroin film), as measured by weight percentage, is between about
1% and 35%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%,
about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about
12%, about 13%, about 14%, about 15%, about 16%, about 17%, about
18%, about 19%, about 20%, about 21%, about 22%, about 23%, about
24%, about 25%, about 26%, about 27%, about 28%, about 29%, about
30%, about 31%, about 32%, about 33%, about 34%, about 35%. In some
embodiments, water content of a silk fibroin material, as measured
by weight fraction, is below 0.2, for example, below 0.1.
[0058] In some embodiments, the rapid imprint lithography technique
described herein may take advantage of rapid, high temperature
treatment (heating) to trigger robust crystallization of silk
fibroin (FIG. 8). In some embodiments, suitable heating is carried
out at temperature of between about 80-140.degree. C., e.g.,
between about 85-135.degree. C., between about 90-130.degree. C.,
between about 95-125.degree. C., between about 100-130.degree. C.,
between about 105-125.degree. C., between about 110-125.degree. C.,
between about 115-125.degree. C., between about 116-124.degree. C.,
between about 117-123.degree. C., between about 118-122.degree. C.,
between about 119-121.degree. C. In some embodiments, heating is
carried out at about 120.degree. C., e.g., about 110.degree. C.,
about 111.degree. C., about 112.degree. C., about 113.degree. C.,
about 114.degree. C., about 115.degree. C., about 116.degree. C.,
about 117.degree. C., about 118.degree. C., about 119.degree. C.,
about 120.degree. C., about 121.degree. C., about 122.degree. C.,
about 123.degree. C., about 124.degree. C., about 125.degree. C.,
about 126.degree. C., about 127.degree. C., about 128.degree. C.,
about 129.degree. C., about 130.degree. C.
[0059] In some embodiments, heating is carried out for a period of
time sufficient to induce crystallization (or beta-sheet formation)
in silk fibroin. Suitable durations in this context depend, but in
some embodiments, heating is carried out for about 1 second, about
2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about
6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about
10 seconds, about 11 seconds, about 12 seconds, about 13 seconds,
about 14 seconds, about 15 seconds, about 16 seconds, about 17
seconds, about 18 seconds, about 19 seconds, about 20 seconds,
about 21 seconds, about 22 seconds, about 23 seconds, about 24
seconds, about 25 seconds, about 26 seconds, about 27 seconds,
about 28 seconds, about 29 seconds, about 30 seconds, about 31
seconds, about 32 seconds, about 33 seconds, about 34 seconds,
about 35 seconds, about 36 seconds, about 37 seconds, about 38
seconds, about 39 seconds, about 40 seconds, about 41 seconds,
about 42 seconds, about 43 seconds, about 44 seconds, about 45
seconds, about 46 seconds, about 47 seconds, about 48 seconds,
about 49 seconds, about 50 seconds, about 51 seconds, about 52
seconds, about 53 seconds, about 54 seconds, about 55 seconds,
about 56 seconds, about 57 seconds, about 58 seconds, about 59
seconds, about 60 seconds, about 61 seconds, about 62 seconds,
about 63 seconds, about 64 seconds, about 65 seconds, about 66
seconds, about 67 seconds, about 68 seconds, about 69 seconds,
about 70 seconds, about 71 seconds, about 72 seconds, about 73
seconds, about 74 seconds, about 75 seconds, about 76 seconds,
about 77 seconds, about 78 seconds, about 79 seconds, about 80
seconds, about 81 seconds, about 82 seconds, about 83 seconds,
about 84 seconds, about 85 seconds, about 86 seconds, about 87
seconds, about 88 seconds, about 89 seconds, about 90 seconds, or
longer.
[0060] To illustrate, as the FTIR spectra on the right show,
heating the film to 120.degree. C. for 60 seconds as in PiP causes
a decrease in the random coil band at 1650 wavenumbers and an
increase in the beta sheet band at 1620 wavenumbers of the Amide I
region of the spectra. Compared to a more "gentile" crystallization
technique, this rapid transition drives water out, causing reflow,
and then as the Tg rises further, locks the pattern in to place via
film crystallization. The improvement came in the realization that
a crystallized patterned film with a high Tg is equivalent to a
metal master for silk imprinting purposes used in previous work.
With this recognition, it was possible to take advantage of the Tg
difference, to use one silk film (master) to imprint another
(blank). The imprinting film (e.g., master), never reaching its Tg
will act as a solid mold for the imprinted film, which will need to
be heated past the transition point, which can be accomplished on
the bench top. This novel concept not only eliminated the
requirement of expensive and fragile metal or silicon master, but
also brought about a whole new avenue of simple yet elegant
processing methods with additional benefits not available in
conventional imprinting methodology.
[0061] Accordingly, in some embodiments of the invention, a
composition comprising a first silk fibroin material comprising a
predetermined nanostructure thereon is provided, in which the first
silk fibroin material has a first beta sheet content and a first
glass transition temperature (T.sub.g1); a second silk fibroin
material, having a second beta sheet content and a second glass
transition temperature (T.sub.g2); and, wherein the first silk
fibroin material is in close contact with the second silk fibroin
material, such that the predetermined nanostructure on the first
fibroin material is substantially replicated onto the second silk
fibroin material to produce an inverse imprint of the predetermined
nanostructure. In some embodiments, wherein the inverse imprint of
the predetermined nanostructure has a resolution of at least 100
nm, at least 250 nm, at least 200 nm, at least 300 nm.
[0062] In some embodiments, the invention provides a composition
comprising a first silk fibroin material comprising a predetermined
nanostructure fabricated thereon; wherein the first silk fibroin
material has a first water content and a first glass transition
temperature (T.sub.g1); a second silk fibroin material, having a
second water content and a second glass transition temperature
(T.sub.g2); wherein the first silk fibroin material is in close
contact with the second silk fibroin material, such that the
predetermined nanostructure on the first fibroin material is
substantially replicated onto the second silk fibroin material to
produce an inverse imprint of the predetermined nanostructure. In
some embodiments, wherein the inverse imprint of the predetermined
nanostructure has a resolution of at least 100 nm, at least 250 nm,
at least 200 nm, at least 300 nm.
PiP Characterization
[0063] This additional benefit comes from the recognition that
since any silk film can become a master, each film can give rise to
a subsequent generation of replica films, and so one imprint from a
fabricated master can potentially be amplified into a very large
number of films in the fashion of a binary tree. In other words,
the use of silk in this way makes it possible for serial
imprinting, while preserving high fidelity.
[0064] In the context of the present disclosure, the preservation
of high fidelity refers to the ability of replicating the structure
(i.e., pattern) of a master plate/film onto a blank (e.g., a second
film) such that the structural features are accurately reproduced
onto the blank to create a mirror image (i.e., inverse) of the
master pattern. Similarly, such step may be repeated for serial
imprinting without losing structural features being replicated at
each round, in a way sufficient to serve the purpose or function of
interest. Furthermore, in some embodiments, high fidelity may also
refer to the ability to generate replicas which are structurally
and functionally equivalent of one another. For example, fidelity
is said to be the highest when one positive imprint is identical to
a second positive imprint, and one negative imprint is identical to
a second negative imprint, while each of the positive imprints is
the exact mirror image of each of the negative imprints.
