U.S. patent application number 11/724178 was filed with the patent office on 2007-12-27 for method and apparatus for use of thermally switching proteins in sensing and detecting devices.
Invention is credited to Lawrence L. Brott, Daniel C. Carter, Rajesh R. Naik, Morley O. Stone.
Application Number | 20070295907 11/724178 |
Document ID | / |
Family ID | 26990060 |
Filed Date | 2007-12-27 |
United States Patent
Application |
20070295907 |
Kind Code |
A1 |
Brott; Lawrence L. ; et
al. |
December 27, 2007 |
Method and apparatus for use of thermally switching proteins in
sensing and detecting devices
Abstract
An apparatus and method for detecting infrared radiation is
provided which comprises a temperature-sensing helical coiled-coil
protein such as TIpA, CC1, collagen or myosin, incorporated into an
electrically conductive film or gel deposited onto an electrically
conductive medium, means for recording changes in conductivity or
resistance of the conductive film or gel caused by the presence of
infrared radiation and its effect on the thermal-sensing protein,
and means to analyze the changes in conductivity or resistance in
the conductive film caused thereby so as to determine if infrared
radiation is present. By virtue of the present invention, a
"biomimetic" infrared sensor is provided which can integrate a
recombinantly produced thermally sensitive protein in a conductive
polymer matrix, such as a film or gel, and provide a low-cost,
lightweight, conformable, and disposable infrared detecting device
having high sensitivity and excellent dynamic range.
Inventors: |
Brott; Lawrence L.; (West
Chester, OH) ; Naik; Rajesh R.; (Dayton, OH) ;
Stone; Morley O.; (Bellbrook, OH) ; Carter; Daniel
C.; (Huntsville, AL) |
Correspondence
Address: |
STITES & HARBISON PLLC
1199 NORTH FAIRFAX STREET
SUITE 900
ALEXANDRIA
VA
22314
US
|
Family ID: |
26990060 |
Appl. No.: |
11/724178 |
Filed: |
March 15, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10313010 |
Dec 6, 2002 |
7193037 |
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11724178 |
Mar 15, 2007 |
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60391089 |
Jun 25, 2002 |
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60336145 |
Dec 6, 2001 |
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Current U.S.
Class: |
250/338.1 |
Current CPC
Class: |
H01L 51/0093 20130101;
G01J 5/20 20130101; H01L 51/4206 20130101; H01L 51/4253 20130101;
B82Y 10/00 20130101; G01N 21/3563 20130101; H01L 51/428 20130101;
Y02E 10/549 20130101; H01L 27/307 20130101; G01N 25/72 20130101;
H01L 27/305 20130101 |
Class at
Publication: |
250/338.1 |
International
Class: |
G01J 5/00 20060101
G01J005/00 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made in the performance of a Cooperative
Research and Development Agreement with the Department of the Air
Force. The Government of the United States has certain rights to
use the invention.
Claims
1. An apparatus for sensing infrared radiation comprising an
electrically conductive film or gel containing therein a
temperature-sensing helical coiled-coil protein capable of
reversible conformational change in the range of -15.degree. C. to
60.degree. C., means for detecting the changes in conductivity or
resistance of the conductive film or gel caused by the effect of
infrared radiation on the protein, and means to determine the
presence of infrared radiation based on the changes in conductivity
or resistance of the conductive film or gel.
2. The apparatus according to claim 1 wherein the means for
detecting the changes in conductivity or resistance of the
conductive film caused by the effect of infrared radiation on the
protein comprises a multimeter capable of sensing changes in
conductivity or resistance.
3. The apparatus according to claim 1 wherein the means to
determine the presence of infrared radiation based on the detected
changes in conductivity or resistance of the conductive film
comprises a computer.
4. The apparatus according to claim 1 wherein the protein is
selected from the group of proteins including TIpA, collagen,
myosin and CC1.
5. The apparatus according to claim 1 wherein the protein is
TIpA8.
6. The apparatus according to claim 5 wherein the protein has the
amino acid sequence of SEQ ID NO:1.
7. The apparatus according to claim 1 wherein the conductive film
or gel comprises a conductive polymer containing the
temperature-sensing helical coiled-coil protein.
8. The apparatus according to claim 1 wherein the conductive film
or gel comprises a polymer having incorporated therein a conductive
material.
9. The apparatus according to claim 8 wherein the polymer is
selected from the group consisting of poly(ethylene oxide),
polyethylene-co-vinyl alcohol), poly(vinyl acetate), gelatin,
acacia, and poly(vinyl alcohol)
10. The apparatus according to claim 8 wherein the conductive
material incorporated into the polymer comprises a material
selected from the group consisting of carbon black, silver flakes,
gold particles, and metal coated ceramics.
11. The apparatus according to claim 1 wherein the electrically
conductive film or gel containing therein a temperature-sensing
helical coiled-coil protein comprises a plurality of films or gels
arranged in a sensing array.
12. An electrically conductive film or gel suitable for use in an
infrared detection device comprising a conductive polymer matrix
having incorporated therein a temperature-sensing helical
coiled-coil protein capable of reversible conformational change in
the range of -15.degree. C. to 60.degree. C.
13. The electrically conductive film according to claim 12 wherein
the protein is selected from the group consisting of TIpA, collagen
and myosin.
14. The electrically conductive film according to claim 12 wherein
the protein is TIpA8.
15. The electrically conductive film or gel according to claim 12
wherein the film or gel is suitable for use in silk-screening.
16. A method of detecting infrared radiation comprising the steps
of positioning the apparatus according to claim 1 so as to be able
to receive infrared radiation, and detecting the presence of
infrared radiation by virtue of the electrical signaling caused by
the conformational changes of the temperature-sensing helical
coiled-coil protein in the apparatus.
17. A method of detecting infrared radiation comprising the steps
of incorporating a temperature-sensing helical coiled-coil protein
capable of reversible conformational change in the range of
-15.degree. C. to 60.degree. C. into a conductive polymeric matrix,
connecting the matrix to a suitable electrical device that can
detect the change in conductivity or resistance caused in the
conductive matrix by virtue of the conformational changes of the
temperature-sensing helical coiled-coil protein, and determining
the presence of infrared radiation on the basis of the change in
conductivity or resistance in the conductive matrix.
18. The method according to claim 17 wherein the protein is
selected from the group of proteins including TIpA, TIpA8,
collagen, myosin and CC1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser No.
10/313,010, filed Dec. 6, 2002, and claims the benefit of U.S.
Provisional Application Ser. No. 60/391,089, filed Jun. 25, 2002,
and of U.S. Provisional Application Ser. No. 60/336,145, filed Dec.
6, 2001, said applications incorporated herein by reference.
FIELD OF THE INVENTION
[0003] This invention relates in general to a method for utilizing
thermally switching proteins in devices which can sense and detect
changes in temperature and thus be used in a variety of sensing
systems, and more particularly to a method and apparatus for
utilizing helical coiled-coil proteins which undergo a
conformational change at particular temperatures, such as the
thermal switching protein TIpA and the modified protein TIpA8, in
conductive polymeric matrices in order to obtain efficient and
inexpensive, but still highly sensitive, temperature and infrared
monitoring detection devices which can be useful in a broad array
of applications calling for IR detection.
BACKGROUND OF THE INVENTION
[0004] Infrared sensing devices are well known and are now utilized
in a wide variety of applications including night vision devices,
enhanced aviation vision systems, fire detection, surveillance and
security, search and rescue devices, and even medical imaging and
diagnostics. Current state-of-the-art devices in this field include
bolometers which are composed of semiconductors using vanadium
oxide as the active component. These devices can provide effective
room temperature infrared detection with a sensitivity in the 8-15
micron range. Devices currently on the market that deal with
infrared detection technology include the Spectrum/RM of Texas
Infrared Inc., the Thermacam.RTM. from FLIR Systems, and the PalmIR
PRO from Raytheon. Still other devices in this field include those
which utilize PtSi and measure infrared radiation by means of
changes in capacitance, and those which utilize PbSe and InSb which
are photoconductive detectors which operate at room temperature
with about a 20% change in response per degree Centigrade.
[0005] Unfortunately, the great limitation on the potential
usefulness and applicability of current infrared devices is their
great expense. Currently, cameras in this field such as the ones
described above have costs in the tens of thousands of dollars, and
prices typically range from about $10,000 to $50,000 depending on
range and detection sensitivity. It is clear that there exists a
distinct need to provide technology by which a low-cost system of
infrared detection can be obtained so that the potential benefits
of infrared detection, such as medical imaging and search and
rescue devices, can become affordable and thus more commonly
available so that the public can benefit from such devices. It is
also clear that there exists a need for providing improved hybrid
organic/inorganic nanostructures utilizing photo polymerization
which can allow for enhanced optical reflectivity and the creation
of holographic or other optical gratings which can be used to form
a broad array of biosensors and other sensing devices.
[0006] Previously, it has been recognized in nature that certain
proteins apparently are configured to have conformational shapes
which allow a particular function when one set of conditions is
present, yet another shape under different conditions or stimuli
which provides for a different function. An example of this
conformationally changing shape is the "coiled-coil" type of
protein which confers a variety of functional capabilities,
including enabling proteins such as myosin to function in the
contractile apparatus associated with muscle cells and associated
non-muscle structures. One such conformationally changing protein
appears to be the TIpA protein encoded by the virulence plasmid of
Salmonella bacteria which is an .alpha.-helical protein that forms
an elongated coiled-coil homodimer. A number of studies regarding
this protein appear to show that it operates in the bacteria as a
temperature-sensing gene regulator. However, it has never been
disclosed or suggested that this protein, or active fragments
therefrom, could be utilized in devices which could monitor and
detect heat in the form of infrared radiation. There is thus a
distinct need in the field to develop devices which can make use of
the thermal conformation shifts in proteins such as TIpA and its
active fragments so as to detect infrared radiation.
SUMMARY OF THE INVENTION
[0007] Accordingly, it is thus an object of the present invention
to create devices which can detect infrared radiation which are
efficient yet inexpensive, and which thus can be used in a variety
of applications.
[0008] It is further an object of the present invention to provide
an apparatus which can utilize a conformational change protein and
translate a change in temperature into electrical conductance and
thus be able to detect heat or infrared radiation.
