U.S. patent application number 10/958452 was filed with the patent office on 2005-07-07 for enzymatic template polymerization.
Invention is credited to Bruno, Ferdinando, Kumar, Jayant, Liu, Wei, Nagarajan, Ramaswamy, Samuelson, Lynne A., Tripathy, Sukant K., Tripathy, Susan.
Application Number | 20050147990 10/958452 |
Document ID | / |
Family ID | 34437748 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050147990 |
Kind Code |
A1 |
Samuelson, Lynne A. ; et
al. |
July 7, 2005 |
Enzymatic template polymerization
Abstract
A conductive polymer is formed enzymatically in the presence of
a polynucleotide template. The method includes combining at least
one redox monomer with a polynucleotide template and a redox
enzyme, such as horseradish peroxidase, to form a reaction mixture.
The monomer aligns along the template before or during the
polymerization. Therefore, the polynucleotide template thereby
affects the molecular weight and conformation of the conductive
polymer. When the conductive polymer is complexed to a
polynucleotide duplex, the conformation of the polynucleotide
duplex can be modulated by changing the oxidation state of the
conductive polymer.
Inventors: |
Samuelson, Lynne A.;
(Marlborough, MA) ; Bruno, Ferdinando; (Andover,
MA) ; Tripathy, Sukant K.; (Acton, MA) ;
Tripathy, Susan; (Acton, MA) ; Nagarajan,
Ramaswamy; (Dracut, MA) ; Kumar, Jayant;
(Westford, MA) ; Liu, Wei; (US) |
Correspondence
Address: |
U.S. Army Soldier Systems Center
Kansas Street
Natick
MA
01760
US
|
Family ID: |
34437748 |
Appl. No.: |
10/958452 |
Filed: |
October 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10958452 |
Oct 5, 2004 |
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10324736 |
Dec 19, 2002 |
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10324736 |
Dec 19, 2002 |
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09447987 |
Nov 23, 1999 |
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09447987 |
Nov 23, 1999 |
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08999542 |
Nov 21, 1997 |
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6018018 |
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08999542 |
Nov 21, 1997 |
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08915827 |
Aug 22, 1997 |
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5994498 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2; 525/54.2 |
Current CPC
Class: |
Y10S 977/704 20130101;
Y10T 436/143333 20150115; H01B 1/128 20130101; C08G 73/0266
20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 525/054.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Goverment Interests
[0002] This invention was made with support from the Government
under ARO Cooperative Grant DAAH04-94-2-003. The Government has
certain rights in this invention.
Claims
What is claimed is:
1. A composition of matter, comprising a polynucleotide template
and a substituted or unsubstituted polyaniline bound together as a
complex, wherein the polyaniline has a chiral formation.
2. The composition of claim 1 wherein the polynucleotide is a
single strand.
3. The composition of claim 1, wherein the polynucleotide is a
double helix.
4. The composition of claim 1, wherein the polynucleotide template
is a deoxyribonucleotide.
5. The composition of claim 1, wherein the polynucleotide is a
ribonucleotide.
6. A method of preparing a polynucleotide/polyaniline complex,
comprising combining a substituted or unsubstituted aniline
monomer, a polynucleotide template and a redox enzyme, whereby the
monomer aligns along the template to form a complex and polymerizes
to form a polyaniline, thereby forming the
polynucleotide/polyaniline complex.
7. The method of claim 6, wherein the polynucleotide is a
deoxyribonucleotide or a ribonucleotide.
8. The method of claim 6, wherein the enzyme is a peroxidase.
9. The method of claim 8, wherein the peroxidase is horseradish
peroxidase.
10. The method of claim 8, wherein hydrogen peroxide is combined
with the aniline monomer, polynucleotide template and a redox
enzyme.
11. The method of claim 8, wherein the polynucleotide template and
redox enzyme are combined in a solution having a pH of about 4 to
about 5.
12. The method of claim 6, wherein the polynucleotide is a single
strand.
13. The method of claim 6, wherein the polynucleotide is a double
helix.
14. The method of claim 13, wherein the polyaniline formed has a
chiral conformation.
15. The method of claim 13, wherein the polyaniline formed has an
achiral conformation.
16. A method of modulating the conformation of a polynucleotide
which is bound to a conductive polymer in a complex, comprising
changing the oxidation state of the conductive polymer.
17. The method of claim 16, wherein the conductive polymer is
selected from the group consisting of: a substituted or
unsubstituted polyaniline or a substituted or unsubstituted
polyphenol.
18. The method of claim 17, wherein the conductive polymer is
polyaniline.
19. The method of claim 18, wherein the polynucleotide is a single
strand.
20. The method of claim 18, wherein the polynucleotide is a double
helix.
21. The method of claim 20, wherein the oxidation state of the
polyaniline is changed by oxidizing said polyaniline, thereby
causing the double helix to have more base pairs per helical repeat
after the polyaniline is oxidized.
22. The method of claim 21, further comprising the step of reducing
the polyaniline, thereby causing the double helix to have less base
pairs per helical repeat after the polyaniline is reduced.
23. The method of claim 22, wherein the oxidation state of
polyaniline is changed electrochemically.
24. The method of claim 20, wherein the oxidation state of the
polyaniline is changed by reducing said polyaniline, thereby
causing the double helix to have fewer base pairs per helical
repeat after the polyaniline is reduced.
25. The method of claim 24, further comprising the step of
oxidizing the polyaniline, thereby causing the double helix to have
more base pairs per helical repeat after the polyaniline is
oxidized.
26. The method of claim 22 or 25, wherein the polyaniline is
cyclically oxidized and reduced.
27. The method of claim 25, wherein the oxidation state of
polyaniline is changed electrochemically.
28. An electrical component, comprising: a) an electrical element;
and b) a nanowire attached to the electrical element, wherein said
nanowire includes a polynucleotide template and a conductive
polymer bound together as a complex.
29. The electrical component of claim 28, wherein the conductive
polymer is selected from the group consisting of: a substituted or
unsubstituted polyaniline or a substituted or unsubstituted
polyphenol.
30. The electrical component of claim 29, wherein the conductive
polymer is polyaniline.
31. The electrical component of claim 30, wherein the
polynucleotide in the polynucleotide/polyaniline complex is a
single strand.
32. The electrical component of claim 30, wherein the
polynucleotide in the polynucleotide/polyaniline complex is a
double helix.
33. The electrical component of claim 30, wherein a portion of the
polynucleotide in the polynucleotide/polyaniline complex is single
stranded and a portion is a double helix.
34. The electrical component of claim 28, further including at
least one other electrical element, and wherein the electrical
elements are connected by said nanowire.
35. The electrical component of claim 34, wherein the
polynucleotide/polyaniline complex includes two or more
polynucleotides that are hybridized.
36. The electrical component of claim 35, wherein the
polynucleotides in the polynucleotide/polyaniline complex are
deoxyribonucleotides.
37. The electrical component of claim 35, wherein the
polynucleotides in the polynucleotide/polyaniline complex are
ribonucleotides.