[0065] Accordingly, in some embodiments, the invention provides a
composition comprising a plurality of negative silk fibroin
imprints and a plurality of positive silk fibroin imprints. In some
embodiments, a positive silk fibroin imprint of such compositions
comprises a nanopattern, and a negative silk fibroin imprint of
such compositions comprises an inverse of the nanopattern present
on the positive silk fibroin imprint. In some embodiments, such
negative silk fibroin imprints are substantially identical replicas
of one another, and such positive silk fibroin imprints are
substantially identical replicas of one another. As used herein,
"substantially identical replicas" mean that such structures are
sufficiently similar that they serve equivalent function for
intended purposes. As an example, such a structure may comprise an
array of photonic crystal that causes structural colors.
Nanopatterns of such photonic crystals created by the PiP technique
described in this application, for example by serial imprinting,
are said to be substantially identical replicas of one another if
each resulting nanopatterned film is able to reproduce visibly same
or similar structural color or colors.
[0066] As a first step in testing this phenomenon, an initial silk
master was taken and was used to create three generations of
imprints. As shown in FIG. 9, the optical, 2D Atomic force
microscopy, and representative AFM cross-sections are provided.
With each subsequent cycle (or generation), an increase in the
sharpness of the peaks can be noted, although it is important to
keep in mind the relative scales of the axes, so the holes are not
as sharp as they appear. Remarkably, though, a marked decrease in
hole depth across generations was not seen, which led to comparable
intensity for all samples tested. A simple calculation provides the
following: assuming a maximum of three generations, N squared+1 and
N squared plus N cubed negative and positive imprints respectively
can be generated. If a modest assumption that three imprints per
generation is made, this results in 10 negative and 36 positive
imprints from a single master use.
[0067] As should be clear from the illustration in FIG. 9, the
terms positive and negative imprints denote s mirror image
relationship to each other, such that if a master pattern is deemed
a positive imprint, an imprint made from the master pattern would
have its mirror image, or negative imprint, and so on. And a second
imprint made from the same master pattern would also be a negative
imprint, or a replica of the first imprint made from the master
pattern.
[0068] With this in hand, further characterization of the technique
was performed, both for optimizing the imprint quality for
desirable conditions, and for investigating and verifying the
limits of the system. With the use of a constant level of pressure,
a 500 nm thick silk film with Tufts written in it using photonic
crystal structures of varying lattice constant, was used as a
master (as shown in FIG. 9) for imprinting. The temperature was
varied between 70 and 120.degree. C., and the time (i.e., duration)
was tested between zero and 60 seconds.
[0069] The FIG. 10 provides an interpolated contour plot of the
results, showing the average post height of the imprints, as per
the AFM images on the right, over tested conditions. These results
are film thicknesses, and hydration states tested, but show a
general trend of optimization, with the best results at
intermediate times and temperatures. This makes logical sense, as
adding additional heat will dry the film quicker, pushing up the Tg
faster, and decreasing the amount of time for reflow, and short
times will not allow sufficient time for reflow. It appears that at
least under these conditions, all silk reflow occurs with the first
30-40 seconds, since there is little change with time after this
point, and so further heating is unnecessary.
[0070] Upon reviewing representative images and cross sections of
these characterization tests, both in optical response and cross
section, it is clear that the two dimensional and optical
replication were both strong. The cross sections in this
non-limiting example are taken from the "T" in Tufts (FIG. 11).
Furthermore, the silk master was able to withstand 18 imprints
without considerable loss in quality, demonstrating that high
fidelity is effectively maintained after a number of cycles of
serial imprinting. Applying this to our calculation from before, in
three generations, 325 negative and 6,156 possible positive
imprints can be generated from the use of a single master.
Extensions and Applications
[0071] However, the advantages of the PiP method extend beyond
rapid, high-throughput nanoscale fabrication. The FIG. 12 provides,
in blue, how this technique could be applied to imprint directly
onto curved surfaces, due to the flexible nature of the silk films
utilized. With the addition of a simply machined aluminum piece to
apply pressure normal to the curved surface, we were able to
imprint this 3,600 dots/mm 2D grating structure on the surface of a
small Plano convex spherical lens with a diameter 15 mm, radius of
curvature 9.8 mm, and 30 mm focal length. The combination of
diffractive features on a refractive device could be useful for
spectrophotometric and beam manipulation applications. The
completed construct was able to select orders of light, and showed
good replication in all three dimensions. Pulling back the transfer
techniques applied to the electromagnetic transduction mechanisms
in the introductory slides, this technique now shows promise for
the application of optical transduction mechanisms to biomedically
relevant surfaces.
[0072] To summarize, the protein-protein imprinting method
described here leverages the variable glass transition temperature
of silk fibroin films to imprint with approximately 100 nm
resolution. In some embodiments, the methods described herein can
achieve resolution of about 500 nm, about 450 nm, about 400 nm,
about 350 nm, about 300 nm, about 250 nm, about 200 nm, about 150
nm, about 100 nm, or higher. This allows for rapid, high throughput
multi-generation imprinting, over at least 3 generations with at
least 18 imprints per generation. Such imprinting can be carried
out on films as thin as 500 nm or less, with temperatures as low as
80.degree. C. or less, and durations of time as short as a few
seconds. Combined with conformal imprinting, this technology can be
of help to work towards increasing the range of applications for
nanoscale fabricated transducers, processed only in water.
EXEMPLIFICATION
[0073] The working examples are presented for illustrative purposes
only, and the embodiments described herein are not construed to be
any way limiting. For detailed disclosures of related art, see, for
example, WO 2008/127404, WO 2008/118211, WO 2008/127402, WO
2008/127403, WO 2008/127401, WO 2008/140562, WO 2008/127405, WO
2009/061823, WO 2009/155397, WO 2010/126640, WO 2011/046652, WO
2011/026101, WO 2011/160098, WO 2012/054121, WO 2011/115643, WO
2011/130335, WO 2011/112931, WO 2012/047682, WO 2012/031282, WO
2012/094436, WO/2012/145739, WO 2010/088585, the contents of each
of which are incorporated herein by reference.
Example 1
Protein-Protein Imprinting (PiP): High Throughput Nanoscale
Imprinting of Silk Fibroin Films for Photonics
[0074] Recent work has shown adaptation of common micro- and
nano-fabrication tools to silk films (R. D. White et al., Journal
of Micromechanics and Microengineering 2011, 21, 115014; K. Tsioris
et al., Advanced Materials 2011, 23, 2015-9; K. Tsioris et al.,
Advanced Functional Materials 2010, 20, 1083-1089; J. P. Mondia et
al., Advanced Materials 2010, 22, 4596-9; H. Tao et al., Advanced
Materials 2011, 23, 3197-201), leading to biocompatible and
degradable electronic and photonic devices which can simultaneously
act as a carrier and stabilizer for protein pharmaceuticals and
other bioactive reagents (J. Zhang et al., Proceedings of the
National Academy of Sciences of the United States of America 2012;
K. Tsioris et al., Advanced Functional Materials 2012, 22, 330-335;
H. Tao et al., Advanced Materials 2012, 24, 2824-37; P. Domachuk et
al., Applied Physics Letters 2009, 95, 253702). In particular, silk
based nanoscale photonic devices face the challenge of
sub-wavelength resolution fabrication on a soft polymeric substrate
(J. P. Mondia et al., Advanced Materials 2010, 22, 4596-9; S. Y.