[0009] It is still further an object of the present invention to
develop proteins and protein fragments which are capable of
achieving reversible conformational changes so as to be useful in
devices which utilize such conformational changes to detect heat
and/or infrared radiation.
[0010] It is even further an object of the present invention to
provide methods of detecting infrared radiation using conductive
polymeric matrices that contain proteins which possess the ability
to reversibly change conformation depending on thermal
characteristics.
[0011] It is yet a further object of the present invention to
provide hybrid organic/inorganic nanostructures with improved
optical reflectivity to create holographic or other optical
gratings which may be utilized in the infrared detectors of the
invention, and which can be integrated with the coiled-coil
proteins of the present invention to form a broad array of
biosensors.
[0012] It is still further an object of the invention to develop
infrared sensing devices that reduce costs of manufacturing by
several orders of magnitude from currently available devices, that
are easy to manufacture and utilize readily available materials,
that reduce and potentially eliminates all cooling requirements
such as are required in many highly sensitive IR detectors, that
are extremely lightweight and can be made disposable, if necessary,
and which have excellent dynamic range and high sensitivity
thresholds.
[0013] These and other objects are achieved by virtue of the
present invention which provides a method and apparatus for sensing
infrared radiation comprising a helical temperature-sensing
coiled-coil (CC) protein such as TIpA, collagen or myosin contained
in an electrically conductive film or gel deposited onto an
electrically conductive medium such as an electrode, means for
recording changes in conductivity or resistance of the conductive
film or gel caused by the presence of infrared radiation and the
effect of the infrared radiation on the CC protein, and means to
analyze the changes in conductivity or resistance in the conductive
film caused by the infrared radiation so as to determine if
infrared radiation is present.
[0014] These and other features of the present invention as set
forth in, or will become obvious from, the detailed description of
the preferred embodiments provided hereinbelow.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0015] FIG. 1 is a graphic representation of changes in ellipticity
of thermal sensing proteins in accordance with the invention at
different temperatures.
[0016] FIG. 2 is a schematic view of the conformational changes of
the helical coiled-coil TIpA protein in accordance with the present
invention.
[0017] FIG. 3 is a schematic view of an infrared detection system
in accordance with the present invention.
[0018] FIG. 4 is a photographic view of an infrared detection
system in accordance with the present invention.
[0019] FIG. 5 is a photographic view of the sensing elements and
printouts in accordance with the present invention.
[0020] FIG. 6 is a schematic view of the effect of the thermal
sensing proteins of the invention on the conductivity of the
polymer matrix of the present invention.
[0021] FIG. 7 is a photographic view of a prototype of an infrared
detecting device of the invention including an 8.times.8 imaging
array.
[0022] FIG. 8 shows the amino acid sequence (SEQ ID NO:1) of the
TIpA8 protein of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] In accordance with the present invention, there is provided
an apparatus for detecting infrared radiation in a variety of
sensing systems which comprises the use of conformationally
changing proteins embedded in a conductive polymer matrix. In a
preferred embodiment, these conformationally changing proteins
comprise any protein capable of significant and reversible
conformational change induced by temperature changes within a range
of -15.degree. C. to 60.degree. C., and include proteins such as
albumin, as well as oligomeric proteins or fragments capable of
such a reversible conformational change induced by temperature. In
addition, these types of conformationally changing proteins in
accordance with the invention include helical coiled-coil (CC)
proteins capable of reversible conformational change induced by
temperature changes within a range of -15.degree. C. to 60.degree.
C. By helical coiled-coil protein is meant any protein or peptide
which undergoes reversible temperature-dependent conformational
changes and thus include those protein fragments from the
coiled-coil proteins or derivatives therefrom which maintain the
conformational change of the full coiled-coil protein. These
coiled-coil proteins include TIpA, CC1, collagen and myosin which
all have similar switching mechanism, and in the description as
follows, the invention is described in terms of the TIpA protein
specifically, although any of the other coiled-coil proteins with
the properties described above can be utilized in the
invention.
[0024] In accordance with the present invention, use is made of the
helical coiled coil TIpA protein, such as the thermal switching
TIpA protein of the type found in Salmonella bacteria, or active
fragments thereof which maintain the ability to undergo the
conformational change which is dependent upon a change of
temperature. Accordingly, by "TIpA protein" is meant those proteins
which undergo the type of conformational change observed in the
TIpA protein such as expressed in Salmonella bacteria, such as
Salmonella typhimurium (1), including those protein fragments
isolated from the TIpA protein, such as TIpA8 as described further
below, or derivatives which maintain the conformational change of
the full TIpA protein, or even further proteins with a similar
switching pattern. In accordance with the invention, as described
in detail further below, the preferred apparatus includes the
incorporation of the TIpA protein or its active fragments into a
conductive polymer matrix so as to be useful in a variety of
sensing systems that utilize detection of infrared radiation. By
virtue of the present invention, a "biomimetic" infrared sensor is
provided which can integrate a recombinantly produced thermally
sensitive TIpA protein in a conductive polymer matrix, such as a
film or gel, so as to provide for the first time a low-cost,
lightweight, conformable, and even possibly disposable, infrared
detecting device having high sensitivity and excellent dynamic
range.
[0025] In the current field of infrared detection, there are
infrared sensing elements which are sensitive to radiation in the
wavelength range from 1 to 15 microns and have numerous commercial
and government applications (including military and law enforcement
agencies). These applications include, but are not limited to night
vision devices, military weapons targeting, aircraft enhanced
vision systems, surveillance/security, fire fighting, fire
detection, search and rescue devices, devices for predictive
maintenance, process control, research and development, driver's
vision enhancers and medical imaging and diagnostics. These highly
sensitive detection systems are generally used in three temperature
dependent formats depending on the sensitivity required by the
application, namely room temperature, cryogenic and near room
temperature cooled detectors. Public available information
indicates that the current market for these technologies in the
U.S. is approximately $1 Billion and growing at a rate of 7% per
annum. The markets are currently limited by the great expense of
highly sensitive imaging systems (usually from 10 to 50K each).
Accordingly, the present invention is designed to provide a much
cheaper alternative to these expensive infrared detection devices,
and thus allow the benefits of infrared detection devices to be
more accessible to the public.
[0026] In the animal kingdom, there have been examples of thermal
or heat detection systems occurring in nature, but prior to the
present invention, no such systems have been usable in creating
infrared detection systems that could be used in commercial
products. For example, it has been known that certain snakes, such
as the python and other venomous snakes, apparently possess
infrared sensing structures which are used for close range
detection only of heat. However, this system appears to be based on
complicated proteins in the snakes' pit organs which are difficult
to produce using recombinant methods (2). The present invention
utilizes helical coiled coil proteins such as the TIpA protein
which has been shown to be connected to heat detection in bacteria.
In many bacteria, there exist such heat sensitive molecular
switches, such as those involved in heat sensing reactions and the
expression of shock proteins. In bacteria, the thermal sensing TIpA
protein has been isolated and now can be produced by recombinant
methods and expressed so as to be useful in the present invention.
FIG. 1 illustrates the optical dispersion plot of two bacterial
thermal sensing proteins, including the TIpA protein of the
invention, versus temperature. In other studies, the thermal switch
properties of the TIpA protein were observed (3). Subsequently, a
modified protein with greater stability, smaller size and enhanced
solubility characteristics, TIpA8, was successfully produced and
incorporated into the infrared devices in accordance with the
present invention as described in more detail below.
[0027] The TIpA protein, along with its active fragments or
derivatives, is a helical protein which forms a dimer at room
temperature apparently via a coiled-coil helical interaction as
shown schematically in FIG. 2. As has been noted by Koski et al.,
J. Biol. Chem. 267:12258-12265 (1992), incorporated herein by
reference, it appears that the last 300 residues of the TIpA
protein are helical, and thus this region appears to encompass the
active thermal switch in accordance with the invention. As set
forth further below, one such derivative from this region (TIpA8)
has maintained the relevant switching portion of this protein and
thus can be utilized in accordance with the present invention.
Still other derivatives from this region which maintain the thermal
switching ability of TIpA can also be utilized in accordance with
the invention as described further below. At temperatures less than
37.degree. C., the coiled-coil dimer associates with a specific
regulatory sequence of the bacterial genome and prevents the
transcription and subsequent expression of a series of proteins. In
the host, or at temperatures greater than 37.degree. C., the dimer
dissociates from the DNA into monomers, allowing the genes to be
transcribed to produce protein. Thus the natural function of TIpA
is to operate as a thermal gene regulation switch. In accordance
with the present invention, the TIpA protein or its active
fragments or derivatives is thus utilized in an infrared detection
device wherein the thermal switching affects the conductivity of a
polymeric film containing the TIpA protein, and that conductivity
is measured so as to determine the presence of the infrared
radiation.
[0028] In accordance with the present invention, the inventors have
developed an improved helical coiled coil protein with enhanced
stability, solubility and tunability of the thermally sensitive
proteins such as the TIpA protein. In this regard, it is possible
to produce a TIpA protein fragment that contains at least one
active portion of the TIpA protein, namely the section of the
protein that undergoes the conformational changes described above.
In accordance with the invention, one of these fragments has been
designated TIpA8, whose protein sequence is shown in FIG. 8 and SEQ
ID NO:1. This sequence corresponds to amino acids 257-299 of the
full TIpA protein, whose sequence has been disclosed in prior
references, including Hurme et al., J. Biol. Chem. 271:12626-12631
(1996) and Koski et al., J. Biol. Chem. 267:12258-12265 (1992),
both incorporated herein by reference. The TIpA8 protein can also
be constructed with a "His" tag, which will comprise one or more
histidine residues added to the N-terminal end as an aid in
purification. The present invention thus contemplates the isolated
and/or purified TIpA8 protein to be utilized preferentially in the
infrared detection devices of the invention.