38. The electrical component of claim 35, wherein the
polynucleotides in the polynucleotide/polyaniline complex are a
combination of deoxyribonucleotides and ribonucleotides.
39. The electrical component of claim 34, wherein the
polynucleotide/polyaniline complex includes two or more
polynucleotides that have been assembled by enzymatic ligation.
40. A method of forming an electrically conductive connection
between electrical elements, comprising the steps of: a) connecting
at least two electrical elements with a polynucleotide; and b)
contacting the polynucleotide with a redox monomer and a redox
enzyme, whereby the monomer aligns along the template to form a
complex and polymerizes to form a conductive polymer, thereby
forming a polynucleotide/conductive polymer complex that
electrically connects the electrical elements, said
polynucleotide/conductive polymer complex being electrically
conductive.
41. The method of claim 40, wherein the conductive polymer is
selected from the group consisting of: a substituted or
unsubstituted polyaniline or a substituted or unsubstituted
polyphenol.
42. The method of claim 41, wherein the conductive polymer is
polyaniline.
43. The method of claim 40, wherein the electrical elements are
connected by hybridization of a first polynucleotide, connected to
a first electrical element, with a second polynucleotide, connected
to a second electrical element.
44. The method of claim 40, wherein the electrical elements are
connected by ligation of a first polynucleotide, connected to a
first electrical element, to a second polynucleotide, connected to
a second electrical element.
45. The method of claim 40, wherein the redox enzyme is a
peroxidase.
46. The method of claim 45, wherein the peroxidase is horseradish
peroxidase.
47. The method of claim 45, wherein hydrogen peroxide is combined
with the polynucleotide, redox monomer and redox enzyme.
48. The method of claim 40, wherein the polymerization of the
aniline monomer is conducted in a solution having a pH of about 4
to about 5.
49. The method of claim 43, wherein the electrical elements are
connected by hybridization of the first and second polynucleotides
before contacting the hybridized polynucleotides with the redox
monomer and the redox enzyme.
50. The method of claim 43, wherein the electrical elements are
connected by hybridization of the first and second polynucleotides
simultaneously with contacting the polynucleotides with the redox
monomer and the redox enzyme.
51. A method for identifying a target polynucleotide, comprising
the steps of: a) combining said target polynucleotide with a probe
that includes a polynucleotide template complexed with a conductive
polymer, whereby said probe hybridizes with the target
polynucleotide, said hybridization modifying at least one
electromagnetic property of the conductive polymer; and b)
detecting said modified electromagnetic property, thereby
identifying the target polynucleotide.
52. The method of claim 51, wherein the conductive polymer is a
substituted or unsubstituted polyaniline.
53. The method of claim 51, wherein the electromagnetic property is
an electrical or optical property.
54. The method of claim 53, wherein the modified electromagnetic
property is detected using UV-Visible absorption, circular
dichroism or cyclic voltammetry.
55. The method of claim 53, wherein the probe is attached to an
electrical element.
Description
RELATED APPLICATION
[0001] This is a continuation of U.S. application Ser. No.
09/447,987, filed Nov. 23, 1999, which is a continuation-in-part of
U.S. application Ser. No. 08/999,542, filed Nov. 21, 1997 (now U.S.
Pat. No. 6,018,018), which is a continuation-in-part of U.S.
application Ser. No. 08/915,827, filed Aug. 21, 1997 (now U.S. Pat.
No. 5,994,498), the entire teachings of which are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0003] Recently, there has been an increased interest in tailored
development of certain classes of polymers, such as electrically
conductive and optically active polymers (e.g. polythiophene,
polypyrrole, polyphenols and polyaniline) for application to wider
ranges of use. Examples of such uses include light-weight energy
storage devices, electrolytic capacitors, anti-static and
anti-corrosive coatings for smart windows, and biological sensors.
However, the potential applications to which polymers can be put
have been limited by their lack of solubility and
processability.
[0004] In particular, interest in developing biosensors has been
stimulated by efforts to sequence the human genome. Analysis and
manipulation of polynucleotides is expected to have genetic
engineering applications and aid in the diagnosis of genetic
disease and in the development and improvement of new drugs. For
example, deoxyribonucleotides (DNA) exist in living organisms
almost exclusively in a double helix conformation. However, many
variations in this conformation has been shown to exist (e.g., A-,
B-, C- and Z-type duplexes). The helical structure of a particular
duplex is related to its sequence and its environment. These
variations in conformation are thought to be responsible for the
binding of molecular species, such as enzymes or regulatory
proteins, to DNA. Therefore, methods of modulating the conformation
of DNA are expected to have applications in the area of biosensors,
molecular recognition and drug development.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a composition of matter in
which a substituted or unsubstituted polyaniline is bound to a
polynucleotide as a complex. The invention also relates to a method
of preparing a polynucleotide/polyaniline complex, wherein the
polynucleotide/polyanilin- e complex is formed by combining a
substituted or unsubstituted aniline monomer, a polynucleotide
template and a redox enzyme, whereby the aniline monomer aligns
along the polynucleotide template to form a complex and polymerizes
to form a polyaniline, thereby forming the
polynucleotide/polyaniline complex.
[0006] Another aspect of the invention is a method of modulating
the conformation of a polynucleotide double helix which is bound to
a conductive polymer as a complex by changing the oxidation state
of the conductive polymer. In a specific embodiment, polyaniline is
bound to a polynucleotide double helix as a complex. Oxidation of
polyaniline (e.g., increasing the positive charge on the
polyaniline) which is complexed to a polynucleotide double helix
causes the double helix to become more tightly wound (i.e., the
double helix will have more base pairs per turn after oxidation of
the polyaniline). Conversely, reducing the polyaniline will cause a
double helix associated with it to become more loosely wound.
Therefore, complexation of polyaniline to a polynucleotide double
helix provides a method of modulating the conformation of the
double helix by changing the oxidation state of the
polyaniline.
[0007] The invention also relates to an electrical element that has
a nanowire extending from it. The nanowire includes a
polynucleotide template and a conductive polymer bound together as
a complex.
[0008] Another aspect of the invention is a method of forming an
electrically conductive connection between electrical elements. The
method includes connecting at least two electrical elements with a
polynucleotide and contacting the polynucleotide with an a redox
monomer and a redox enzyme. The monomer aligns along the template
to form a complex and polymerizes to form a conductive polymer that
is complexed to the polynucleotide that connects the electrical
elements. The polynucleotide/conductive polymer complex is
electrically conductive and, therefore, forms an electrically
conductive connection between the electrical elements.
[0009] Another embodiment of the invention is a method of
identifying a target polynucleotide by contacting the target
polynucleotide with a probe that includes a polynucleotide template
complexed with a conductive polymer. The probe hybridizes with the
target polynucleotide which causes at least one electromagnetic
property of the conductive polymer to be modified. The target
polynucleotide is identified by detecting the modified
electromagnetic property.