Lee et al., Proceedings of the National Academy of Sciences of the
United States of America 2010, 107, 12086-90). Previous work
introduced the possibility of direct, rapid nanoimprinting in silk
for the fabrication of photonic structures by leveraging the
material properties of this protein (J. J. Amsden et al., Advanced
Materials 2010, 22, 1746-9).
[0075] In this context, the glass transition temperature (T.sub.g)
of the protein is a parameter of particular relevance. In a silk
film dried under ambient conditions, the water retained by the film
acts as a plasticizer, significantly lowering the glass transition
from 178.degree. C. to .about.78.degree. C. (X. Hu et al.,
Thermochimica Acta 2007, 461, 137-144). The actual T.sub.g depends
inversely on the water content and can be modeled as a function of
the fractions of silk and water in the dried construct (N. Agarwal
et al., Journal of Applied Polymer Science 1998, 63, 401-410). In
imprinting, the application of heat and pressure to a silk fibroin
film layered on a hard mask rapidly pushes the film above T.sub.g,
causing it to transition from a glassy state to a liquid-like
rubber, allowing reflow of polymer on the nanoscale (J. J. Amsden
et al., Advanced Materials 2010, 22, 1746-9).
[0076] In this work, we expand upon this technique by exchanging
the conventionally fabricated hard silicon or glass masters with a
second silk fibroin film, in a process we term protein-protein
imprinting (or PiP for short). This is accomplished by control of
the water content and beta-sheet crystallinity of each of the two
films. Though the heat and pressure application are similar to the
previous technique, using a dry, crystallized fibroin master and a
wet, uncrystallized "blank" film allows for reflow of the template
film only and limits the adhesion between the two layers for easy
release. As an improvement to the current technique, this method
significantly increases throughput, reduces dependence on the hard
mask, and, additionally, allows for direct conformal imprinting on
curved surfaces due to the flexibility of the nanoimprinted silk
master.
[0077] Nanoimprint lithography on nonplanar surfaces has been
investigated recently for applications in spectroscopy (Y. Xie et
al., Optics Express 2003, 11, 992-5), superhydrophobic structures
(L. Mishchenko et al., ACS Nano 2010, 4, 7699-7707), and microlens
arrays (K. Hoshino et al., IEE Conference on Micro-Electro
Mechanical Systems 1999, 429-434; H. O. Jacobs et al., Science
2002, 296, 323-5), and has shown resolution on the order of
.about.100 nm. Here, a wide variety of approaches have been
attempted, ranging from nanoimprint lithography with flexible
materials to chemical vapor deposition assisted methods. Most of
these currently available techniques suffer from complex procedures
or destruction of the imprint master (W. M. Choi & O. O. Park,
Nanotechnology 2004, 15, 1767-1770; R. Mukherjee et al., Industrial
Engineering Chemical Research 2009, 8812-8818; B. Farshchian et
al., Microelectronic Engineering 2011, 88, 3287-3292). The required
photoresists and imprinted materials are often not biocompatible,
limiting their use in biomedical applications (B. Farshchian et
al., Microelectronic Engineering 2011, 88, 3287-3292). Furthermore,
most of those processes are relatively time consuming, with a
required imprinting duration ranging from a few minutes to several
hours. For example, a common conformal printing technique using
thin poly(dimethylsiloxane) (PDMS) membranes usually requires
.about.5 min imprint times for a resolution of .about.500 nm, and
lacks long term repeatability due to master deformation after
repeated heating for the long imprinting time (W. M. Choi & O.
O. Park, Nanotechnology 2004, 15, 1767-1770). Further, none of
these prior approaches provides facile modes to incorporate
bioactive components with retention of function, a feature that
permits broader utility in medical devices and other domains.
[0078] For the application of PiP to nonplanar surfaces we
demonstrate resolution on the order of currently available
nonplanar imprint technologies, with higher throughput and fewer
concerns about master durability. In addition, due to the
biocompatibility of silk, nonplanar PiP can be used to apply
nanophotonic structures to curved biological surfaces (D.-H. Kim et
al., Science 2011, 333, 838-43; J. Viventi et al., Nature
Neuroscience 2011, 14, 1599-605). Together, this approach provides
a versatile and simple fabrication method for biointegrated silk
devices, further expanding the utility of silk fibroin as a bridge
between high technology and biomedical applications.
Method and Mechanism
[0079] The PiP process is shown in FIG. 1a. A patterned silk
master, shown in yellow, was placed underneath the film to be
protein imprinted, shown in blue, on a smooth polished Ni surface
(for uniform thermal distribution) heated to 120.degree. C. About
50 Psi of pressure was applied to the top and held for between 5
and 60 seconds, during which reflow occurred. This produced the
inverse pattern in the blank film, as shown in the scanning
electron microscope (SEM) images. If crystallization occurs in the
imprinted film, this new film could be utilized as a master for a
subsequent generation, which would then have the original pattern,
as shown in step two. Such a methodology could lead to multiplexing
the production of films (for instance two imprints per generation
would yield 4 positive and 8 negative imprints of the master in
only three generations).
[0080] Indeed, crystallization of the imprinted films does take
place, as is shown in FIG. 1c. Here, Fourier-transform infrared
(FTIR) spectra were taken for heated films at 5-second intervals,
and beta-sheet content of the films was quantified using previously
established curve-fitting procedures (V. a Lorenz-Fonfria & E.
Padros, Spectrochimica Acta. Part A, Molecular and Biomolecular
Spectroscopy 2004, 60, 2703-10; X. Hu et al., Macromolecules 2006,
39, 6161-6170; Q.-N. Wei et al., Journal of Applied Polymer Science
2012, 125, E477-E484). At 120.degree. C. and .about.50 Psi, the
kinetics of crystallization occurred rapidly, with the films
reaching a crystalline plateau after only 15 seconds of pressure.
This plateau was found to be equivalent, if not higher, in
crystalline content to conventional silk film annealing treatments
such as exposure to methanol. Additionally, the hydration state of
the films was monitored using Thermal Gravimetric Analysis (TGA),
in a similar procedure. Films dried under ambient conditions were
found to have .about.10.4% residual water, which rapidly decreased
to .about.7% as the crystallinity increased, as shown in FIG. 1c.
This corresponds to little change in the T.sub.g of the plasticized
film, with a glass transition close to 80.degree. C. in all
cases.
[0081] The adhesion of the two layers after imprinting is essential
to the success of the process, as difficulty in separating the two
films could cause damage to either nanoscale pattern, decreasing
yield. Investigation of the inter-layer adhesion was examined, by
performing tensile testing (Instron 3360, Instron Inc., Norwood,
Mass.) both for untreated films, and films crystallized via the PiP
method. The figure shows a 6-fold decrease in interfacial strength,
to a value of less than 500 kPa, indicating easy separation of the
two layers after PiP, because of the crystallization of the films.