[0029] As with the full TIpA protein, TIpA8 can be expressed
recombinantly using conventional methods well known in this field
and purified at large scale demonstrating the ability to produce
sufficient quantity and quality of this material so as to support
the commercial development of useful products. In accordance with
the present invention, TIpA8 can be integrated into signal
transduction means such as electrically conductive films as set
forth in detail below which can meet the theoretical limit of 50 mK
in sensitivity for semiconductor based infrared detectors operating
at room temperature, and which possibly can be used in detectors
operating with 3 mK in sensitivity, thus improving upon the
sensitivity of detectors currently on the market
[0030] In accordance with the present invention, two basic
approaches may be utilized in creating signal transduction systems
which can incorporate the helical coiled coil proteins such as heat
sensing TIpA proteins of the present invention. The first such
successfully developed system comprises thin films incorporated
with the protein on which diffraction gratings can be created using
precision lasers and similar newly developed technology. Examples
of the production of such diffraction gratings are disclosed in
Brott et al. (4) and Naik et al. (5), and these references are
incorporated herein as if set forth in full. In addition,
two-photon-induced photopolymerization may be used to create
holographic patterns and other devices useful in three dimensional
optical storage, such as described in Kirkpatrick et al. (6), and
in pending U.S. patent application Ser. No. 09/657,169, filed Sep.
7, 2000, incorporated herein by reference, and these devices using
optical signal transduction may also be utilized as substrates in
the infrared detection systems of the present invention. It is also
possible that holographic patterning can be used in accordance with
the invention to form Fresnel lenses providing an alternate
possibly more compact optical path to integrate the films into the
electronic devices of the invention.
[0031] In the preferred embodiment of the invention, an infrared
sensing device is provided which comprises an electrically
conductive polymeric film which has the helical coiled coil
proteins such as the thermally-sensing TIpA protein as described
above incorporated therein, and this film is itself deposited onto
an electrically conductive medium such as electrodes or a
commercially available electric chip. In the preferred apparatus,
variations in temperature are thus recorded by measuring the
changes in conductivity or resistance of the polymer film as caused
by the thermally-sensitive conformational changes of the
coiled-coil protein in accordance with the invention. In the
preferred embodiment, the electrically conductive polymer film will
thus comprise a suitable polymer, either an electrically conductive
polymer, and/or a polymeric material that has been doped with an
electrically conductive particle, such as carbon black, and the
thermosensitive protein such as TIpA in accordance with the
invention as described above. In addition, optional surfactants and
plasticizers may be added as necessary when so desired. Since the
modulation of the oligomerization of carbon black is accomplished
through the addition of protein, the protein of the invention is
serving as a novel plasticizer. Finally, the
electrically-conductive, thermal-sensing protein containing film
will be connected through suitable electrical means such as
electrodes or electrical chips, and suitable signal transduction
means, to a suitable means such as a multimeter and/or a computer,
so that the change in electrical conductivity and/or resistance can
be measured and translated so as to detect the presence of infrared
radiation in the target area of the detection device in accordance
with the invention. In the preferred embodiment, the electrical
properties of the film or gel can be read out using standard
electrical readout equipment such as a multimeter, and the signal
can also be translated by means of a computer so as to provide a
readout that can be readily understood by the user of the device.
This sensing concept may be applied using a single detection
element, or can even be used in an array format, such as shown and
described below. Such arrays based on integrated circuits which
read out suspended bridge thermal sensing elements, can be
fabricated from existing inexpensive components to form imaging
systems for cameras, night vision equipment, etc.
[0032] In the preferred embodiment, the polymer used in the
conductive polymer film which contains the thermally sensitive
protein in accordance with the invention can be any suitable
polymer that dissolves in a solvent miscible with the solvent used
to dissolve the protein, and that the solvent does not adversely
affect the functionality of the protein. Examples of useful
solvents include but are not limited to dimethyl sulfoxide,
acetonitrile and water, although numerous other solvents as would
be contemplated by one of ordinary skill in this art may also be
used. In accordance with the invention, a number of suitable
polymers can be used to form the electrically conductive film in
accordance with the invention, including polymers which have their
own conductivity, or polymers into which a conductive material such
as carbon black may be added. Such a suitable conductive
carbon-containing polymer is disclosed, for example, in U.S. Pat.
No. 6,290,911, incorporated herein by reference. Additional
examples of suitable polymers include poly(ethylene oxide),
poly(ethylene-co-vinyl alcohol), poly(vinyl acetate), gelatin,
acacia, or poly(vinyl alcohol). Although not required for suitable
functioning of the film in the infrared devices of the invention,
it may be desired in some applications for the polymer to have
functional groups for cross-linking. Accordingly, the polymers used
in the conductive films of the invention may also have
cross-linking, and thus cross-linking can be achieved through a
number of suitable means, including chemically, photochemically, by
gamma irradiation, or simply through strong hydrogen bonding across
side groups of the polymer.
[0033] As indicated above, since it is desired to create a
measurable resistance across the polymer film so as to translate
the action of the conformational thermal sensing protein into an
electrical signal, it is preferred that the polymeric film be
constructed so that an electrically conductive material is
incorporated into the matrix. In addition to the use of carbon
black as described above, other suitable examples of the conductive
particles useful in the invention include conductive silver flakes,
gold particles, and metal coated ceramics. The amount of conductive
material should preferably be in the percolation threshold range of
the polymer matrix system so that even slight changes in conductive
particle distances result in large changes of resistance.
[0034] As indicated above, the electrically conductive films of the
invention include a thermally-sensing helical coiled-coil protein,
and this protein is preferably used by dissolution in a solvent
that will not inhibit the functionality of the protein.
Additionally, if the solvent used is water, it is desirable that
distilled water be used and not a buffered solution. Further
considerations in the conductive polymeric matrix of the invention
include the preferred requirement that the glass transition
(T.sub.g) of the matrix be lower than the operating temperature of
the device. If the T.sub.g of the polymer host is above the
operating temperature, it can be lowered through the use of
plasticizers or modulated with any suitable means of active
temperature control. Examples of suitable plasticizers include
poly(ethylene glycol), glycerol or propylene glycol.
[0035] Additionally, it is preferred that the electronically
conductive particles be homogeneously dispersed in the polymer
matrix. When carbon black is used, a nonionic surfactant may be
used to stabilize the dispersion of the conductive particles. A
typical example of a surfactant is polyethylene(10)
isooctylcyclohexyl ether, commercially sold as Triton X-100.
[0036] In general, the infrared detection devices of the invention
can be constructed in a variety of suitable ways, as can the
conductive film of the invention which contains the thermally
sensitive coiled-coil protein such as the TIpA protein. In one
suitable embodiment, the conductive film is obtained by dissolving
a suitable polymer, such as poly(vinyl alcohol) in distilled water,
and then heating and stirring for several hours at a suitable
temperature (e.g., about 90-100.degree. C.). In a separate flask, a
suitable amount of carbon black (e.g., Vulcan XC-72, Cabot) is
suspended in water and a suitable surfactant (e.g., Triton X-100)
is added. Next, the carbon black and surfactant may be added to the
polymer, e.g., by placing the vial in a water bath sonicator for 10
minutes, and then adding the contents to the polymer solution, and
enough heat is generally supplied to evaporate the total water
content to a suitable amount, e.g., 25 ml. Next, an optional
plasticizing ingredient such as glycerol may be added to the
polymer matrix, which is again stirred for an additional period
under heat. Finally, the conductive polymeric matrix of the
invention is obtained by removing the matrix from the heat source
and allowed to cool to room temperature.
[0037] In the next step of preparing the thermally-sensitive
conductive polymeric film of the invention, the polymer matrix such
as obtained above is weighed out into a vial, and a suitable amount
of the thermosensitive protein solution (e.g., in water) is added
and mixed until homogeneous. A suitable wet film layer of this
protein polymer mixture is then allowed to form, and may also be
deposited onto an appropriate electrically conductive material,
such as an electrode, and allowed to dry overnight to produce the
completed thermal detector. Still further, it is also possible to
deposit the protein polymer mixture containing the thermally
sensitive protein of the invention onto other suitable devices
which can be used to receive and translate optical signals, such as
the nanopatterned peptide/silica hybrid structure such as disclosed
in Brott et al., Nature 413:291-293 (2001), incorporated herein by
reference. In other embodiments, the polymeric film or gel
composite with protein and an amorphous silicon chip for use as an
active element in a detector system.
[0038] In one preferred embodiment of the invention, the thermal
detector as obtained above may be utilized as a sensor array in
imaging systems in which multiple detectors are utilized in one
device, e.g., an integrated circuit which reads out suspended
bridge thermal sensing elements. In the preferred embodiment, this
receiving means can comprise a two-probe multimeter which is
coupled to the thermal detector, and a computer which receives the
output from the multimeter. In the preferred embodiment, the
computer can graph the resistance of the detector as a function of
time, so that fluctuations in temperature can be monitored in real
time or recorded for subsequent review of the detection pattern.
Suitable devices in accordance with the invention may be utilized
to detect infrared radiation from a number of sources, and in a
flashlight test performed in using a prototype of the present
invention, the prototype clearly detected the infrared radiation
from a small battery operated flashlight approximately 20 feet
away. In this manner, a highly sensitive infrared detector may be
formed which will be useful in a wide variety of applications such
as described above. In addition, the conductive films of the
invention may also be used in other devices and applications, such
as uses in newly developed electronics manufacturing processes
involving silk-screening.
[0039] A summary of the elements of the preferred apparatus of the
invention is shown in the schematic drawing FIG. 3. As shown in
that figure, apparatus 10 is generally on the right side of the
figure and is broken down into three general units, namely the
imager 12 wherein the infrared radiation is received, focused and
detected by the detector or detector array of the invention, the
multimeter 14 which obtains a standard electrical readout from the
imager, and the computer 16 which in this case can be utilized to
interpret the electrical signal from the multimeter and present a
suitable readout, either through digital, graphic or visual means,
so that a user of the device can determine that infrared radiation
has been detected from heat source 20. As shown in FIG. 3, the
imager 12 of the invention can be constructed with a lens 15, such
as a germanium lens as shown in the drawing, which can focus
radiation from the heat source 20 onto the thermochip 17 which is
comprised of the conductive polymeric matrix of the invention and
includes a thermally sensitive coiled-coil protein as described
above. The imager 12 also includes a "breadboard" 19 or
electrode-type device, such as a standard commercially available
electric chip which will transmit the electrical signal, i.e., the
change in conductance or resistance caused by infrared radiation on
the thermally-sensing protein in the conductive matrix as described
above. This "breadboard" 19 is thus capable of transmitting a
signal to a suitable electrical reading device such as multimeter
14, which then transmits the signal to a suitable analytical device
such as a computer which will determine that infrared radiation has
been detected based on the electrical signal. Actual versions of
the elements of the present invention, including imager 12,
multimeter 14, computer 16, thermal sensing chip 17, along with the
heat source 20, are shown in FIG. 4.