[0010] In this invention, the polynucleotide can serve at least
three critical functions. First, the polynucleotide can serve as a
template upon wlich the monomers preferentially align themselves to
form a complex, such as a charge-transfer complex, thereby limiting
parasitic branching and controlling the shape of the polymer. In
the case of polyaniline, the mechanism of polymerization is
primarily para-directed and results in formation of the
electrically active form of polyaniline. This preferential
alignment provides improved electrical and optical properties of
the final polymer complex. Second, the polynucleotide can serve as
a large molecular dopant species which is complexed and essentially
locked to the polyaniline chains. Current limitations to the actual
use of polyaniline in electronic and optical applications largely
has been due to poor dopant stability. Small ionic dopants or
chromophores that are currently used are known to diffuse away with
time and/or conditions. Locking of a large polyelectrolyte dopant
(e.g., a polynucleotide) to the polymer is significant in that it
ensures that the electronic nature of the conjugated backbone
structure of the polymer is maintained, and hence the desired
electronic and optical properties are stabilized. Third, the
polynucleotide template can improve water solubility of the final
polynucleotide/polyaniline complex for environmentally friendly,
facile, and inexpensive processing.
[0011] In addition to the above advantages, complexation of a
polynucleotide duplex, such as DNA, to an electrically conductive
polymer provides a method by which the conformation of the duplex
can be modulated, thereby providing possible application in the
area of biosensors and drug development. For example, changing the
oxidation state of polyaniline bound to a DNA duplex changes the
linear length of a helical turn and, therefore, could be used to
study the binding properties of DNA regulatory proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows the general mechanism of enzymatic
polymerization of aniline in the absence of the polynucleotide,
promoting ortho- and para-directed reactions.
[0013] FIG. 2 shows the chemical structure of oxidized (conducting)
and reduced (insulating) forms of the polyaniline which is formed
during enzymatic template guided polymerization.
[0014] FIG. 3 shows the visible absorption spectra of the
polyaniline template complex (0.05M aniline to 0.1M sulfonated
polystyrene (SPS)) formed at various pH's.
[0015] FIG. 4 shows a plot of absorbance versus (SPS)/aniline ratio
to find the optimum dopant-to-monomer ratio.
[0016] FIG. 5A shows the visible absorption and redox behavior of
polyaniline/SPS prepared at pH 4.0 with increasing pH.
[0017] FIG. 5B shows the visible absorbance and redox behavior of
polyanilines/SPS prepared at pH 4.0 with decreasing pH.
[0018] FIG. 6A shows the visible absorbance and redox behavior of a
50 bilayer film of poly(diallyl dimethyl ammonium chloride) (PDAC)
alternating with SPS/polyaniline (prepared at pH 4.0) with
increasing pH.
[0019] FIG. 6B shows the visible absorbance and redox behavior of a
50 bilayer film of SPS/polyaniline (prepared at pH 4.0) with
decreasing pH.
[0020] FIG. 7A shows the visible absorbance of polyphenol without
SPS versus phenol monomer. Polyphenol precipitated out of solution
as a result of polymerization.
[0021] FIG. 7B shows the visible absorbance of polyphenol/SPS
template versus phenol monomer. The polyphenol did not precipitate
out of solution.
[0022] FIG. 8 is a schematic representation of polyaniline bound to
a DNA double helix.
[0023] FIG. 9 shows the UV-Vis spectra of DNA and DNA/polyaniline
(Pani) during polymerization.
[0024] FIG. 10 shows the CD spectra of DNA and DNA/polyaniline
during polymerization.
[0025] FIG. 11 shows the CD spectra of DNA; a mixture of DNA,
aniline monomer and horseradish peroxidase (HRP); DNA and
NH.sub.4F; and DNA/polyaniline.
[0026] FIG. 12A shows the CD spectra of DNA/polyaniline as the pH
is increased from 4 to 10.
[0027] FIG. 12B shows the CD spectra of DNA/polyaniline as the pH
is decreased from 10 to 4.
[0028] FIG. 13A shows the UV-Vis spectra of DNA/polyaniline as the
pH is increased from 4 to 10.
[0029] FIG. 13B shows the UV-Vis spectra of DNA/polyaniline as the
pH is decreased from 10 to 4.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The features and other details of the apparatus and method
of the invention will now be more particularly described and
pointed out in the claims. It will be understood that the
particular embodiments of the invention are shown by way of
illustration and not as limitations of the invention. The principal
features of this invention can be employed in various embodiments
without departing from the scope of the invention. All parts and
percentages are by weight unless otherwise specified.
[0031] Enzyme-catalyzed polymerization of aniline typically
involves reaction at the ortho and para positions of the aromatic
ring as shown in FIG. 1. This mechanism often results in branched
polymeric materials which are intractable and have negligible
electrical and optical properties. This invention describes a novel
template assisted enzymatic polymerization which results in a new
class of polyanilines. In general, the polyanilines formed by the
enzymatic template guided polymerization described herein are
linked at the para-position (see FIG. 2) and, therefore, are less
branched than they generally would be as a result of some other
polymerization method.
[0032] In one embodiment, the invention is a composition of matter
that includes a polynucleotide template and a polyaniline bound
together as a complex. A method of the invention includes preparing
a polynucleotide/polyaniline complex by combining an aniline
monomer, a polynucleotide template and a redox enzyme. The monomer
aligns along the template to form a complex and polymerizes to form
polyaniline, thereby forming the polynucleotide/polyaniline
complex.
[0033] Typically, enzymatic template guided polymerization
reactions of the invention are carried out in water. However, other
solvents can be include, for example, dimethyl formamide, dimethyl
sulfoxide, methanol, ethanol, dioxane, etc. The pH of the solvent
is adjusted to a pH in a range of between about 4.0 and about 10.0.
Preferably, the pH is between about 4.0 and about 5.0 for aniline
monomer. Examples of suitable buffers include Tris-HCl buffer,
sodium phosphate, sodium citrate, etc. When the template used is a
polynucleotide, the preferable buffer is sodium citrate.
[0034] A suitable redox enzyme is added to the reaction mixture.
The concentration of enzyme in the reaction mixture is sufficient
to significantly increase the polymerization rate of the monomer in
the reaction solution. Typically, the concentration of enzyme in
the reaction mixture is in a range of between about one unit/ml and
about five units/ml where one unit will form 1.0 mg purpurogallin
from pyrogallol in 20 seconds at pH 6.0 at 20.degree. C. Examples
of suitable enzymes include peroxidases, laccase, etc. Preferred
enzymes are peroxidases. A particularly preferred enzyme is
horseradish peroxidase.
[0035] Monomers which are suitable for the template polymerization
reaction are monomers which can be polymerized by enzymatic, redox
polymerization to form a conductive and/or optical active polymer.
Such monomers are defined herein as "redox monomers". Examples of
suitable redox monomers include substituted or unsubstituted
anilines and substituted or unsubstituted phenols. Therefore, the
polyaniline or polyphenol formed can be substituted or
unsubstituted. The monomer can be a neutral compound, a cation or
an anion. Further, the monomer can be, for example, a dye, such as
an azo compound, or a ligand. Alternatively, an oligomer can be
employed rather than a monomer. Mixtures of different monomers,
oligomers, or of monomers and oligomers, can also be employed. In
one embodiment, oligomers can form from the monomer prior to
association or complexation with a template. The concentration of
monomer in the reaction mixture generally is in a range of between
about 0.05 mM and about 100 mM.