This property is advantageous, as both high and low adhesion
strengths are possible in the same material through the control of
the thermal transition, thus enabling imprinting.
[0082] Additionally, the data indicate that the crystallization
plateau is responsible for locking the imprinted pattern into
place, as the T.sub.g does not change appreciably with the modest
(.about.3%) reduction in water content seen here (N. Agarwal et
al., Journal of Applied Polymer Science 1998, 63, 401-410). The
kinetics of crystallization, and thus reflow, occurs rapidly, which
means PiP imprints may be possible on an even shorter timescale.
This confirms that it should be possible to directly imprint
subsequent generations utilizing imprinted owing to the high degree
of crystallinity imparted by the process.
Characterization
[0083] The PiP mechanism suggests the possibility of
multi-generation imprinting, which was confirmed by evaluating the
pattern quality (e.g., resolution) with subsequent imprinted
generations. For this, electron beam lithography written periodic
photonic crystal structures of 100 nm tall, 200 nm diameter Cr
pillars on Si--which serves as the original (non-silk) master and a
baseline for imprinting quality comparison--was utilized, with
lattice constants varying from 200-700 nm. The optical response
under darkfield illumination (Olympus IX71, Center Valley, Pa.) as
well as the surface profile measured via atomic force microscopy
(AFM)(Veeco Dimension 3100, Plainview, N.Y.) have been collected,
as shown on the right hand side of FIG. 2d. The mean cross section
of the image is shown on the left. This master was utilized to
create an inverse silk master through the previous rapid
nanoimprinting technique (FIG. 2c). The results show good spatial
and optical fidelity, with a slight reduction in pattern depth (57
nm vs. 80 nm), accompanied by sharper features at the extremes.
[0084] The silk master was then utilized in PiP to create a first
generation imprint (FIG. 2b), which in turn imprinted the second
generation, which then produced the third (FIG. 2a). The results of
these imprints show a modest reduction in reproduction of the
nanoscale features with each subsequent generation, marked by
rounding of the sharp edges of the features. No further decrease in
feature depth was seen, and more importantly, no appreciable change
in optical response was noted. Based on this the possibility of at
least three generations of imprints via the PiP method seems to be
a reasonable assertion. Given this, the number of possible samples
becomes N.sup.2+1 negative and N.sup.2+N.sup.3 positive imprints,
where N is the number of imprints per generation. As further
generations beyond this are produced, the quality will degrade,
eventually affecting the macroscale properties and efficacy of the
imprinted patterns.
[0085] The exploration of the PiP mechanism above also indicates
the possibility of optimization of the PiP procedure for specific
applications by altering the temperature and time used for
imprinting. This was assessed via a similar experiment within a
broad range of these two primary imprinting parameters. Here, a
silk master of a similar lattice, with optical response shown in
FIG. 3b-d, was utilized to imprint a single generation, which was
then investigated via AFM. This master however, was produced to
have an initial hole depth of .about.500 nm, with a similar
diameter of 200 nm and lattice constants varying from 200-700 nm.
The results of the imprinting process are shown in FIG. 3a. Once
again, a reduction in hole depth was seen with the first imprint,
and rounding of the features was noted. Imprints were made at
temperatures between 70 and 120.degree. C. for times between 5 and
60 seconds.
[0086] An interpolated contour plot of the results is shown in FIG.
3e. The results show a maximum depth of above 400 nm, reached at
.about.20 seconds for 120.degree. C. and .about.30 seconds near
100.degree. C. the depth then rapidly decreased, with no imprints
made below 80.degree. C. This response makes sense given the PiP
mechanism, where no reflow occurs below T.sub.g. The kinetics of
crystallization, determined by the temperature used, would be
slower at lower temperatures leading to the slower reflow response
noted here (X. Hu et al., Biomacromolecules 2011, 12, 1686-96).
This broad parameter space for imprinting allows the PiP parameters
to be tailored to the application.
[0087] The same master was used though the course of this
experiment, and was analyzed before and after use. The results are
shown in FIG. 3b-d, with 3b and 3d representing the same master
before and after being used for 18 imprints, and 3c showing a
characteristic image of one of those imprints. As before, the
imprint showed rounding of the features, with little measured
variation in depth. The master, however, shows little to no change
after 18 uses, specifically on the nanoscale, as the cross sections
show. This indicates that 18 imprints is a reasonable expectation
for each generation, which would yield a maximum of N.sup.2+1=325
negative and N.sup.2+N.sup.3=6,156 positive imprints from a single
use of the original master. With each of these imprints occurring
in as little as 20 seconds, and accounting for the possibility of
multiplexing (FIG. 1a(2)), PiP is a viable option for
high-throughput nanofabrication on silk fibroin films.
Conformal Imprinting and Extensions
[0088] In addition to increasing throughput, the relative
flexibility of the silk fibroin masters as compared to a hard
silicon or metal mask was leveraged to directly achieve conformal
imprinting. The modified scheme for this method is shown in FIG.
4a. PiP was used to imprint directly on a plano-convex lens with
diameter of 15 mm and radius of curvature of 9.8 mm, whose surface
was coated with a thin layer of silk (FIG. 4b). After PiP at
120.degree. C. for 60 sec, the surface was evaluated with AFM (FIG.
4d,e). The results showed good reproduction of the master pattern
on the microscale of features on the order of 100 nm, which is
comparable to currently available options (W. M. Choi & O. O.
Park, Nanotechnology 2004, 15, 1767-1770; R. Mukherjee et al.,
Industrial Engineering Chemical Research 2009, 8812-8818; B.
Farshchian et al., Microelectronic Engineering 2011, 88,
3287-3292), while the optical response (FIG. 4b, inset) shows
coverage over the entire curved lens surface. The combination of
this technique with previously reported silk transfer techniques
for arbitrary surface transfer (H. Tao et al., Advanced Materials
2012, 24, 1067-72; D.-H. Kim et al., Science 2011, 333, 838-43; D.
Kim et al., Nature Materials 2010, 9, 511-517) can yield conformal
contact on curved tissue surfaces, as is demonstrated in FIG. 4c.
Here, the film was removed from the lens and transferred and
adhered to the skin. By this method, PiP can be used to manufacture
molded curvilinear silk protein based sensing devices that could
integrate with the human body. The possibility of a skin-skin
mounted sensor fabricated using PiP was explored as a proof of
concept demonstration by monitoring the shift in the colorimetric
response of the device with the addition of a thin coat of ethanol
(FIG. 4c, inset).
CONCLUSIONS
[0089] PiP is introduced as a rapid, high-throughput method for the
fabrication of nanoscale structures in silk films. The possibility
of at least 3 generations of imprints with at least 18 imprints per
generation was demonstrated, making this a truly high throughput
technique. Application to conformal surfaces is possible with minor
adjustments to the system, at resolutions comparable to other
currently available non-planar nanoimprint lithography techniques.