[0040] As indicated above, the thermal sensing chip 17, including
the thermal sensing protein incorporated into the conductive
polymer matrix, can be used alone or in a broad array of similar
detectors. In these embodiments, as shown in FIG. 5, the thermal
chip 17 can constitute a single element (left side), or in any
number of suitable arrays wherein a plurality of detectors are
used, including an array in a 1.times.7 pattern (FIG. 5, middle)
and an array wherein a pattern of 4.times.4 sensing chips (right
side) are used. Even further, other arrays such as 8.times.8, etc.
may also be used. As one skilled in the art would recognize, the
particular array that will be used will depend on the nature of the
source to be detected and the nature or type of device that is
being used for any particular application. As would be readily
understood by one skilled in this art, the more detector chips that
are utilized in an array, the more precise the detection of IR will
become. At the same time, greater arrays of chips will often
necessitate an increase in the power and scope of the analytical
device, i.e., a computer with greater memory may be required in
those applications having a broad array of detecting chips.
[0041] The general functioning of the thermal proteins of the
present invention and their effect on the conductive film is shown
in the schematic drawing of FIG. 6. As shown in this drawing, at
low temperatures (left side), the polymer and thermal biomolecule
tightly bind the conductive material (in this case, carbon black)
in place. However, at higher temperatures (right side), the polymer
and biomolecule expand, thereby allowing the conductive carbon
black to rearrange and agglomerate, and as a result, the
conductivity increases. It is this change in conductivity or
resistance in the conductive polymer matrix of the invention which
allows for the device to be used as a highly sensitive infrared
detector as described above.
[0042] It is also the case that peptides or proteins are provided
in accordance with the invention that contain an .alpha.-helical
Coiled-coil (CC) structural motif, i.e., proteins or peptides which
contain the heptapeptide repeating motif a-b-c-d-e-f-g with
preferentially a polar or hydrophobic amino acids at positions `a`
and `d` (Pauling, L. and Corey, R. B. 1953 Nature 171, 59-61), and
these peptides or proteins can also be useful in the invention.
With regard to these peptides and proteins, residues `a` and `d` of
the "a-b-c-d-e-f-g" repeating motif form a hydrophobic backbone
structure which drives and stabilizes the coiled-coil interactions
between pairs of alpha helices. CC proteins are ubiquitous in
nature and include collagen, myosin, TIpA, and many others. It
would then be understood that CC proteins may have their respective
sensitivity and wavelength response altered by the direct
manipulation of residues `a` and `d` involved in forming the
superhelical coil. Residues substituted at `a` and `d` which
thermally stabilize the CC motif to higher temperature will
decrease sensitivity and shift spectral response to shorter
wavelengths. Conversely, residues substituted at `a` and `d` which
destabilize the superhelical coil to increasing temperature will
increase sensitivity and shift spectral response to longer
wavelengths. It would also be understood that by decreasing the
length of any CC protein at some point will destabilize the
oligomeric superhelical interaction (increase sensitivity and shift
spectral response to longer wavelengths) conversely increasing the
length of the CC structure will decrease sensitivity and shift
spectral response to shorter wavelengths.
[0043] In accordance with another aspect of the present invention,
the substrate of the invention may comprise a hybrid
organic/inorganic ordered nanostructure of silica spheres obtained
through the incorporation of a polycationic peptide (such as
derived from the C. fusiformis silaffin-1 protein) into a polymer
hologram created by two-photon-induced photopolymerization. In this
structure, such as disclosed in Brott et al., Nature 413, 291-293,
(2001), incorporated by reference, the thermally sensing
coiled-coil proteins of the invention may be incorporated and
utilized in highly sensitive infrared detection devices.
[0044] In accordance with the present invention, the nanostructures
of the invention may be prepared using diatoms to form intricate
silica ordered nanostructures that comprise silica spheres obtained
through the incorporation of a polycationic peptide (derived from
the C. fusiformis silaffin-1 protein) into a polymer hologram
created by two-photon-induced photopolymerization. When these
peptide nanopatterned holographic structures are exposed to a
silicic acid, an ordered array of silica nanospheres is deposited
onto the clear polymer substrate. These structures exhibit a nearly
fifty-fold increase in diffraction efficiency over a comparable
polymer hologram without silica. This approach, combining the ease
of processability of an organic polymer with the improved
mechanical and optical properties of an inorganic material, will
thus be useful in the present infrared detection devices disclosed
above, as well as in other photonic devices.
[0045] A holographic two-photon-induced photopolymerization
(H-TPIP) process as described in Kirkpatrick, S. M. et al., Appl
Phys. A 69, 461-464 (1999), incorporated herein by reference, may
be utilized to prepare the ordered silica holograms of the present
invention which can incorporate the thermally-sensing coiled-coil
proteins described above. Unlike conventional holograms formed
through the use of ultraviolet lasers, holograms created through
the two-photon process use an ultrafast infrared laser. Because
infrared wavelengths typically do not alter the functionality of
biological compounds, monomer formulations containing peptides can
be polymerized without affecting the biological activity. We
incorporated a peptide that has recently been shown to be
responsible for biosilification into a formulation to be cured by a
holographic two-photon-induced photopolymerization with the
expectation that the peptide would be segregated into regions of
low crosslinking density. The approach of using ultraviolet lasers
to phase separate small liquid crystal molecules in a polymer-based
hologram has been used extensively and this technique is also
applicable to the H-TPIP process. As predicted by the inventors,
exposing the peptide-containing structure to a liquid silane causes
silica to form in the holographic nanopattern and this hybrid
organic/inorganic device has a higher degree of order leading to a
superior device compared to randomly ordered monolayers of silica
on indium-tin oxide (ITO) coated glass.
[0046] A short 19-amino-acid R5 peptide unit (SSKKSGSYSGSKGSKRRIL)
(SEQ ID NO:2) of the silaffin-1 precursor polypeptide from C.
fusiformis can be used to catalyze the formation of silica
nanospheres within minutes when added to silicic acid to neutral pH
and ambient temperature. A chemically synthesized R5 peptide that
lacks a post-translational modification of its lysine residues was
used in the present work. The post-translational modification of
lysine residues is required for silica formation under acidic pH
conditions. However, in this case, the modification of the lysine
residues was unnecessary. Consequently, the process started by
incorporating this peptide (e.g., in water) into a monomer
formulation. This formulation consisted of SR-9035, a
trimethylolpropane triacrylate, and SR-399, a dipentaerythritol
pentaacrylate obtained from Sartomer (which were used without the
removal of inhibitor), along with triethanol amine and isopropyl
thioxanthone; followed by heating the entire mixture to aid in
dissolution. The triacrylate is preferred because of its high water
miscibility which is due to its numerous ethylene glycol units, and
the pentaacrylate was used to create a highly crosslinked system.
The triethanol amine functions as a co-initiator and thioxanthone
as the initiator. Typically, in a two-photon-initiated
polymerization, a fluorescent chromophore is also required to
absorb two photons of near-infrared laser light. The excited
chromophore transfers its energy to the initiator which begins the
polymerization process. However, we have found that the
thioxanthone used in this formulation does not require highly
colored chromophores, and consequently, extremely large curing
depths and exceptionally clear and colorless polymers are
produced.
[0047] A thin layer (e.g., about 178 .mu.m) of the monomer/peptide
formulation was deposited onto a clean glass slide, which was then
placed in a miniature atmospheric chamber fitted with glass windows
and flushed with nitrogen. The sample was cured in a two-beam
transmission holographic arrangement using a 790-nm
titanium-sapphire laser (90-fs pulse width with a repetition rate
of 500 Hz) for 30 s. The intensity distribution of the volume
hologram drives the local polymerization rate as a function of the
local field intensity, which results in alternating areas of high
and low crosslink density. Because certain areas of the sample cure
more rapidly than others, the smaller molecules (namely water and
peptide) phase separate from the areas of higher crosslink density
and migrate into areas of lower density. This phenomenon has been
observed in similar systems using liquid crystals as the small
molecule. An alternative explanation of this phase separation could
be that as the hydrophilic monomer is converted into a more
hydrophobic polymer, the peptide is driven into the monomer-rich
regions. As a result, peptide-rich domains are created in the
polymer sample with the periodicity of the hologram. After the
curing process, the sample was briefly rinsed with water to remove
any uncured monomer. Atomic force microscopy (AFM) revealed that
the hologram had a periodicity of 1.33 .mu.m.
[0048] The silane precursor (1 M tetrahydroxysilane) was
synthesized by dissolving tetramethyl orthosilicate (TMOS) in 1 mM
HCl. This product was then added to a sodium phosphate-citrate
buffer (pH 8) to produce a final concentration of 113 mM, and this
dilute solution remains stable for over two hours, after which it
slowly converts into a clear amorphous gel. Freshly prepared
hydrolyzed silane was slowly applied to the hologram and allowed to
react with the R5 peptide embedded in the hologram for 10 min.
before being rinsed with water to remove any unreacted silane. A
control hologram lacking the R5 peptide was also treated with the
tetrahydroxysilane solution but did not exhibit any nanosphere
formation. However, when a sample that included the peptide and was
treated with the silane was analyzed by scanning electron
microscope, it was revealed that silica spheres formed a regular
two-dimensional array with the periodicity of the hologram. A study
of the size distribution of the silica spheres reveals that the
average nanosphere diameter is 452 nm (.+-.81 nm). The silica
content of the spheres was confirmed using electron dispersive
spectroscopy (EDS). Additionally, analysis using the AFM indicated
that the hologram had a periodicity of 1.60 .mu.m with the silica
spheres embedded in the troughs of the surface relief pattern. The
difference in the spacing between the holograms treated with and
without the tetrahydroxysilane solution can be explained by the
fact that the control grating shrinks as it dries out owing to
water evaporation, whereas the shrinkage in the hybrid hologram is
inhibited owing to the added mechanical strength of the silica
spheres, preventing the ridges of the hologram from moving closer
together. Consequently, the untreated grating exhibited nearly 17%
more shrinkage than the treated grating. Also, the silica spheres
are the most prominent feature of the hologram and the troughs in
the structure are actually the peaks of the polymer.