[0036] A polynucleotide template preferably is present in an amount
that at least causes a portion of the aniline monomer present to
bind to the polynucleotide template and that causes at least a
portion of the monomer to polymerize while bound to the template. A
"polynucleotide template," as that term is employed herein, is
defined as a nucleotide polymer or oligomer that can bind, such as
by ionic binding, a redox monomer before and during polymerization
of the monomer, whereby the monomer polymerizes. When aniline is
the redox monomer, the template facilitates coupling of the aniline
monomers predominately at the para position of the aromatic ring.
It is believed that binding of aniline monomers can affect
polymerization of adjacent monomers along the polynucleotide
template, thereby controlling polymerization, and that the
negatively charged polynucleotide backbone forms electrostatic
bonds with aniline monomers, thus causing them to align along the
backbone of the polynucleotide template. When the redox monomers
are polymerized a polynucleotide/conductive polymer complex is
formed. When aniline monomers are polymerized in the presence of a
polynucleotide template a polynucleotide/polyaniline complex is
formed.
[0037] In alternative embodiments, other polymers or oligomers can
be employed as templates for polymerization. For example,
sulfonated polystyrene, sulfonated polystyrene polyion salts,
polypeptides, proteins, biological receptors, zeolites, caged
compounds, azopolymers, and vinyl polymers, such as polyvinyl
benzoic acid, polystyrene sulfonic acid and polyvinyl phosphonates,
poly(vinyl phosphonic acid), etc can be suitable templates. The
template can be an anion or cation, such as a polyanion or a
polycation. Further, the template can be an optically active
polyelectrolyte, for example, azo polymers. The template can also
be a dendrimer or a compound that forms micelles, for example,
dodecyl benzene sulfonic acid. The monomer or oligomer associates
with the template to form, for example, a complex. After
polymerization, the complex can be electrically or optically
active.
[0038] The polymerization reaction is a redox reaction and
typically is initiated by adding a suitable oxidant, such as a
hydrogen peroxide solution, etc. In one embodiment, the hydrogen
peroxide has a concentration in the polymerization solution in a
range of between about one millimolar and about five millimolar. To
avoid or minimize denaturation of the enzyme, a dilute solution of
hydrogen peroxide can be prepared from a 30% stock solution and
added slowly to the reaction with stirring. Preferably, the dilute
solution of hydrogen peroxide is about 0.1 M to about 0.001 M.
Typically, the reaction mixture is maintained at a temperature in a
range of between about 10.degree. C. and about 25.degree. C. during
polymerization. More preferably, the temperature of the reaction
mixture is maintained at a temperature of about 20.degree. C.
during polymerization.
[0039] The resulting polymer can be, for example, a linear polymer,
such as an extended linear polymer intertwined with the template.
Alternatively, the polymer can be dendritic, or branched. In any
case, the polymer can have a conformation that would not be
produced in the absence of the template.
[0040] In one embodiment, the polymer can be polyaniline complexed
with a polynucleotide template, wherein the polyaniline is an
extended linear, helical or branched polymer intertwined with the
polynucleotide template. In a specific embodiment, the polyaniline
is a component of a water soluble electrically conducting
complex.
[0041] Optionally, the method of the invention includes forming a
layer of the polymer on a surface. In this embodiment, the pH of
the polymer solution is reduced to a suitable pH, such as a pH in a
range of between about 2.0 and about 8.0, by adding a suitable
acid, such as hydrochloric acid, etc. A suitable surface, such as a
glass slide treated with an alkali, such as Chemsolv.RTM. alkali,
is immersed in a polymer solution for a sufficient period of time
to cause the polymer to accumulate at the surface. In one
embodiment, a glass slide is immersed in a polymer solution for
about ten minutes and then removed. The surface can then be washed
with water at a pH of about 2.5 in order to remove unbound polymer
from the surface.
[0042] Distinct layers of polymers can be applied to a surface by
this method. For example, an initial layer can be formed by
exposing a suitable surface to a polymer formed by the method of
the invention that is a polyanion and then subsequently exposing
the same surface, having the polyanion deposited upon it, into a
solution of a polycation. In one specific embodiment, a glass slide
treated with Chemsolv.RTM. alkali is exposed to a one
milligram/milliliter solution of poly(diallyl dimethyl ammonium
chloride) at a pH of 2.5 as a polycation, and then exposed to a one
milligram/milliliter solution of SPS/polyaniline formed by the
method of the invention, as a polyanion. A bilayer of polymers is
thereby formed. Additional layers of these or other polymers can
subsequently be applied.
[0043] Polymerization of the template can be initiated
simultaneously with, or subsequent to alignment and polymerization
of the bound monomer or oligomer. In one embodiment, the template
can be removed from the resulting polymer, such as by
decomposition, dissolution, or enzymatic degradation to leave
behind a polymer shell.
[0044] In one specific embodiment of the method of the invention,
the template-assisted enzymatic polymerization of aniline can be
carried out in an aqueous solution using 0.1M sodium phosphate or
tris-HCl buffer and a pH ranging from about 4.0 to about 10.0.
Aniline monomer typically can be added in a range of between about
10 mM and about 1001M, and an appropriate amount of a template, in
this case sulphonated polystyrene (SPS) (molecular weight of
70,000), can be added in ratios ranging from about 1:10 to about
10:1 SPS/aniline. The enzyme horseradish peroxidase then can be
added to the reaction mixture in a range of about one unit/ml to
about five units/ml. To initiate the reaction, an oxidizer, such as
a 30% solution of hydrogen peroxide, can be added slowly in 10
.mu.l increments over a reaction time of 3 hours, with constant
stirring to a final concentration ranging from about 10 mM to about
100 mM.
[0045] In another specific embodiment of the invention, the
template guided polymerization of aniline can be carried out using
a polynucleotide template. The polynucleotide is dissolved in an
aqueous buffer, such as a 10 mM sodium citrate buffer, having a pH
of about 4 to about 10. Preferably, the buffer pH is about 4 to
about 5. The concentration of the polynucleotide can be determined
by UV absorption using the molar extinction coefficient of the
polynucleotide at a particular wavelength. This gives the
concentration of nucleotide bases which make up the polynucleotide.
The molar extinction concentration of a polynucleotide in the
polymerization reaction is typically about 1 mM to about 5 mM.
Aniline monomer is added to the polynucleotide solution in a
concentration such that the ratio of aniline to nucleotide bases in
the polynucleotide is about 1:10 to about 10:1, more preferably
about 1:1. Horseradish peroxidase (HRP) is added to the solution to
a concentration of about 1 unit/mL to about 5 units/mL, then an
oxidizer, such as hydrogen peroxide, is added slowly to the
reaction mixture to initiate the reaction. The amount of hydrogen
peroxide added is about one-fifth equivalent to about 1.0
equivalents of the aniline monomer in the reaction mixture. The
reaction time is typically about 80 min.