The addition of PiP to the silk materials fabrication toolbox opens
new avenues for biocompatible device fabrication, which will
further expand the utility of silk as a bridge between high
technology and biomedical applications.
Experimental
[0090] Silk Processing:
[0091] Aqueous silk solution was produced by regeneration from
Bombyx mori cocoons according to established procedures (S. Sofia
et al., Journal of Biomedical Materials Research 2001, 54, 139-48).
Briefly, the cocoons were boiled for 30 min in a 0.2 M solution of
sodium carbonate to remove sericin, glue like protein holding the
cocoon together. The fibers were dried overnight, and then
dissolved in a 9 M solution of lithium bromide. Dialysis against
Milli-Q for .about.72 hours yielded a roughly 6% aqueous solution
of silk fibroin. All casting work was carried out on a PDMS surface
or directly on a patterned silicon master. The films were dried at
ambient conditions (.about.25.degree. C., .about.40% RH) and
resulted in films that were .about.100 .mu.m thick.
[0092] Silk masters were produced using these films, and applying
the existing silk nanoimprint lithography technique (H. Perry et
al., Advanced Materials 2008, 20, 3070-3072; J J. Amsden et al.,
Advanced Materials 2010, 22, 1746-9), by heating to
.about.120.degree. C. for 60 sec against a metal master. Heat or
methanol treatments have been previously reported to be able to
tune the .beta.-sheet crystallinity of silk fibroin and were used
to crystallize the master in this work.
[0093] FTIR Spectroscopy:
[0094] FTIR scans were taken on a (Jasco FTIR 6200, Easton, Md.)
spectrometer with attached ATR detector. A total of 64 scans at a
resolution of 4.0 cm.sup.-1 were co-added to produce spectra
ranging from 400-4000 cm.sup.-1. A cosine apodization was
simultaneously applied by the software. From these scans, the amide
III region (1200-1350 cm.sup.-1) was selected for its sensitivity
to protein secondary structure and lack of sensitive to water
content. Amide III curves were normalized and baseline corrected,
and then fit to 12 Gaussian curves, according to the work of Wei et
al. (Journal of Applied Polymer Science 2012, 125, E477-E484).
Bands corresponding to .beta.-sheet secondary structure motifs were
then added to give a relative value for the .beta.-sheet
crystalline content of the films.
[0095] Thermal Gravimetric Analysis (TGA):
[0096] Water content of the silk films was assessed through TGA (TA
Instruments Q500, New Castle, Del.). Films were heated to
200.degree. C. at a rate of 10.degree. C. min.sup.-1 with a
constant mass measurement. All of the mass lost during this
procedure was determined to be water evaporation, according to
published results. All water is removed by 187.degree. C., and
total water removal is independent of heating rate for silk fibroin
films (X. Hu et al., Thermochimica Acta 2007, 461, 137-144).
[0097] Tensile Testing:
[0098] Tensile testing (Instron 3360, Instron Inc.) of the
interface between the two films was performed for mode 2 failure
via a conventional lap shear configuration. This process was
similar to ASTM D3163, with modified geometry due to specimen
limitations
[0099] Conformal-PiP Die Fabrication:
[0100] An aluminum piece was machined on a lathe to the inverse
specifications of the surface to be imprinted, in order to ensure
pressure was normal to the surface in all locations. Aluminum was
selected as the material for this piece due to its high thermal
conductivity (.about.230 W m.sup.-1 K.sup.-1), to ensure that the
heat transfer properties of the system were minimally affected.
[0101] Silk Film Transfer:
[0102] Silk films were transferred onto skin via a previously
established transfer procedure (H. Tao et al., Advanced Materials
2012, 24, 1067-72). Briefly, the side of the film to be attached
was exposed to a high (.about.90%) relative humidity environment
for a few seconds to partially solubilize the film. The film was
then applied to the transfer surface with light pressure and
allowed to dry, thereby attaching it to the surface of the
skin.
Example 2
Development of Silk Embosser
[0103] An exemplary embossing system was developed for silk film
imprinting and stamping. This system features temperature
controlled embossing surfaces, adjustable embossing pressures, and
variable embossing times. The device can also be fitted with
interchangeable temperature controlled embossing and stamping
tools. The design, development, fabrication, applications, and
device future are explored for both systems. These devices may
facilitate new discoveries and in the case of the embosser, may
provide a path towards high volume production of silk film based
technologies.
[0104] Silk films have been shown to be useful as optical
instruments. These films can be used to replicate fine features at
the nanoscale level. Simple hot embossing techniques were developed
to imprint nanoscale patterns into silk films. These techniques
involved using a simple hot plate and applying pressure by hand.
These processes, although successful, could be made simpler by
developing some simple devices.
[0105] A simple embossing device was first created using a simple
office desktop embosser. This device proved useful for a series of
experiments, but had some shortcomings. Since the device was
controlled manually, with the exception of the temperature control,
there was little control over embossing pressures and times. So an
improved design was created based on the lessons learned from the
first design iteration. The second design included provisions for
programmable embossing time and programmable pressures. These
parameters can be keyed in using a simple computer interface. This
design required the use of mechanical CAD software, a series of
industrial controls, and extensive programming.
Silk Film Embossing System
[0106] Embossing is the process of creating an impression of a
design or pattern on another surface. Hot embossing is the process
of heating a material to reach its glass transition temperature
(Tg) and applying sufficient pressure to imprint a pattern. This
process allows for the rapid replication of features. A
nano-embosser is an embossing device capable of imprinting patterns
at a small scale. These machines can replicate many fine features
but require high temperatures and pressures. Using silk films, it
is possible to imprint patterns with features on the nanoscale
using low pressures and low temperatures. Rather than spending many
thousands of dollars on such a device, a simplified version of the
tool was developed.
[0107] Nano embossing has become a useful process that offers
relatively low cost patterning of nano-scale features over large
areas. This technology has applications in the field of MEMs,
optoelectronics, biomedical, and micropackaging (Worgull M. Hot
embossing [electronic resource]: Theory and technology of
microreplication. 2009: 1 online resource (xix, 345).
[0108] Creating a low cost reproduction method for silk film
devices could facilitate silk's wide acceptance for use in
biomedical and photonic applications. This represents the first
step towards a high volume production system for silk film
patterned devices. The concept of silk film embossing has been
described previously (Amsden J J, Domachuk P, Gopinath A, White R
D, Negro L D, Kaplan D L, Omenetto F G. Rapid nanoimprinting of
silk fibroin films for biophotonic applications. Adv Mater. 2010;
22(15): 1746-1749). It was reported that silk films can be used to
replicate nanoscale features. Tests were performed with a hot plate
that heated a patterned nickel shim. A transparent silk film was
placed on top of the heated shim and some pressure was exerted.
This caused the silk film to form to the master at a very small
scale. It was desirable to create a system with repeatable
temperature and pressure profiles. In this section two embossing
system designs are presented. As described in further detail below,
the first is a modified desktop embosser, while the second is a
custom device designed and fabricated in-house.