[0049] Finally, to test the improvement that this technique can
impart to an optical device, the first-order diffraction efficiency
of the treated hologram was compared to that of the untreated
sample. These measurements were performed by transmitting a
helium-neon laser through each sample and measuring the diffraction
pattern in the far field. A measurement of the incident and
transmitted power in the first-order diffraction spot showed a
substantial increase in the diffraction efficiency of the grating
with silica versus the grating without, as would be expected from
the difference in index and shrinkage. The untreated grating
exhibited a diffraction efficiency of approximately 0.02%, while
the grating with the silica spheres showed an efficiency of
approximately 0.95%. This large increase can be attributed to the
fact that the spheres form an almost continuous line of silica
along the valleys of the hologram, achieving a high fill
factor.
[0050] We have thus shown that the incorporation of the peptide
responsible for biosilification into a microfabricated structure
using H-TPIP can result in an unusual composite organic/inorganic
device that has significantly improved optical performance and
superior mechanical properties compared to those of a corresponding
polymeric device without silica. Although we have used a
polymer/silica hybrid structure, this technique is universally
applicable for any catalyst or binding agent that can be
incorporated into a polymer. For example, as different catalysts
are identified, a wide variety of unique hybrid structures are now
possible with differing shapes and mechanical properties.
Additionally, antibodies can be incorporated into the hologram and
potentially used to optically identify specific antigens.
Consequently, this technique allows a simple yet general and easily
modifiable method for nanopatterning, and thus can provide a
substrate for incorporation of the thermally sensitive proteins of
the present invention.
[0051] It is thus submitted that the foregoing embodiments are only
illustrative of the claimed invention and not limiting of the
invention in any way, and alternative embodiments that would be
obvious to one skilled in the art not specifically set forth above
also fall within the scope of the claims.
REFERENCES
[0052] The following references as utilized in the detailed
description above are incorporated by reference as if set forth in
the above specification in full: [0053] 1. R. Hurme, K. D. Berndt,
S. J. Normark, and M. Rhen, "A Proteinaceous Gene Regulatory
Thermometer in Salmonella," Cell 90, 55-64 (1997). [0054] 2. A. L.
Campbell, T. J. Bunning, M. O, Stone, D. Church, and M. S. Grace,
"Surface Ultrastructure of Pit Organ, Spectacle, and Non Pit Organ
Epidermis of Infrared Imaging Boid Snakes: A Scanning Probe and
Scanning Electron Microscopy Study, Journal of Structural Biology
126, 105-120 (1999). [0055] 3. R. R. Naik, S. M. Kirkpatrick and M.
O, Stone, "The Thermostability of an Alpha-helical Coiled-coil
Protein and Its Potential use in Sensor Applications," Biosensors
& Bioelectronics, 16, 1051-1057 (2001). [0056] 4. L. L. Brott,
R. R. Naik, D. J. Pikas, S. M. Kirkpatric, D. W. Tomlin, P. W.
Whitlock, S. J. Clarson, and M. O, Stone, "Ultrafast Holographic
Nanopatterning of Biocatalytically Formed Silica," Nature 413,
291-293, (2001). [0057] 5. R. R. Naik, L. L. Brott, S. M.
Kirkpatrick and M. O, Stone, "Functional Biomimetic Optical
Devices," SPIE meeting in Australia Dec. 17-19 (2001). [0058] 6. S.
M. Kirkpatrick et al., "Holographic Recording Using
Two-photon-induced Photopolymerization", App. Phys. A., 69, 461-464
(1999).
[0059] The following examples are presented as illustrative of the
present invention or methods of carrying out the invention, and are
not restrictive or limiting of the scope of the invention in any
manner.
EXAMPLES
Example 1
Preparation of a Sensing Device in Accordance with the
Invention
[0060] A prototype of a device in accordance with the present
invention, as shown in FIG. 7, was prepared as follows: Poly(vinyl
alcohol) (3.750 g, 98-99% hydrolyzed, average MW=85,000-146,000,
Aldrich) is dissolved in distilled water (25 ml) through heating to
96.degree. C. and stirring for three hours. In a separate flask,
0.563 g of carbon black (Vulcan XC-72, Cabot) is suspended in water
(15 ml) and surfactant (21.0 .mu.l, Triton X-100) by placing the
vial in a water bath sonicator for 10 minutes and then added to the
polymer solution and enough heat is supplied to evaporate the total
water content to 25 ml. Glycerol (1.419 g Aldrich) is then added to
the polymer matrix and stirred an additional 15 minutes. The
polymer matrix is removed from the heat source and allowed to cool
to room temperature.
[0061] The polymer matrix (0.666 g) is weighed into a vial and a
thermosensitive protein solution (335 .mu.l of 3 .mu.g/.mu.l TIpA
in water) is added and mixed until homogeneous. A 254 .mu.m wet
layer if this protein polymer mixture is deposited onto two
electrodes and allowed to dry overnight to produce the completed
thermal detector.
[0062] A two-probe multi-meter is then coupled to the detector with
its output fed into a computer. By graphing the resistance of the
chip as a function of time, fluctuations in temperature can be
monitored. In a flashlight test performed in a laboratory, the
alpha prototype (8.times.8 sensing array) clearly detected and
imaged the infrared radiation from a small battery operated
flashlight approximately 20 feet across the laboratory. In a
further test, a hand waved across the front of the detector clearly
detected and crudely imaged the infrared radiation.
Performance Specifications of Alpha Prototype
[0063] The performance specifications of the alpha prototype in
accordance with the invention are as follows:
Spectral Response: 3 to 5 Microns
Thermal Sensitivity Approximately 50 mK
Response Time: 100 milliseconds (currently limited by electronics
of prototype)
Operating Range: -16.degree. C. to 65.degree. C. (limited by glass
phase transition temperature, T.sub.g)
Dynamic Range: 15% Signal Change/.degree. K
Optics & Readout: Standard
Example 2
Isolation and Testing of the TIpA Protein
Overview:
[0064] Coiled-coil proteins are assemblies of two to four
.alpha.-helices that pack together in a parallel or anti-parallel
fashion. Coiled-coil structure can confer a variety of functional
capabilities, which include enabling proteins such as myosin to
function in the contractile apparatus of muscle and non-muscle
cells. The TIpA protein encoded by the virulence plasmid of
Salmonella is an .alpha.-helical protein that forms an elongated
coiled-coil homodimer. A number of studies have clearly established
the role of TIpA as a temperature-sensing gene regulator, however
the potential use of a TIpA in a thermo-sensor application outside
of the organism has not previously been done. In the following
example, we demonstrate that TIpA has several characteristics that
are common with .alpha.-helical coiled-coils and its thermal
folding and unfolding is reversible and rapid. TIpA is extremely
sensitive to changes in temperature. We also have compared the
heat-stability of TIpA with other structurally similar proteins.
Using a folding reporter, in which TIpA is expressed as a
C-terminal fusion with green fluorescent protein (GFP), we were
able to use fluorescence as an indicator of folding and unfolding
of the fusion protein. Our results on the rapid conformational
changes inherent in TIpA support the previous findings and we
present here preliminary data on the use of a GFP-TIpA fusion
protein as temperature sensor.
Introduction:
[0065] The .alpha.-helical coiled-coil motif was first described in
1953 as the main structural element of a large class proteins,
which include muscle proteins and transcription factors (Pauling
and Corey, 1953; Lupas, 1996). Coiled-coils and helical bundles are
two common .alpha.-helical motifs found in native proteins.
Coiled-coil proteins are composed of two or more .alpha.-helices
that are wound into a left-handed superhelix (Lupas, 1996). The
.alpha.-helices are packed in a parallel or anti-parallel
orientation with respect to one another. The formation of coiled
coils are indicated by a heptad sequence repeat denoted [abcdefg],
where positions a and d are occupied by hydrophobic residues, with
polar residues at other positions. The coiled-coil structure in
proteins confers a variety of functional capabilities: they form
large rigid structures (keratins), molecular stalks (kinesins),
levers (myosins) and can also act as scaffolds (tropomyosins)
(Lupas, 1996).
[0066] The TIpA gene of Salmonella encodes an autoregulatory
repressor protein that makes use of the coiled-coil motif to sense
temperature changes and subsequently modulate transcription (Hurme
et al., 1997). TIpA contains an N-terminal DNA-binding region and a
large coiled-coil domain and has a tendency to form homodimers
(Koski et al., 1992). It has been postulated that the molecular
basis for thermosensing in TIpA is the dynamic coiled-coil to
monomer structural transition that is directly coupled to
differences in temperature. At temperatures below 37.degree. C.,
TIpA assumes a coiled-coil formation that is capable of binding to
sequence-specific DNA. Temperatures above 37.degree. C. promote
unfolding of TIpA, and these random coil monomers are unable to
remain bound to DNA (Hurme et al., 1996). TIpA represents a novel
class of molecules that has adapted the coiled-coil motif to
function; coupling its transcriptional activity with protein
folding, in response to temperature cues. The overproduction of
TIpA in E. coli results in the formation of an ordered intermediate
filament-like structure, which suggests that TIpA is able to
aggregate into a higher order structure (Hurme et al., 1994).
[0067] Apart from TIpA, a number of native proteins as well as de
novo designed coiled-coil peptides can undergo dramatic
conformational changes in response to a variety of stimuli, such as
pH, ionic strength and so forth. A number of papers have
demonstrated the use of coiled-coil peptides in biotechnology and
materials science research (Cho et al., 1998; Peak et al., 1998;
Wang et al., 1999). For example, hybrid hydrogels, assembled from
synthetic polymers and proteins, have potential in bioengineering
applications such as cellular encapsulation and controlled reagent
delivery systems. Hybrid hydrogels assembled from synthetic
polymers and coiled-coil protein domains have been shown to be
responsive to stimuli, but are hampered by slow recovery times
(Wang et al., 1999). Although a number of design principles have
been used in the de novo synthesis of peptides, it is quite clear
that much can be learned from the folding interactions, stability
and topology of native proteins. In this paper we demonstrate that
the temperature-dependent reversible folding/unfolding of TIpA is
rapid, and few proteins exhibit similar kinetics of
folding/unfolding. In addition, using a GFP-TIpA fusion protein, we
demonstrate the use of fluorescence as an indicator of structural
transitions. The results presented here should allow us in the
future to assemble hybrid hydrogels that display reversible and
rapid temperature-dependent gel structural transitions, which would
otherwise be difficult to achieve using hydrogels of synthetic
polymers alone.