[0046] In yet another specific embodiment of the method of the
invention, the template-assisted enzymatic polymerization of phenol
can be carried out in an aqueous solution using 0.1M sodium
phosphate or tris-HCl buffer and pH ranging from 4.0 to 10.0.
Phenol monomer typically can be added in a range of between about
10 mM and about 100 mM and an appropriate amount of the template,
sulphonated polystyrene (molecular weight of 70,000), can be added
in ratios ranging from about 1:10 to about 10:1 SPS/phenol. The
enzyme horseradish peroxidase then can be added to the reaction
mixture in a range of about one unit/ml to about five units/ml. To
initiate the reaction, an oxidizer, such as a 30% solution of
hydrogen peroxide, can be added slowly in about 10 .mu.l increments
over a reaction time of about 3 hours with constant stirring to a
final concentration ranging from about 10 mM to about 100 nM.
[0047] It is to be understood that polymers formed by the method of
the invention can be formed in an oxidized, electrically conducting
form or in a reduced, insulating form of the polymer (see FIG. 2).
Other physical properties of the polymers that can be affected by
the method of the invention include the molecular weight and shape
of the polymer. It is also to be understood that the polymers
formed by the method of the invention can be modified after
polymerization. For example, modification can be made at amine
functional groups to form amides or imine groups.
[0048] Dissolved polymers formed by the method of the invention can
be precipitated from solution by adjusting the pH with a suitable
acid or base. Examples of suitable acids or bases include
hydrochloric acid, sodium hydroxide, etc.
[0049] In a preferred embodiment, a polynucleotide duplex can be
used as a template for polymerization of polyaniline.
Polynucleotide duplexes have a handedness and, therefore, may
impose a chirality on the polyaniline to which they are complexed.
Alternatively, the polyaniline complexed to a polynucleotide duplex
may be achiral.
[0050] The conformation of the polynucleotide duplex complexed to a
conductive polymer can be controlled by controlling the degree of
oxidation of the conductive polymer. In general, the more positive
charges the backbone of the conductive polymer carries, the more
tightly wound the double helix will be. For example, the
conformation of the polynucleotide duplex in a
polynucleotide/polyaniline complex can be controlled by controlling
the degree of oxidation of the polyaniline. When polyaniline is in
the conducting, or oxidized, form, where the polyaniline is
protonated (see FIG. 2), the polynucleotide duplex is more tightly
wound (e.g., has more base pairs per helical repeat) than when
polyaniline is in the insulating, or reduced form, where the
polyaniline is neutral. Polyaniline can be converted from the
insulating form to the conducting form by adding protons to (or
subtracting electrons from) the polyaniline backbone. This
oxidation process is called "doping" the polyaniline. Conversely,
the polyaniline can be dedoped, or reduced, by subtracting protons
from (or adding electrons to) the polyaniline backbone. When a
polynucleotide/polyaniline complex is in solution, it can be doped
(oxidized) by decreasing the pH of the solution, or dedoped
(reduced) by increasing the pH of the solution.
[0051] Alternatively, the polyaniline in the complex can be doped
or dedoped electrochemically using, for example, a
potentiostat/galvanostat set-up. The potentiostat/galvanostat
set-up can have, for example, a three-electrode cell with platinum
wire as the working electrode, Ag/AgCl as the reference electrode
and platinum mesh as the counter-electrode. The
polynucleotide/polyaniline complex can be contained in an
electrolyte solution, such as a sodium citrate buffer having about
0.1 M ammonium chloride in contact with the working electrode. The
doping and dedoping process may be observed by cycling the
potential between about -0.02 V and 0.8 V with respect to the
Ag/AgCl electrode.
[0052] Therefore, the conformation of the polynucleotide duplex in
a polynucleotide/polyaniline complex can be modulated by changing
the oxidation state of the polyaniline. After oxidation or
reduction of the polyaniline, the polynucleotide duplex can recover
its original conformation, or substantially the same conformation
as its original conformation, by returning the polyaniline to its
original, or near its original oxidation state. A polynucleotide
duplex has substantially the same conformation if the conformation,
as determined by circular dichroism (hereinafter "CD"), is at least
about 75% the same.
[0053] Another embodiment of the invention is an electrical
component. The electrical component includes an electrical element,
such as, a voltage source, a resistor, a capacitor, an inductor, a
diode, a switch or a transistor, which is attached to one or more
nanowires. A nanowire, as that term is employed herein, includes a
polynucleotide and a conductive polymer, such as polyaniline, bound
together as a complex.
[0054] The polynucleotide in the nanowire can be a single strand, a
double helix or a portion of the polynucleotide can be a single
strand and a portion of the polynucleotide can be a double helix.
In addition, the polynucleotide can be a deoxyribonucleotide, a
ribonucleotide, a polynucleotide analog, a modified polynucleotide
or an oligonucleotide. Also encompassed within the invention are
polynucleotides that are a combination of deoxyribonucleotide, a
ribonucleotide, a polynucleotide analog, a modified polynucleotide
and an oligonucleotide.
[0055] An electrical element can be connected to one or more other
electrical elements by a nanowire. The electrical elements can be
connected to each other in a closed electrical path, or a path that
can be closed by an electrical switching element, to form a
circuit. When the nanowire is used to connect two or more
electrical elements, the nanowire can be self-assembled by
hybridization. In this embodiment, each electrical element has one
or more polynucleotides attached to it, a portion of which can
hybridize to a portion of a polynucleotide on a different
electrical element. Alternatively, a polynucleotide connector which
can hybridize a portion of the sequence of two or more
polynucleotides that are attached to different electrical elements
can be use to connect the two elements. The polynucleotide
connector can be one or more polynucleotides. When the connector is
composed of more than one polynucleotide, at least a portion of
each polynucleotide that makes up the connector is hybridized to
one or more other polynucleotides in the connector.
[0056] The polynucleotide is attached to a surface of the
electrical element by derivatizing the polynucleotide with a group
that can bind to the surface. Therefore, selection of a functional
group with which the polynucleotide is to be derivatized is
dependent on the type of material to which the polynucleotide is to
be attached. When the polynucleotide is to be attached to a surface
on an electrical element which is gold, silver, copper, cadmium,
zinc, palladium, platinum, mercury, lead, iron, chromium,
manganese, tungsten, or any alloys of the above metals, the
polynucleotide to be attached is preferably derivatized with a
thiol, sulfide or disulfide group. When the surface to which the
polynucleotide is to be attached is doped or undoped silica,
alumina, quartz or glass, the polynucleotide is preferably
derivatized with a carboxylic acid. When the surface to which the
polynucleotide is to be attached is platinum or palladium, the
polynucleotide is preferably derivatized with a nitrile or
isonitrile group. Finally, when the surface to which the
polynucleotide is to be attached is copper, the polynucleotide is
preferably derivatized with a hydroxamic acid group.