[0109] A portable embossing system was developed using a standard
desktop embosser and some off the shelf industrial automation
components. In the system the smooth bottom plate was used as a
controlled heated surface. An Ultra Thin Sheet Heater
(McMaster-Carr Part Number 35475K12) with an adhesive backing was
trimmed to size and placed on the top side of the plate. The heater
contains a meandering conductive strip sandwiched between thin
insulating sheets. The wire leads required to run the heater were
terminated with a bayonet style connector. On the underside of the
plate a thermocouple is mounted to monitor the temperature. The
thermocouple leads are terminated with a thermocouple plug. Both
the heater and thermocouple connect to a small control box which
houses the necessary control electronics to make any necessary
temperature adjustments. Within the enclosure lies an OMRON
temperature controller and a Solid State Relay to control the
output (E5CSV temperature controllers [homepage on the Internet].
Sep. 24, 2012 [cited Nov. 30, 2012]. Available from:
http://www(dot)ia(dot)omron(dot)com/products/family/1624/). The
controller is capable of running a PID control loop and includes an
auto tuning features which eliminates any need to manually alter
the controller's settings. The solid state relay allows the plate
to be heated at intervals of minutes to milliseconds. The ability
to turn the heater output on and off at precise intervals is what
allows the heated plate to sustain its temperature over a long
period of time. FIG. 13 shows this system built and running with a
controlled temperature of 80 Degrees Celsius.
[0110] The device has been used for several nanoimprinting
experiments. To do so a nickel shim master is placed on top of the
heated plate. The temperature is set using the buttons on the front
panel of the temperature control device. Once the temperature has
been keyed in, the metal plate is heated until the set point is
reached. The silk film can then be placed on top of the master.
Pressure is applied by pushing down on the lever. Typically, the
pressure is applied for a period of five seconds to a minute and
temperatures range from 50 to 120 degrees Celsius. An example of
what can be achieved using the portable embosser is shown in FIG.
15.
Design
[0111] The portable desktop embosser was quite useful, but an
effort was undertaken to develop an embossing system that was more
flexible, had two heated surfaces instead of one, and had an
adjustable range of pressures. The following section details the
design and development of a semi-automated desktop embossing
system.
[0112] A drawing of the improved embossing systems is shown in FIG.
16. The system employs the use of a pneumatic cylinder as the means
to provide the embossing pressure. The cylinder coupled with a
proportional valve system allows for controlled embossing
pressures. Three 8 inch by 8 inch aluminum plates each with a
thickness of 1/2 in along with four 16 inch long metal extrusions
make up the body of the embosser. These components provide a stable
platform that holds the cylinder in place and provides a
cross-member for the embossing. The bottom plate is shown in design
is detailed in FIG. 17.
[0113] The center plate sits on top of the cylinder to provide
additional stability to the device. One inch notches have been cut
out of each corner to accommodate metal extrusions that run up
along each corner of the structure. This design is shown in FIG.
30.
[0114] The top plate is similar to the bottom plate in that it has
holes to accommodate the screws that fasten the extrusion pieces to
the rest of the structure. Instead of a large hole for the
cylinder, four holes with a counterbore are supplied at the center
of the plate. These holes allow for the mounting of the
interchangeable heating plates. The plate is attached by fastening
it with four M10 screws. The design of this plate is shown in FIG.
31.
[0115] Two metal plates are used as the embossing surfaces. Each
plate has been machined so that two 4 inch long 1/4 in diameter
cartridge heaters can be inserted. Each cartridge can provide 100 W
of heat to the metal plate. These cartridges are connected to a
solid state relay that is controlled by a programmable logic
controller. Using this controller along with an attached
thermocouple, the plate temperatures can be regulated. There are
two independently controlled plates in the design. This can be seen
in the front view of the device in FIG. 32. These plates can be
changed in the future if it is determined that different plate
geometries are needed. Four screws hold each plate in place. All
that must be done is to remove the four screws and replace the
plate with one that matches the threaded hole pattern.
[0116] The system is controlled with an Allen-Bradley Micro830
Programmable Logic Controller. Additional information about the
Micro830 control system may be obtained from:
http://ab(dot)rockwellautomation(dot)com/Programmable-Controllers/Micro83-
0. The controller has the capability to handle a number of digital
inputs and outputs, serial connectivity, and can handle
simultaneous PID loops. The Micro830 also has three available slots
for plug-in modules. The capabilities are extended with the use of
three Plug-in modules. The first module is the TC-2 module that
allows for the use of up to two thermocouples. The second module
(OF-2) provides two channels of analog voltage or current output.
Each output can be configured as a voltage or current source. As a
voltage source, the module can supply between 0 and 10 VDC. As a
current source, the module can supply a 4 to 20 mA output. Lastly,
the IF4 module is used to allow for analog voltages or currents to
be measured. This is intended to allow for the inclusion of sensors
in a further revision of the system. The system requires an
additional power supply provided by Allen-Bradley. A 24 VDC power
supply is required to provide the necessary power to the control
electronics.
[0117] The controller is programmed using the manufacturer's
Connected Components Workbench software. The software development
environment allows the developer to create a standalone product
utilizing ladder logic, structured text, or functional block
diagrams. The program for the embosser uses a combination of
structured text, functional block diagrams, and ladder logic
modules (Bishop R H, editor. Mechatronic system control, logic, and
data acquisition [electronic resource]. Boca Raton: Taylor &
Francis; 2008; Bolton W. Programmable logic controllers (5th
edition); Parr E A. Programmable controllers. 3rd ed. Oxford;
Burlington, Mass.: Newnes; 2003).
[0118] To control the two heated plates, an output of the
controller is connected to a solid state relay. The solid state
relay output is connected to a 120 VAC supply and two 100 W
cartridge heaters. Each heater has its own receptacle drilled into
the back of the metal embossing surface. A solid state relay allows
the controller to pulse the output at almost any interval to
accurately control the heat applied to each plate. A connected
thermocouple provides the feedback necessary for the temperature
control loop. Each heated plate in the design has its own
thermocouple, solid state relay, and associated cartridge heaters.
A drawing of the heated plate is shown below in FIG. 34. A
thermocouple bayonet adapter is threaded into the center hole. The
hole extends half way into the plate to read the current
temperature at the plate's center. Two 4 inch deep holes have been
drilled on each side of the thermocouple to provide a place for the
cartridge heaters. The cartridge heaters are each 4 inches long and
have a nominal diameter of 1/4 in. At the end of each heater are
two leads which must be connected to an appropriate power source.
In this design both heaters are connected in parallel so that both
heaters will energize when the PLC commands the solid state relay
to provide power. A view of the back side of the design is shown in
FIG. 35. A pneumatic cylinder has been chosen as the method for
applying pressure between the two plates. One plate is stationary
and is fastened to the top crossbar of the embosser. The other
plate is fastened to the end of the piston of the pneumatic
cylinder. Control of the cylinder is achieved by the use of a
proportional valve (Festo VPPM-6L-L-1-G18-0L6H-V1N). A suitable air
supply is connected to the inlet port of the proportional valve.
The outlet port is connected to one of the ports of the pneumatic
cylinder. The pneumatic cylinder is equipped with two ports.