Materials and Methods:
Bacterial Strains and Growth Conditions.
[0068] Escherichia coli strains were grown in Luria Broth medium;
for plasmid selection, ampicillin (100 mg/ml) was added (Sambrook
et al., 1989). Recombinant proteins were expressed in E. coli
BL21(DE3) strain (Novagen, Madison, Wis.).
Standard DNA Techniques
[0069] Standard procedures for plasmid isolations, restriction
enzyme digestions, ligations, gel purification and transformation
in bacteria were performed as described (Sambrook et al., 1989).
Polymerase chain reaction (PCR) was performed using standard
methods in a Perkin-Elmer Thermal Cycler. DNA sequence analysis was
carried out on ABI Prism 310 Genetic Analyzer (Perkin-Elmer Applied
Biosystems, Foster City Calif.).
Construction of Expression Vectors
[0070] TIpA and the coiled-coil domain (CC1) of Tar were amplified
by conventional PCR from S. typhimurium and E. coli genomic DNA,
respectively. Primers flanking the open reading frame of S.
typhimurium TIpA gene and the coiled-coil domain of E. coli Tar
gene were synthesized using published sequences (Genbank accession
numbers: IM88208 and P07017). The amplification product was
directly cloned into the TOPO vector (Invitrogen, Carlsbad,
Calif.). The fragments were then digested out of the TOPO vector
with appropriate restriction enzymes and cloned into the expression
vector pET21 b (Novagen, Madison, Wis.). The GFP-TIpA expression
plasmid was constructed by inserting in-frame the GFP (cycle 3
mutant) open reading frame upstream of TIpA in pET21 b. DNA
sequence analysis was performed on GFP-TIpA, as well as all other
expression plasmids.
Protein Expression and Purification
[0071] pET21 b. DNA sequence analysis was performed on GFP-TIpA, as
well as all other expression plasmids. The histidine-tagged
recombinant proteins were expressed in E. coli BL21(DE3) after
induction with 1 mM isopropyl R-thiogalactosidase (IPTG) at
30.degree. C. for 3-4 hrs. The cell pellet was resuspended in
Novagen binding buffer and sonicated with a microtip at the
following settings: power level between 2-3 at 30% duty cycle for
10 secs. Bacterial lysates were pre-cleared at 25,000.times.g for
20 min, and applied directly onto a Ni-NTA metal affinity resin.
The histidine tag permits the purification of the recombinant
protein using standard procedures with a nickel-chelating resin.
The purified protein was dialyzed extensively against 10 mM sodium
phosphate buffer pH 7.0 and stored at -20.degree. C. Protein purity
was greater than 90% as determined by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). Protein concentration was determined by
the method of Bradford using a commercially available kit (Pierce,
Rockford, Ill.).
Circular Dichorism Spectroscopy
[0072] CD spectra were measured in the range of 180 to 250 nm on a
Jasco J-720 spectropolarimeter equipped with a temperature control
unit. The ellipticity and wavelength were calibrated with a
standard solution of L-10 camphosulfonic acid. The measurements
were carried out at the indicated temperature using a 0.1 cm path
length water jacketed quartz cuvette. Raw data were acquired at an
interval of 1 nm and a bandwidth of 2 nm, and 3 consecutive scans
were accumulated with a scan speed of 120 nm/min and averaged.
Fluorescence Spectroscopy
[0073] Fluorescence measurements of cell lysates prepared from
equivalent number of cells, as measured by OD.sub.500, were carried
out on a Perkin-Elmer LS50B spectrofluorometer using 1 cm quartz
cuvette. The excitation and emission wavelengths were set to 395
and 509 nm respectively, with a slit width set between 5 and 12
nm.
Results and Discussion:
Circular Dichroism Analysis of Coiled-Coil Proteins.
[0074] TIpA from Salmonella was expressed in E. coli and purified
as described (see materials and methods). The circular dichroism
(CD) spectrum of TIpA has the characteristic feature of an
.alpha.-helical coiled-coil protein with negative ellipticity peaks
at 222 and 208 nm. The evidence for the .alpha.-helical content
(85%) and coiled-coil motif in TIpA has been previously well
documented by Hurme et al., (1996). TIpA exhibits a significant
structural transition as the temperature is increased from
25.degree. C. to 55.degree. C. Heating of TIpA to 55.degree. C.
results in the complete loss of .alpha.-helicity at 222 nm. This
indicates that the folded coiled-coil protein undergoes a
conformational change to an unfolded random coil.
[0075] The Tar gene which encodes for the aspartate receptor in E.
coli is involved in thermotatic response, but the thermosensing
mechanism is not well understood (Mizuno and Imae, 1984; Nara et
al., 1996). We speculated that like TIpA, N terminal coiled-coil
domain of Tar may serve a similar thermosensing function. We
expressed and purified the coiled-coil domain (amino acid residues
34-190) as a histidine-tagged fusion protein in E. coli. The far-UV
CD spectrum of purified CC1 shows a similar helical coiled-coil
structure with negative ellipticity at 208 and 222 nm. CC1 is able
to unfold to a random coil upon heating to 55.degree. C. We believe
that the structural transition of TIpA from a folded coiled-coil to
an unfolded random coil conformation is a two-state equilibrium. A
stepwise increase in temperature from 20.degree. C. to 65.degree.
C. leads to the cooperative unfolding of TIpA as indicated by the
loss of .alpha.-helicity at 222 nm. The presence of an isodichroic
point at approximately 203 nm in the spectra obtained at different
temperatures is indicative of a two-state .alpha.-helical to random
coil transition (Hurme et al., 1997).
[0076] We took our analysis a step further by investigating whether
the structural transition (.alpha.-helix to a random coil) induced
by the thermal unfolding of TIpA or CC1 was reversible. Our
research showed the reversibility of the structural transition
(.alpha.-helix H random coil) as demonstrated by the recovery of
the initial DE222 nm following cooling of the thermally unfolded
protein. Previous research suggests that the unfolded TIpA is able
to fold completely following heating to 55.degree. C. (Hurme et
al., 1997). We demonstrated that TIpA is able to completely recover
following heating to 55.degree. C. and 65.degree. C. Additionally,
the recovery was rapid (less than a minute) as observed by
restoration of its .alpha.-helicity when cooled back to 10.degree.
C. In contrast, CC1 was unable to recover following cooling of the
thermally unfolded protein to 10.degree. C. Considering the
physiological temperature range (37.degree. C.-42.degree. C.) at
which bacteria are able to function, we observed very little
recovery in the unfolding of CC1 following heating to temperatures
above 42.degree. C. The rapid recovery of TIpA following heating to
65.degree. C. back to its native coiled-coil conformation is
remarkable since structurally similar proteins either denature
irreversibly at high temperatures or recover with much slower
kinetics. The rapid recovery of TIpA following heating back to its
native coiled-coil conformation is further supported by the fact
that the loss of .alpha.-helicity during heating is reversible. As
the temperature increases, a significant loss in the
.alpha.-helical content of TIpA is observed. The .alpha.-helical
structure is regained when the temperature is decreased. Our
results showed that the kinetics of refolding was superimposable
upon the kinetics of unfolding. These results in combination with
those of Hurme et al., (1997) confirm that TIpA can serve as an
active thermosensing device based on the properties of its
coiled-coil domain. The rapid response of TIpA to temperature
changes makes TIpA an ideal proteinaceous thermometer.
[0077] The irreversibility of the structural transition of CC1
suggests that the N-terminal coiled-coil domain may not serve as a
thermosensor but only as a recognition site for aspartate. This is
most likely the case since studies have implicated the cytoplasmic
coiled-coil domain to be essential for thermosensing in vivo
(Nishiyama et al., 1999). We are currently investigating whether
the cytoplasmic coiled-coil domain of Tar can serve as a
thermosensing device. In addition, using de novo protein design, we
have been able to construct a small 16 amino acid coiled-coil
peptide that exhibited some of the same thermodynamic properties of
TIpA and the results of which will be published elsewhere.
GFP-TIpA Fusion Protein as Indicator of Structural Transitions
[0078] Green fluorescent protein (GFP) has become an important tool
in cell biology and has been extensively used as a reporter
molecule in biology (Misteli and Spector, 1997). De Angelis et al.,
(1998) demonstrated that tagging a protein with GFP can provide
information about the protein's conformational state. When two GFP
molecules are brought into close proximity, changes in the
fluorescence intensity occurs when excited with 395 nm or 475 nm
light. This phenomenon is called proximity imaging (PRIM). They
demonstrated that homodimerization of a candidate protein, which is
fused to GFP at either the amino- or carboxy-terminal, causes
fluorescence quenching when excited at 395 nm. Waldo et al., (1999)
used GFP as a fluorescent indicator of protein folding. It was
shown that the fluorescence intensity of GFP is related to the
productive folding of protein modules fused to GFP. Aggregation of
the protein modules led to a decrease in the fluorescence intensity
of the GFP chromophore. We decided to test whether fusing GFP to
TIpA would act as fluorescent indicator of the structural
transitions that occur in response to temperature changes. As
mentioned earlier, TIpA is able to undergo structural transitions
from a dimer to a monomer via its change from a coiled-coil to
random coil conformation in response to thermal changes.
[0079] We constructed a GFP-TIpA folding reporter vector, wherein
GFP is expressed as a N-terminal fusion with TIpA. The GFP-TIpA
plasmid was transformed into and expressed in E. coli BL21(DE3).