[0057] The invention also relates to a method of forming an
electrically conductive connection between one or more electrical
elements. The electrically conductive connection is formed by
connecting two or more electrical elements with a polynucleotide.
The polynucleotide is contacted with a redox monomer, such as an
aniline monomer, and a redox enzyme, whereby the redox monomer
aligns along the polynucleotide and is polymerized. The
polynucleotide/conductive polymer complex (nanowire) formed
connects the electrical elements and is electrically
conductive.
[0058] The polynucleotide connecting two or more electrical
elements can self-assemble by hybridization. In this embodiment,
the entire polynucleotide, or a portion thereof, that is attached
to an electrical element, hybridizes to a complementary, or
substantially complementary, polynucleotide, or a portion of a
polynucleotide, attached to a different electrical element. The
specificity of hybridization allows specific connections between
electrical elements to be predetermined by determining the sequence
of the polynucleotide attached to each electrical element. The
polynucleotides hybridize forming the predetermined connections
when they are combined in a solution having the appropriate
conditions of temperature and chemical composition.
[0059] In another embodiment, a polynucleotide attached to an
electrical element can be enzymatically ligated to a polynucleotide
attached to another element. Ligases are enzymes which repair
damaged DNA. An example of a suitable ligase is T4 DNA ligase. In
this embodiment, the polynucleotides attached to each electrical
element are double helixes which preferable have at least one
cohesive, or sticky end, on the end of the duplex which is not
attached to the electrical element. The term "cohesive end" refers
to a single stranded polynucleotide which occurs at the terminal
end of a double helix. The cohesive end is typically from three to
twenty, preferably three to eight, bases long and can hybridize to
a complementary cohesive end. Once two complementary cohesive ends
have hybridized a ligase can form covalent bonds between the two
duplexes.
[0060] The term "self-assembled polynucleotide" refers to either
specific hybridization between polynucleotides attached to two
different electrical elements or to hybridization of two cohesive
ends followed by ligation. A nanowire formed using a self-assembled
polynucleotide template is a self-assembled nanowire. Self-assembly
of nanowires to form specific connection between electrical
elements is expected to facilitate the construction of
nanometer-scale devices.
[0061] A redox monomer and a redox enzyme are added to the solution
containing the electrical elements having attached polynucleotide
templates either simultaneously with the electrical elements or,
preferably, after the polynucleotides attached to the electrical
elements have hybridized. The redox monomers, for example, aniline
monomers align along the hybridized polynucleotides to form a
complex and are polymerized, thereby connecting the electrical
elements with a nanowire having a conductive and/or optically
active polymer complexed to a polynucleotide.
[0062] Another embodiment of the invention is a method of
identifying a target polynucleotide by contacting the target
polynucleotide with a probe which includes a
polynucleotide/conductive polymer complex that can bind to a target
polynucleotide by hybridization. Hybridization of the probe to the
target polynucleotide modifies at least one electromagnetic
property of the conductive polymer. Preferably, substituted or
unsubstituted polyaniline is the conductive polymer. The probe can
bind by hybridization to a target polynucleotide which is
complementary, or substantially complementary, to the sequence, or
a portion of the sequence, of the probe polynucleotide. The
electromagnetic property of the conductive polymer which is
modified is an optical and/or electrical property. The change in
optical and/or electrical properties of the conductive polymer
during or after hybridization with the target can be used in
discriminating between perfectly complementary targets and targets
that have one or more mismatches. In another embodiment, the
conductive polymer can be complexed to the target polynucleotide,
as well as the probe. In this embodiment, the conductive polymer is
enzymatically polymerized on the target polynucleotic. The modified
electromagnetic property can be detected by a combination of
characterization methods that may include, but are not limited to,
UV-visible absorption, circular dichroism and cyclic voltammetry.
These characterization methods can be employed to estimate the
extent of hybridization. Discrimination between targets which are
perfectly complementary to the probe and those which have one or
more mismatches may also be discerned by monitoring the optical
and/or electrical properties during thermal melting of the
hybridization complex of the probe and the target. In addition, the
polynucleotide/conductive polymer complex of the probe may be may
be attached to an electrical element.
[0063] A "polynucleotide" as used herein refers to single, double
and triple stranded polynucleotides, as well as, quadruplexes. The
polynucleotide in a polynucleotide/polyaniline complex can be a
deoxyribonucleotides, ribonucleotides (hereinafter, "RNA"),
modified polynucleotides, and polynucleotide analogs such as
peptide nucleic acid (hereinafter, "PNA") and morpholino nucleic
acids. In addition, a polynucleotide can be composed of more than
one polynucleotide molecule. For example, a polynucleotide duplex
can be composed of two polynucleotides of the same type (e.g., both
ribonucleotides or both deoxyribonucleotides), or it can be
composed of a mixture of different types of polynucleotides (e.g.,
a combination of a ribonucleotide and a deoxyribonucleotide).
[0064] Complementary binding, or hybridization, is generally
understood to occur in an antiparallel manner, however, there are
occasions in which hybridization can occur in a parallel fashion,
such as in a triple helix, and this arrangement is also within the
scope of the present invention. Hybridization is understood to
essentially follow a complementary pattern wherein a purine pairs
with a pyrimidine via hydrogen bonds. More particularly, it is
understood that when hybridization occurs, complementary
base-pairing of individual base pairs generally follows Chargaff's
Rule wherein an adenine pairs with a thymine (or uracil) and
guanine pairs with cytosine. However, hybridization can occur
between less than perfectly complementary sequences provided a
stable binding complex is formed. The stability of a binding
complex is dependent on ionic strength, temperature and the
concentration of destabilizing agents such as urea and formamide in
the hybridization medium, as well as, on factors such as the length
of the polynucleotide sequence, base composition, and percent
mismatch between hybridizing sequences.
[0065] In addition, modified bases can account for unconventional
base-pairing. A modified polynucleotide is understood to mean
herein a DNA or RNA polynucleotide that contains chemically
modified nucleotides. The term "polynucleotide analogue" is
understood herein to denote non-nucleic acid molecules such as PNA
(see Egholm, et al., J. Am. Chem. Soc. (1992), 114: 1895, the
teachings of which are incorporated herein by reference in their
entirety) and morpholino antisense oligomers (see Summerton and
Weller, Antisense and Nucleic Acid Drug Dev. (1997), 7: 187, the
teachings of which are incorporated herein by reference in their
entirety) that can engage in base-pairing interactions with
conventional nucleic acids. These modified bases and polynucleotide
analogues are considered to be within the scope of the instant
invention. For example, polynucleotides containing deazaguaine and
uracil bases can be used in place of guanine and thymine,
respectively, to decrease the thermal stability of hybridized
complex. Similarly, 5-methylcytosine can be substituted for
cytosine in hybrids if increased thermal stability is desired.
Modification to the sugar moiety can also occur and is embraced by
the present invention. For example, modification to the ribose
sugar moiety through the addition of 2'-O-methyl groups which can
be used to reduce the nuclease susceptibility of RNA molecules.