Supplying air into one of the ports will push the piston outward
while supplying air into the other port would cause the piston to
retract. In this design, the piston will be forced out and the
pressure will be maintained by the proportional valve component.
The analog output module of the Micro830 PLC provides the necessary
voltage signal to control the proportional valve. Any necessary
scaling of the output pressure is performed by the valve system
electronics.
Electrical and Software
[0119] This project involved both hardware and software design. The
software design was done in two parts. Programming had to be done
within the PLC control system and an additional software interface
had to be programmed so that a user could set all the necessary
embossing parameters.
[0120] In order to program the Allen-Bradley PLC system, a free
software development package was used. Connected Components
Workbench (CCW) from Rockwell automation serves as the software
development suite for the entire family of Allen-Bradley Micro800
series of controllers.
[0121] A series of tasks must be performed by the PLC. Among them
are the management of two temperature control loops, commanding a
scaled voltage to drive the proportional valve and cylinder, and
the handling of both incoming and outgoing serial
communications.
[0122] The management of the temperature controls is done in
software by monitoring the current temperature using a thermocouple
and adjusting the amount of heat. As the plate heats up and
approaches the target temperature, the PLC starts pulsing the
heater output. The duty cycle of this pulsing goes from 90% down to
zero. A simple on/off control for heating would cause very large
overshoot of the target temperature. In a future iteration there
could be an addition of a PID control loop. The PID control loop
proved difficult to tune and it was decided to implement a simpler
control scheme. The rung of the ladder diagram for temperature
control is shown in FIG. 13.
[0123] The PLC stores a value for each thermocouple reading. The
value location depends on the module's physical position on the
PLC. In this design the module has been placed in the first
physical location. Within the programming environment, the
programmer must identify the existence and location of each module.
A screenshot of this process is shown in FIG. 15. By selecting the
TC-2 module in the first slot, the user has the option to change
the type of thermocouple used. A configuration panel appears below
the picture of the device. The design uses J Type thermocouples and
a scan frequency of 16.7 Hz for each channel. With the two channels
configured, it's now possible to read the temperature of each
thermocouple.
[0124] Thermocouple measurements are performed periodically by the
PLC. The programmer only needs to retrieve the value from the
appropriate location and do some necessary math to obtain a useable
value. The thermocouple reading is stored in a two byte register as
an unsigned number. This number will range from 0 to 65535 and is
found by accessing the variable _IO_P1_AI.sub.--00 for channel 0
and _IO_P1_AI.sub.--01 for channel 1. To convert the value to
degrees Celsius, 2700 must be subtracted and that result must be
divided by 10. An example follows:
.sub.--IO.sub.--P1.sub.--AI.sub.--00=2991
Degrees C.=(.sub.--IO.sub.--P1.sub.--AI.sub.--00-2700)/10
(2991-2700)/10=29.1 Degrees C.
Channel 1 is read in the same manner.
[0125] In order to command the proportional valve, a voltage of 0
to 10V must be supplied. This is done using the 2080-OF2 module.
First, the module must be configured within the CCW development
environment. The design uses one analog output channel. By
selecting the 2080-OF2 module in the second location, a menu will
appear at below the picture of the controller. Each channel can be
configured as a voltage or current output. In this design, channel
0 will be configured as a voltage output and channel 1 will be
disabled. This configuration is shown in FIG. 17.
[0126] To command a voltage at the terminals of the analog output
module, an unsigned 16 bit number must be written to the proper
location. With an analog module in the second module location,
channel 0 will be addressed using the system variable
_IO_P2_AO.sub.--00. A number ranging from 0 to 65535 is written to
the system variable. In this case writing a 0 to the variable will
provide a 0V output and writing 65535 will provide a 10V
output.
[0127] The following example demonstrates how to command a voltage
of 3.0V using the analog voltage output of the PLC.
3V/10V*65535=19661
Writing the value of 19661 to the system variable
_IO_P2_AO.sub.--00 will produce a 3.0V output.
[0128] The analog output module commands the proportional valve.
The user interface on the PC must first send a control message to
the PLC to communicate the scale of the output and the duration.
The whole embossing sequence has been programmed as a single rung
in a ladder diagram. The sequence is shown in FIG. 18.
[0129] When an Emboss flag is set within the PLC software, it
initiates the Embossing process. First a preprogrammed 4% output
control voltage is sent to the proportional valve for a period of 1
second. This allows the pneumatic cylinder to rise slowly before
rising to higher pressures. By using the SCALER function block, a
4.0% output can be scaled to an integer to command the valve. The
value is converted from a real type to an integer using an
ANY_TO_UINT function block. The output of this block writes a value
to the register commanding the voltage output. Then the TON
function block is used to delay for 1 second before the pneumatic
cylinder is commanded to a higher pressure. The sequence is the
same, the pressure is scaled for the output module and a delay
timer is engaged for the specified number of seconds. After the
programmed time has elapsed, the pneumatic valve is commanded to a
0% (0V) output. This causes the piston to drop under its own
weight. The Emboss flag is then reset so that the process can be
performed again.
[0130] There is an additional module installed in the third
location. An analog input module is not currently used, but could
provide the necessary interface for any analog sensors.
[0131] Onboard digital outputs are used to control the solid state
relays in the design. There is no need for additional modules for
standard I/O. The unit provides 14 input channels and 10 output
channels.
[0132] An on-board serial port is used to communicate with a host
computer. The Micro830 has its own dedicated serial port, but a
proprietary connector must be used to connect to a standard 9 pin
serial computer connection. Depending on the type of host computer
that is used, a USB to serial converter might be required. Serial
ports have been configured to run at 19200 bps. In order to
configure the serial port on the PLC, it must be selected in CCW
and configured. FIG. 19 illustrates the configuration of the
onboard serial port for the embossing system.
[0133] Serial messages are sent from the host computer to the PLC
to configure the necessary temperature set point for the top and
bottom plates, proportional valve control, and embossing timing.
The PLC sends periodic messages to report each plate's current
temperature throughout the process. This information is captured
and parsed by the PC and displayed to the user.
[0134] A communication protocol was developed to communicate
between the host program and the software running on the PLC.
Communication was achieved by using the serial port object within
Visual Studio (Del Sole A. Visual basic 2010 unleashed.
Indianapolis, Ind.: Sams; 2010; Bai Y. The windows serial port
programming handbook. Boca Raton, Fla.: Auerbach Publications;
2005). Messages initiated from the PC application have the
following form:
[0135] $UTEMP40LTEMP40PRESS65TIME30EMBOSSNOHALTNO#??
[0136] Carriage return and linefeed characters are appended to each
message. Messages are trapped between two delimiters $ and #. A
short word identifies each field of the message. The above message
would command both the upper and lower plate temperatures to be set
at 40 degrees Celsius. The proportional valve would be set at 65%
of its maximum. Embossing time will be set at 30 seconds. The value
following the EMBOSS field reads NO, which indicates embossing will
not start. Also, the NO following the HALT field indicates that the
embossing does not need to be interrupted.
[0137] The same above command can be used to initiate the embossing
process with one change.