Induction with IPTG resulted in the appearance of fluorescent cells
within 4 hours in liquid culture. Interestingly, longer incubation
times lead to a decrease in fluorescence, which is most likely due
to aggregation of the overproduced fusion protein (Naik and Stone,
2000). We postulated that at lower temperatures, formation of TIpA
dimers would result in fluorescence quenching of the fused GFP
modules. At higher temperatures, unfolding of the coiled-coil
domains of TIpA leads to the dissociation of the dimers into
monomers, which enhances the fluorescence of GFP. Our results
showed that the fluorescent intensity of the GFP-TIpA fusion
protein when excited at 395 nm increases by about 30% as the
temperature is raised from 10.degree. C. to 55.degree. C. In
contrast, when the temperature of GFP alone was increased from
10.degree. C. to 55.degree. C., we observed little change in the
fluorescence intensity when excited at 395 nm. In fact, we observed
that the fluorescence of GFP alone showed a decrease of
approximately 10% at 55.degree. C. The change in the fluorescence
intensity of GFP-TIpA as a function of temperature is most likely
due to the structural conformation of the downstream coiled-coil
domain of TIpA. Further analysis of the effect of temperature on
the fluorescence intensity of the GFP-TIpA fusion protein
demonstrates that increasing the temperature from 10.degree. C. to
37.degree. C. leads to an intermediate increase in the fluorescent
intensity when excited with 395 nm light. An additional increase in
the fluorescent intensity is observed when the sample is heated
further to 55.degree. C. This process is reversible as shown by the
decrease in the fluorescence intensity of the GFP-TIpA fusion
protein as the temperature is lowered to 10.degree. C. Although a
single cycle is shown, the response was stable and reproducible
over numerous cycles. In order to determine that fluorescent
intensity of the GFP-TIpA fusion protein increases linearly with
temperature, we plotted the fluorescence intensity versus
temperature. The fluorescence intensity of the fusion protein
increases linearly within the temperature range tested. These
results clearly show that GFP functions as an indicator of the
structural transitions in TIpA. The low fluorescence of the
GFP-TIpA fusion protein at 10.degree. C. is most likely due to the
aggregation property of the TIpA dimers (Hurme et al., 1994). It is
known that TIpA dimers are nonexchanging at room temperature or on
ice, whereas they are dynamic chain exchanging structures at
temperatures above 37.degree. C. (Hurme et al., 1994). The gradual
increase in fluorescence intensity of the GFP-TIpA fusion protein
may be most arguably due to the dimer to monomer transition, since
the addition of a reducing agent to the GFP-TIpA fusion protein
sample causes the fluorescence to be unresponsive to temperature
changes (data not shown). However, it is also likely that unfolding
of the coiled-coil domain in response to an increase in temperature
may indirectly affect the structure of the upstream GFP module. We
are currently designing experiments that would differentiate
between these two possibilities. Irrespective of the outcome of
these experiments, it is quite clear from the results presented
here that the fluorescence of GFP acts as an indicator of the
structural transitions in TIpA in response to temperature.
CONCLUSIONS
[0080] Here we present our results on the thermostability of an
.alpha.-helical coiled-coil bacterial thermosensor. TIpA exhibits a
high degree of thermostability and an unusual reversible refolding
ability. The kinetics of folding/unfolding of TIpA is rapid and
different from that of other coiled-coil proteins. Increasing the
temperature leads to the cooperative unfolding of the coiled-coil
domain to an unfolded random coil. The unfolding of TIpA correlates
quite well with increasing temperature, and the protein assumes a
complete random coil conformation at temperatures higher than
55.degree. C. Based on previous results on the use of GFP as a
fluorescent indicator of protein folding (Waldo et al., 1999; De
Angelis et al., 1998), we constructed a GFP-TIpA fusion protein
that is responsive to temperature changes. We perceive the changes
in the fluorescence of GFP to be a direct measure of changes in the
structural conformation of TIpA. At low temperatures, GFP-TIpA
forms dimers which results in fluorescence quenching. Increasing
the temperature causes dissociation of dimers, in part due to the
unfolding of the coiled-coil domain of TIpA, and an enhancement in
fluorescence intensity.
[0081] Since signal transduction is perhaps the biggest obstacle to
biosensor development, the TIpA proteins of the invention will be
extremely useful in overcoming this problem. Biological signal
transduction usually involves an extremely complicated sequence of
molecular recognition and binding packaged in a specific set of
steps. By utilizing the TIpA protein, we have isolated an initial
sensing molecule i.e., the "trigger", and can thus use this protein
in achieving an optical readout as a measure of the transduction
process.
[0082] Previously, one weakness of protein-based biological sensors
was the speed of response, especially in a thermal process because
proteins almost always exhibit a prolonged unfolding-refolding
cycle, i.e., several minutes, that is too slow for most sensing
applications. However, the use of the TIpA protein in accordance
with the invention overcomes these problems because of its
unfolding/refolding cycle which is incredibly fast. It remains
unclear as to exactly how quickly TIpA can respond to thermal
stimuli, but the protein has responded faster than we can shift the
temperature and produce thermal equilibrium. Albeit that the
response time of the GFP-TIpA fusion protein was slower than that
of TIpA alone, two different techniques (CD spectroscopy versus
Fluorescence spectroscopy) were used in measuring responses to
temperature changes.
[0083] In accordance with the invention, the TIpA protein can thus
be incorporated into polymer hydrogels in order to exploit the
thermodynamic properties of TIpA in a manner previously not
obtained, and these hybrid hydrogels will be assembled from
synthetic polymers and TIpA. The dissociation of the coiled-coil
aggregates of TIpA through elevation of temperature cause
dissolution of the gel network and a return to a more porous state.
Such materials or hybrid hydrogels in accordance with the present
invention thus have potential in a number of biosensor applications
requiring controlled release of molecules embedded in the
hydrogel.
REFERENCES
[0084] The following references as utilized in the Example above
are incorporated by reference as if set forth in the above
specification in full: [0085] Chao, H., D. L. Bautista, J.
Litowski, R. T. Irvin, and R. S. Hodges. 1998. Use of a heteromeric
coiled-coil system for biosensor application and affinity
purification. J. Chromatogr. B Biomed. Appl. 715:307-329. [0086] De
Angelis, D. A., G. Miesenbock, B. V. Zemelman, and J. E. Rothman.
1998. PRIM: Proximity imaging of green fluorescent protein-tagged
polypeptides. Proc. Natl. Acad. Sci. USA. 95:12312-12316. [0087]
Hurme, R., K. D. Berndt, E. Namork, and M. Rhen. 1996. DNA binding
exerted by a bacterial gene regulator with an extensive coiled-coil
domain. J. Biol. Chem. 271:12626-12631. [0088] Hurme, R., K. D.
Berndt, S. J. Normark, and M. Rhen. 1997. A proteinaceous gene
regulatory thermometer in Salmonella. Cell. 90:55-64. [0089] Hurme,
R., E. Namork, E. Nurmiaho-Lassila, and M. Rhen. 1994. Intermediate
filament-like network formed in vitro by a bacterial coiled-coil
protein. J. Biol. Chem. 269:10675-10682. [0090] Koski, P., H.
Saarilahti, S. Sukupolyi, S. Taira, P. Riikonen, K. Osterlund, R.
Hurme, and M. Rhen. 1992. A new .alpha.-helical coiled-coil protein
encoded by the Salmonella typhimurium virulence plasmid. J. Biol.
Chem. 267:12258-12265. [0091] Lupas, A. 1996. Coiled coils: new
structures and functions. Trends Biochem. Sci. 21:375-382. [0092]
Misteli, T., and D. L. Spector. Applications of the green
fluorescent protein in cell biology and biotechnology. Nat.
Biotechnol. 15:961-964. [0093] Mizuno, T., and Y. Imae. 1984.
Conditional inversion of the thermoresponse in Escherichia coli. J.
Bacteriol. 159:360-367. [0094] Naik, R. R., and M. O, Stone 2000.
The use of green fluorescent protein as an indicator in protein
expression. In preparation. [0095] Nara, T., I. Kawagishi, S,
Nishiyama, M. Homma, and Y. Imae. 1996. Modulation of the
thermosensing profile of the Escherichia coli aspartate receptor
Tar by covalent modification of the methyl-accepting site. J. Biol.
Chem. 271:17932-17936. [0096] Nishiyama, S., I. N. Maruyama, M.
Homma, and 1. Kawagishi. 1999. Inversion of the thermosensing
property of the bacterial receptor Tar by mutations in the second
transmembrane region. J. Mol. Biol. 286:1275-1284. [0097] Pauling,
L., and R. B. Corey. 1953. Compound helical configurations of
polypeptide chains: structure of proteins of the x-keratin type.
Nature. 171:59-61. [0098] Petka, W. A., J. L. Harden, K. P.
McGrath, D. Wirtz, and D. A. Tirrell. 1998. Reversible hydrogels
from self-assembling artificial proteins. Science. 281:389-392.
[0099] Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. [0100] Waldo, G. S., B.
M. Standish, J. Berendzen, and T. C. Terwilliger. 1999. Rapid
protein-folding assay using green fluorescent protein. Nat.
Biotechnol. 17:691-695. [0101] Wang, C., R. J. Stewart, and J.
Kopecek. 1999. Hybrid hydrogels assembled from synthetic polymers
and coiled-coil protein domains. Nature. 397:417-420.
Example 3
Preparation of a Holographic Nanopattern of Biocatalytically Formed
Silica for Use in the Present Invention
[0102] In accordance with the present invention, a structure for
use with the thermally sensing proteins of the present invention
was developed from another form of nature: diatoms.
[0103] Diatoms are of interest to the materials research community
because of their ability to create highly complex and intricate
silica structures under physiological conditions: what these
single-cell organisms accomplish so elegantly in nature requires
extreme laboratory conditions to duplicate.sup.1,2-this is true for
even the simplest of structures. Following the identification of
polycationic peptides from the diatom Cylindrotheca fusiformis,
simple silica nanospheres can now be synthesized in vitro from
silanes at nearly neutral pH and at ambient temperatures and
pressures.sup.3,4. Here we describe a method for creating a hybrid
organic/inorganic ordered nanostructure of silica spheres through
the incorporation of a polycationic peptide (derived from the C.
fusiformis silaffin-1 protein) into a polymer hologram created by
two-photon-induced photopolymerization. When these peptide
nanopatterned holographic structures are exposed to a silicic acid,
an ordered array of silica nanospheres is deposited onto the clear
polymer substrate. These structures exhibit a nearly fifty-fold
increase in diffraction efficiency over a comparable polymer
hologram without silica. This approach, combining the ease of
processability of an organic polymer with the improved mechanical
and optical properties of an inorganic material, could be of
practical use for the fabrication of photonic devices.