Modifications occurring with different moieties of the nucleic acid
backbone are also within the scope of this invention. For example,
the use of methyl phosphate or methyl phosphonate linkages to
remove negative charges from the phosphodiester backbone can be
used.
[0066] Polynucleotides can bind to each other to form specific
binding complexes by complementary base-pairing interactions
between the polynucleotides. Possible base-pairing interactions
useful in the method include duplexes that have canonical
Watson-Crick base-pairing (reviewed in Cantor and Schimmel,
Biophysical Chemistry, Part I. The Conformation of Biological
Macromolecules, Ch. 3 and 6, Freeman, San Francisco, 1980, the
teachings of which are incorporated herein by reference in their
entirety) or noncanonical base-pairing schemes such as triple helix
formation (Felsenfeld, Davies, and Rich, J. Am. Chem. Soc. (1957)
79: 2023; for review see Doronina and Behr, Chem. Soc. Rev. (1997),
p. 63-71, the teachings of which are incorporated herein by
reference in their entirety) and quadruplex formation (Sen and
Gilbert, Nature (1990) 344: 410-414; Sen and Gilbert, Methods
Enzymol. (1992) 211: 191-9, the teachings of which are incorporated
herein by reference in their entirety). Methods for determining the
thermal stability of a particular hybridization complex are well
known in the literature (see Wetmur, Critical Reviews in
Biochemistry and Molecular Biology, 26: 227-259 (1991); Quartin and
Wetmur, Biochemistry, 28: 1040-1047 (1989), the teachings of which
are incorporated herein by reference in their entirety).
[0067] The invention will now be further and more specifically
described by the following examples. All parts and percentages are
by weight unless otherwise specified.
EXAMPLES
Example 1
[0068] A. Materials and Methods
[0069] Horseradish peroxidase (HRP) (enzyme classification number
(EC) 1.11.1.7), phosphate and Tris-HCl buffers were obtained from
Sigma Chemicals Company, St. Louis, Mo. Aniline, sulfonated
polystyrene (SPS) and hydrogen peroxide (30%) were obtained from
Aldrich Chemicals, Inc., Milwaukee, Wis. All the chemicals were
used as received.
[0070] B. Results and Discussion
[0071] The progress of a template-assisted polymerization reaction
of aniline in the presence of the polyelectrolyte, sulfonated
polystyrene (SPS) in a 1:1 ratio, was monitored by the change in
visible absorbance. A Perkin-Elmer Lambda-9.RTM. UV-Vis-near IR
spectrophotometer was used for the spectral characterization of the
polymer. FIG. 3 shows the visible absorption spectra of the
sulfonated polystyrene/polyaniline (SPS/PA) complex prepared under
various pH conditions of 4, 6, 8, and 10. As shown in FIG. 3,
SPS/PA, prepared at a pH of 4, exhibited a strong absorbance
maximum at approximately 780 nm. This was indicative of the
emeraldine, or oxidized, electrically conducting form of
polyaniline. Polymerization at higher pH resulted in an absorption
maximum of about 600 nm, indicating a more insulating form of
polyaniline. In all cases, the polymer complex did not precipitate
out of solution, indicating that complexation of the polyaniline to
the SPS had occurred.
[0072] Optimization of the molar ratio of monomer to
polyelectrolyte template (repeat unit) was carried out. FIG. 4
shows a plot of absorption maxima for various SPS/aniline ratios.
As shown, a ratio of 1:2, SPS/aniline was the minimum ratio
required to obtain the electrically conducting form of polyaniline,
which had an absorption maximum at approximately 780 nm at a pH in
a range of between about 4 and about 5.
[0073] The reversible reduction/oxidation (redox) behavior of the
SPS/PA complex was monitored by measuring visible absorption of the
complex under various pH conditions. In all cases the polymer
complex was prepared at pH 4.0 to obtain the electrically active
form of the polyaniline and then the pH of the solution was
adjusted for the absorption maxima measurements. As shown in FIG.
5A, the SPS/PA complex shifted in absorption maxima to shorter
wavelengths as the pH of the solution was increased. This was
indicative of reduction of the polyaniline backbone to a more
insulating state. FIG. 5B shows the reverse behavior where the
absorption maximum was found to shift back to longer wavelengths
with decreasing pH conditions. This was indicative of oxidation of
the polyaniline backbone back to a more electrically conductive
state. This reversible redox behavior was repeatable and confirms
that an electrically active form of polyaniline was present in the
final SPS/PA template complex. Molecular weight determination was
carried out by column chromatography using Protein PAK 300
SW.RTM.-Waters Association columns. Molecular weights of
approximately 74,000 Daltons were measured indicating
polymerization of the aniline and complexation to the SPS
template.
[0074] C. Thin Films by Layer-by-Layer Technique
[0075] Self-assembly of the SPS/PA complex onto glass slides was
carried out by the layer-by-layer electrostatic deposition
technique (Ferreira, M., et al., Thin Solid Films (1995), 244: 806
and Decher, G., et al., Thin Solid Film, (1992), 210-211, the
teachings of which are incorporated herein by reference in their
entirety). A glass slide treated with alkali (Chemsolv.RTM.
alkaline) was exposed to polycation and polyanion solutions
repeatedly to transfer monolayers of these polyelectrolytes per
every exposure. 1 mg/ml solution of poly(diallyl dimethyl ammonium
chloride) (PDAC) at pH 2.5 was used as the polycation while
approximately 1 mg/ml solution of SPS/PA at pH 2.5 was used as the
polyanion. The glass slide was exposed to each polyelectrolyte
solution for 10 minutes and washed with water at the same pH to
remove the unbound polymer from the surface. This process was
repeated to obtain the desired number of layers.
[0076] FIGS. 6a and 6b show the visible absorption spectra of a
film of fifty bilayers wherein PDAC layers alternate with SPS/PA
layers, under various pH conditions. As shown in the figures, the
multilayer film exhibited similar redox behavior as was observed
previously with the solution absorption spectra. This confirmed
that facile electrostatic deposition was feasible with the SPS/PA
polymer complex and that the electrical activity was maintained
after deposition. In addition, multilayer and bulk films were
prepared on indium tin oxide (ITO) slides and four-point probe
conductivity measurements were taken. The results gave
polymer-complex conductivities in the range of 10.sup.-3 to
10.sup.2 S/cm.
Example 2
[0077] A. Materials and Methods
[0078] Horseradish peroxidase (HRP) (enzyme classification number
(EC) 1.11.1.7), phosphate and Tris-HCl buffers were obtained from
Sigma Chemicals Company, St. Louis, Mo. Phenol, sulfonated
polystyrene (SPS) and hydrogen peroxide (30%) were obtained from
Aldrich Chemicals, Inc., Milwaukee, Wis. All the chemicals were
used as received.