[0138] $UTEMP40LTEMP40PRESS65TIME30EMBOSSYESHALTNO#??
[0139] Although it may seem wasteful to send the entire message
each time, the overhead is quite low and allows for a simpler
parsing routine on the PLC. Attached to each message is a checksum.
This is denoted by the two question marks following the control
message. This checksum is generated by the PC side software and
checked on the PLC side. Using a checksum to validate the message
ensures that commands are not compromised. The checksum is an
exclusive or of each character in the command starting with the `$`
character and ending with the `#` character. If an improperly
formatted message is detected on the PLC, no action will be taken.
Future enhancements to the system could include additional commands
for other controls tasks.
[0140] A table of valid commands is listed in Table 1.
TABLE-US-00001 TABLE 1 Valid Control Messages Field Valid Values
UTEMP 0 to 200 LTEMP 0 to 200 PRESS 0 to 100 TIME 1 to 60 EMBOSS
YES or NO HALT YES or NO
[0141] A function block diagram was created to validate the
checksum and parse the message coming from the PC. The function
block is shown in FIG. 20.
[0142] The function block has only one input. The InString variable
captures a properly terminated control string from the serial port
buffer. It then validates the message's checksum and parses the
control message. Parameters from the message are then transferred
to the proper variables. The code for this function block is
Appendix A.
[0143] The code begins by locating the `#` character which denotes
the end of the message. It then calculates a checksum by using an
exclusive or for all the characters including the `#` character. A
comparison is made to see if the message checksum matches the
calculated checksum. If the checksum is correct, the message is
parsed and the output variables are populated. If there is a
problem with the checksum, no action will be taken. This ensures
that malformed messages do not adversely affect the machine.
[0144] A status message is sent periodically from the PLC
communicating the current temperature of the plates. An example
message is shown below.
[0145] $UT43LT37#??
[0146] This indicates that the upper heated plate currently reads a
temperature of 43 degrees Celsius and the lower plate reads a
temperature of 37 degrees Celsius. Again, this message is trapped
between the two delimiters $ and #. This message is read by the
computer and parsed to inform the user of current temperatures.
TABLE-US-00002 TABLE 2 Valid Control Messages Field Valid Values UT
0 to 200 LT 0 to 200
[0147] The function block for this is shown in FIG. 21. Both TC_IN1
and TC_IN2 are inputs to thermocouples. These inputs are read and
scaled within the function block code. The outputs are NumChars and
Output_String. NumChars is the number of characters present in the
Output_String and Output_String is the series of characters
generated by the function block code.
[0148] The Report_Temp code is listed in Appendix B. Both
thermocouple inputs are read and scaled. A string of characters is
created using the format explained previously. Once the string is
complete an exclusive or checksum is generated and appended to the
string of characters. Finally, the number of characters in the
string is calculated and the final string is generated.
[0149] A computer interface was created using Microsoft Visual
Studio. Once the user is connected to the PLC via a serial
communications link, the current temperature is reported by the
interface. Then the user may make temperature adjustments, pressure
adjustments, and change the embossing time. The user interface is
show in FIG. 22.
Fabrication
[0150] The system was simple to build given its overall simplicity
and low part count. The assembly process took approximately 30
minutes. A photo of the completed embosser is shown in FIG. 23. The
completed embosser and the controls are shown in FIG. 24.
Applications
[0151] The desktop embosser was used in experiments regarding
protein-protein imprinting and the creation of silk pockets. The
embosser has been used in early experiments regarding the
fabrication of silk based batteries. An embossing temperature of
100 degrees Celsius, an embossing time of 90 seconds, and maximum
pressure was used. For an initial experiment the following
arrangement was employed: Aluminum-silver battery as an initial
model system: silk, aluminum foil (anode), potassium nitrate in
silk (salt bridge), silver nitrate in silk (cathode), aluminum foil
(charge collector), and silk. The result is a silk sandwich with
two exposed leads. Voltage was measured at the terminals as shown
in FIG. 37.
TABLE-US-00003 APPENDIX A checksum := 0; NumChars :=FIND(InString,
`#`); FORIndex :=0TONumCharsBY1DO checksum := XOR_MASK(checksum,
ANY_TO_DINT(ASCII(InString,Index))); END_FOR; IF checksum =
ANY_TO_DINT(MID(InString,FIND(InString, `$R`)-(FIND(InString,
`#`)+1),(NumChars+1))) THEN checksumOK := TRUE; UpperTempSet :=
ANY_TO_DINT(MID(InString,FIND(InString, `LTEMP`)-(FIND(InString,
`$$UTEMP`)+5),FIND(InString, `$$UTEMP`)+ 6)); LowerTempSet :=
ANY_TO_DINT(MID(InString,FIND(InString, `PRESS`)-(FIND(InString,
`LTEMP`)+4),FIND(InString, `LTEMP`)+ 5)); PressSet :=
ANY_TO_REAL(MID(InString,FIND(InString, `TIME`)-(FIND(InString,
`PRESS`)+4),FIND(InString, `PRESS`)+ 5)); TimeSet :=
ANY_TO_TIME(ANY_TO_UINT((MID(InString,FIND(InString, `EMBOSS`)-
(FIND(InString, `TIME`)+3),FIND(InString, `TIME`)+ 4))) * 1000);
IFFIND(MID(InString,FIND(InString, `HALT`)-(FIND(InString,
`EMBOSS`)+5),FIND(InString, `EMBOSS`)+ 6), `YES`) >0THEN Emboss
:=TRUE; ELSE Emboss :=False; END_IF;
IFFIND(MID(InString,FIND(InString, `#`)-(FIND(InString,
`HALT`)+3),FIND(InString, `HALT`)+ 4), `YES`) >0THEN Halt
:=TRUE; ELSE Halt :=False; END_IF; ELSE checksumOK := FALSE;
END_IF;
TABLE-US-00004 APPENDIX B checksum := 0; Current_Temp_1 := (TC_IN1
- 2700)/10; Current_Temp_2 := (TC_IN2 - 2700)/10;
Temp_Output_String := ANY_TO_STRING(Current_Temp_1);
Temp_Output_String := INSERT(Temp_Output_String,`$$UT`,1);
Temp_Output_String := INSERT(Temp_Output_String, `LT`,
MLEN(Temp_Output_String)+1); Temp_Output_String :=
INSERT(Temp_Output_String,ANY_TO_STRING(Current_Temp_2),MLEN(Temp_Output_S-
tring)+1); Temp_Output_String := INSERT(Temp_Output_String, `#`,
MLEN(Temp_Output_String)+1); NumChars :=
ANY_TO_DINT(MLEN(Temp_Output_String)); FORIndex :=0TONumCharsBY1DO
checksum := XOR_MASK(checksum,
ANY_TO_DINT(ASCII(Temp_Output_String,Index))); END_FOR;
Temp_Output_String := INSERT(Temp_Output_String,
ANY_TO_STRING(checksum), MLEN(Temp_Output_String)+1); NumChars :=
ANY_TO_DINT(MLEN(Temp_Output_String)); OutputString
:=Temp_Output_String;
* * * * *
References