[0104] We have recently developed a holographic two-photon-induced
photopolymerization (H-TPIP) process.sup.5 and here we describe how
this technique can be used to prepare nanopatterned structures that
contain biological macromolecules such as the TIpA proteins of the
present invention. Unlike conventional holograms formed through the
use of ultraviolet lasers, holograms created through the two-photon
process use an ultrafast infrared laser. Because infrared
wavelengths typically do not alter the functionality of biological
compounds, monomer formulations containing peptides can be
polymerized without affecting the biological activity. We
incorporated a peptide that has recently been shown to be
responsible for biosilification into a formulation to be cured by a
holographic two-photon-induced photopolymerization with the
expectation that the peptide would be segregated into regions of
low crosslinking density. The approach of using ultraviolet lasers
to phase separate small liquid crystal molecules in a polymer-based
hologram has been used extensively.sup.6 and we hypothesized that
this technique would also be applicable to the H-TPIP process. We
predicted that exposing the peptide-containing structure to a
liquid silane would cause silica to form in the holographic
nanopattern and that this hybrid organic/inorganic device would
have a higher degree of order leading to a superior device compared
to randomly ordered monolayers of silica on indium-tin oxide (ITO)
coated glass.sup.7.
[0105] A short 19-amino-acid R5 peptide unit (SSKKSGSYSGSKGSKRRIL)
(SEQ ID NO:2) of the silaffin-1 precursor polypeptide from C.
fusiformis is able to catalyze the formation of silica nanospheres
within minutes when added to silicic acid to neutral pH and ambient
temperature.sup.3. A chemically synthesized R5 peptide that lacks a
post-translational modification of its lysine residues was used in
the present work. The post-translational modification of lysine
residues is required for silica formation under acidic pH
conditions.sup.3,8. However, because our research was conducted
under slightly basic conditions, the modification of the lysine
residues was unnecessary. Consequently, work began by incorporating
this peptide (0.8 mg in 16 .mu.l of water) into a monomer
formulation. This formulation consisted of 160 .mu.l SR-9035, 0.022
g SR-399 (SR-9035 is a trimethylolpropane triacrylate and SR-399 is
a dipentaerythritol pentaacrylate obtained from Sartomer which were
used without the removal of inhibitor), 0.006 g triethanol amine
and 0.005 g isopropyl thioxanthone; the entire mixture was heated
for 15 min at 50.degree. C. to aid in dissolution. The triacrylate
was chosen for its high water miscibility which is due to its
numerous ethylene glycol units, and the pentaacrylate was used to
create a highly crosslinked system. The triethanol amine functions
as a co-initiator and thioxanthone as the initiator. Typically, in
a two-photon-initiated polymerization, a fluorescent chromophore is
also required to absorb two photons of near-infrared laser light.
The excited chromophore transfers its energy to the initiator which
begins the polymerization process. However, we have found that the
thioxanthone used in this formulation does not require highly
colored chromophores, and consequently, extremely large curing
depths and exceptionally clear and colorless polymers are
produced.sup.9,10.
[0106] A thin layer (178 .mu.m) of the monomer/peptide formulation
was deposited onto a clean glass slide, which was then placed in a
miniature atmospheric chamber fitted with glass windows and flushed
with nitrogen. The sample was cured in a two-beam transmission
holographic arrangement using a 790-nm titanium-sapphire laser
(90-fs pulse width with a repetition rate of 500 Hz) for 30 s. The
intensity distribution of the volume hologram drives the local
polymerization rate as a function of the local field intensity,
which results in alternating areas of high and low crosslink
density. Because certain areas of the sample cure more rapidly than
others, the smaller molecules (namely water and peptide) phase
separate from the areas of higher crosslink density and migrate
into areas of lower density. This phenomenon has been observed in
similar systems using liquid crystals as the small molecule. An
alternative explanation of this phase separation could be that as
the hydrophilic monomer is converted into a more hydrophobic
polymer, the peptide is driven into the monomer-rich regions. As a
result, peptide-rich domains are created in the polymer sample with
the periodicity of the hologram. After the curing process, the
sample was briefly rinsed with water to remove any uncured monomer.
Atomic force microscopy (AFM) revealed that the hologram had a
periodicity of 1.33 .mu.m.
[0107] The silane precursor (1 M tetrahydroxysilane) was
synthesized by dissolving tetramethyl orthosilicate (TMOS) in 1 mM
HCl. This product was then added to a sodium phosphate-citrate
buffer (pH 8) to produce a final concentration of 113 mM. We note
that this dilute solution remains stable for over two hours, after
which it slowly converts into a clear amorphous gel. Freshly
prepared hydrolyzed silane was slowly applied to the hologram and
allowed to react with the R5 peptide embedded in the hologram for
10 min. before being rinsed with water to remove any unreacted
silane. A control hologram lacking the R5 peptide was also treated
with the tetrahydroxysilane solution but did not exhibit any
nanosphere formation. However, when a sample that included the
peptide and was treated with the silane was analyzed by scanning
electron microscope, it was revealed that silica spheres formed a
regular two-dimensional array with the periodicity of the hologram.
A study of the size distribution of the silica spheres reveals that
the average nanosphere diameter is 452 nm (.+-.81 nm). The silica
content of the spheres was confirmed using electron dispersive
spectroscopy (EDS). Additionally, analysis using the AFM indicated
that the hologram had a periodicity of 1.60 .mu.m with the silica
spheres embedded in the troughs of the surface relief pattern. The
difference in the spacing between the holograms treated with and
without the tetrahydroxysilane solution can be explained by the
fact that the control grating shrinks as it dries out owing to
water evaporation, whereas the shrinkage in the hybrid hologram is
inhibited owing to the added mechanical strength of the silica
spheres, preventing the ridges of the hologram from moving closer
together. Consequently, the untreated grating exhibited nearly 17%
more shrinkage than the treated grating. Also, the silica spheres
are the most prominent feature of the hologram and the troughs in
the structure are actually the peaks of the polymer.
[0108] Finally, to test the improvement that this technique can
impart to an optical device, the first-order diffraction efficiency
of the treated hologram was compared to that of the untreated
sample. These measurements were performed by transmitting a
helium-neon laser through each sample and measuring the diffraction
pattern in the far field. A measurement of the incident and
transmitted power in the first-order diffraction spot showed a
substantial increase in the diffraction efficiency of the grating
with silica versus the grating without, as would be expected from
the difference in index and shrinkage. The untreated grating
exhibited a diffraction efficiency of approximately 0.02%, while
the grating with the silica spheres showed an efficiency of
approximately 0.95%. This large increase can be attributed to the
fact that the spheres form an almost continuous line of silica
along the valleys of the hologram, achieving a high fill
factor.
[0109] We have thus shown that the incorporation of the peptide
responsible for biosilification into a microfabricated structure
using H-TPIP can result in an unusual composite organic/inorganic
device that has significantly improved optical performance and
superior mechanical properties compared to those of a corresponding
polymeric device without silica. Although we have used a
polymer/silica hybrid structure, this technique is universally
applicable for any catalyst or binding agent that can be
incorporated into a polymer. For example, as different catalysts
are identified, a wide variety of unique hybrid structures are now
possible with differing shapes and mechanical properties.
Additionally, antibodies can be incorporated into the hologram and
potentially used to optically identify specific antigens.
Consequently, this technique allows a simple yet general and easily
modifiable method for nanopatterning, and thus can provide a
substrate for incorporation of the thermally sensitive TIpA
proteins of the present invention.
REFERENCES
[0110] The following references as utilized in the Example above
are incorporated by reference as if set forth in the above
specification in full: [0111] 1. Parkinson, J. & Gordon, R.
Beyond micromachining: the potential of diatoms. Trends Biotechnol.
17, 190-196 (1999). [0112] 2. Morse, D. E. Silicon biotechnology:
harnessing biological silica production to construct new materials.
Trends Biotechnol. 17, 230-232 (1999). [0113] 3. Kroger, N.,
Deutzmann, R. & Sumper, M. Polycationic peptides from diatom
biosilica that direct silica nanosphere formation. Science 286,
1129-1132 (1999). [0114] 4. Cha, J. N., Stucky, G. D., Morse, D. E.
& Deming, T. J. Biomimetic synthesis of ordered silica
structures mediated by block copolypeptides. Nature 403, 289-292
(2000). [0115] 5. Kirkpatrick, S. M. et al. Holographic recording
using two-photon-induced photopolymerization. Appl. Phys. A 69,
461-464 (1999). [0116] 6. Bunning, T. J. et al. The morphology and
performance of holographic transmission gratings recorded in
polymer dispersed liquid, crystals. Polymer 36, 2699-2708 (1995).
[0117] 7. Wang, C. et al. Two-dimensional ordered arrays of silica
nanoparticles. Chem. Mater. 12, 3662-3666 (2000). [0118] 8. Kroger,
N., Deutzmann, R. & Sumper, M. Silica-precipitating peptides
from diatoms, the chemical, structure of silaffin-1a from
Cylindrotheca fusiformis. J. Biol. Chem. 276, 26066-26070 (2001).
[0119] 9. Belfield, K. D. et al. Multiphoton-absorbing organic
materials for microfabrication, emerging optical applications and
non-destructive three-dimensional imaging. J. Phys. Org. Chem. 13,
837-849 (2000). [0120] 10. Brott, L. L. Naik, R. R., Kirkpatrick,
S. M. Pikas, D. J. & Stone, M. O. Near-IR two-photon induced
polymerizations using either benzophenone or thioxanthone-based
photoinitiators. Polymer Preprints 42, 675-676 (2001).
Sequence CWU 1
1
2 1 43 PRT Salmonella typhimurium 1 Thr Arg Glu Thr Leu Gln Gln Arg
Leu Glu Gln Ala Ile Ala Asp Thr 1 5 10 15 Gln Ala Arg Ala Gly Glu
Ile Ala Leu Glu Arg Asp Arg Val Ser Ser 20 25 30 Leu Thr Ala Arg
Leu Glu Ser Gln Glu Lys Ala 35 40 2 19 PRT Cylindrotheca fusiformis
2 Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys Gly Ser Lys Arg 1
5 10 15 Arg Ile Leu
* * * * *