[0079] B. Results and Discussion
[0080] The progress of a template-assisted polymerization reaction
of phenol in the presence of the polyelectrolyte, sulfonated
polystyrene (SPS) in a 1:1 ratio, was monitored by the change in
visible absorbance. Perkin-Elmer Lambda-9.RTM. UV-Vis-near IR
spectrophotometer was used for the spectral characterization of the
polymer. FIG. 7A shows the visible absorption of polyphenol without
SPS, versus phenol monomer. As shown, there was a significant
absorption maximum in the visible spectrum upon polymerization,
indicating formation of polyphenol. However, with time the polymer
began to precipitate out of solution. FIG. 7B shows the visible
absorption of polyphenol with SPS, versus phenol monomer. As shown
again, there was a significant absorption maximum of the
polymerized system in the visible spectrum. In this case, there was
no observed precipitation of the polymer complex out of
solution.
[0081] Molecular weight determination was carried out by column
chromatography using Protein PAK 300 SW.RTM. columns manufactured
by Waters Association. Molecular weights as large as 136,000
Daltons were measured, indicating polymerization of the phenol and
complexation to the SPS template.
Example 3
[0082] A. Preparation of DNA-Polyaniline Complex
[0083] Horseradish peroxidase (HRP, EC 1.11.1.7) type II, (150-200
units/mg) solid was purchased from Sigma Chemical Co. (St. Louis,
Mo.). Calf Thymus DNA was purchased from Worthington Biochemical
Corporation (Freehold, N. J.). Aniline monomer (purity 99.5%) and
hydrogen peroxide (30% by weight) were purchased from Aldrich
Chemicals, Inc., Milwaukee, WI, and were used as received. All
other chemicals were of reagent grade or better. All glassware and
plasticware were sterilized by autoclaving for 30 minutes.
[0084] A stock solution of calf Thymus DNA (19.9 mg) was dissolved
in 40 ml of sterile, 10 mM sodium citrate buffer maintained at pH
4. The solution was stored in the refrigerator for 48 hours before
reaction. The concentration of DNA was determined by the UV
absorbance at 258 nm. The molar extinction coefficient (O) at 258
nm was taken as 6420 1 mol.sup.-1 cm.sup.-1, as reported by
Sprecher, et al., Biopolymers (1979), 18: 1009. The reaction
mixture had 10 ml of DNA stock solution, aniline in an amount
equivalent to twice the molar concentration of DNA present in 10 ml
and catalytic amount of HRP (0.15 mg). The polymerization was
carried out by the dropwise addition of hydrogen peroxide (0.098
M), over a period of 240 seconds. The total amount of hydrogen
peroxide was limited to 1/5.sup.th of the stoichiometric amount,
calculated with respect to aniline concentration. For synthesis in
bulk, the polymerization was carried to completion and a
stoichiometric amount of hydrogen peroxide was added. The
DNA-polyaniline complex precipitates out from the mixture. The
precipitate was washed several times using 1:1 mixture of acetone
and pH 4 water in order to remove residual aniline and low
molecular weight polyaniline. The gravimetric yield was 75%.
Results of elemental analysis of the DNA-polyaniline complex
indicated C (46.8%), H (4.4%), P (5.45%). This indicated a ratio of
2.5:1 for DNA to aniline in the complex. The theoretically values
calculated based on this ratio are C (43.2%), H (5.0%), N (13.1%),
P (4.8%).
[0085] UV-Vis spectra and circular dichroism (CD) spectra were
obtained simultaneously using Hewlett-Packard diode array detector
photometer (type HP8452A) and Jasco CD spectrometer J-720,
respectively. The elemental analysis was performed by Schwarzkopf
Microanalytical Laboratory, Woodside, N. Y.
[0086] B. Results and Discussion
[0087] At pH 4, the aniline molecules are protonated, and the
electrostatic attraction between protonated aniline and the
phosphate groups of the DNA helps in aligning the monomer on DNA
(FIG. 8). The alignment of the monomer on the DNA promotes
para-directed coupling of aniline molecules during polymerization
(FIG. 2). The phosphate groups in the DNA matrix provide a
proton-rich environment that helps in accomplishing polymerization
of aniline at a significantly higher pH condition than is possible
in the absence of the DNA template. The polymerization is catalyzed
by HRP, and the polyaniline formed remains bound through ionic
interactions to the DNA.
[0088] C. Formation of Polyaniline on DNA (UV-Vis Spectra)
[0089] The UV-Vis spectra of DNA, DNA with aniline and HRP before
and during polymerization are shown in FIG. 9. The addition of
aniline and HRP increased the absorption in the 200-280 nm range,
while the absorption in the visible region remained constant.
UV-Vis spectra were recorded 5 minutes after the addition of
hydrogen peroxide and subsequently after 20, 40, 60 and 80 minutes.
The UV-Vis spectrum obtained after 5 minutes indicated absorption
bands centered around 420 nm polaron bands and 750 nm bipolaron
band. As time proceeded, the bipolaron band at 750 nm diminished
while the 420 nm and 310-320 nm bands increased in intensity. The
750 mm band have been attributed to presence of pernigraniline
(quinoid form) of polyaniline. The pernigraniline formed in the
initial stages of the reaction was reduced to emeraldine salt by
the addition of aniline to growing polymer chain. This change was
also accompanied by an increase in absorbance in the region of 1000
nm. After 80 minutes, the solution turned completely green, and the
absorption spectra indicated the presence of polyaniline in the
emeraldine salt form, which provided further evidence for the
presence of polyaniline in the oxidized state. The DNA, thus
provides the counter-ion and acts as a dopant for the
polyaniline.
[0090] D. Change in CD Spectra During Polymerization
[0091] The CD spectra of Calf thymus DNA at pH 4, shown in FIG. 10,
compared well with the already reported spectra, of DNA polymorph
`B`. The spectrum did not change with the addition of aniline and
horseradish peroxidase. Very significant changes in the CD spectra
were noticed after 5 minutes, subsequent to the addition of
hydrogen peroxide. The 220 nm positive peak increased in intensity,
while the 245 nm negative peak reduced in intensity. The positive
(.DELTA..epsilon.) shoulder at 270 nm changes to a new negative
peak with fine structure. The positive peak at 275 nm reduced in
intensity significantly. The CD spectra in the visible region
showed the appearance of broad bands centered at 367 nm and 444
nm.
[0092] The CD spectra were measured until 80 minutes after the
addition of hydrogen peroxide. A comparison of the spectra of
DNA-polyaniline obtained at 5 minutes and 80 minutes indicated very
little changes in the 200-320 nm region. It was concluded that
changes in the secondary structure of DNA occurred earlier than 5
minutes after the addition of hydrogen peroxide. However, the
positive, broad bands centered around 367 nm and 444 nm increased
in intensity steadily over time.
[0093] At pH 4, the polyaniline to remain charged and the phosphate
groups in DNA provided the counterion for maintaining charge
neutrality. The shielding of phosphate groups by polyaniline
induced a change in the secondary structure of DNA leading to the
formation of the over-wound polymorph. On comparison with the
earlier reports pertaining to change in secondary structure of Calf
thymus DNA induced by the nature of solvent and concentration of
salt (see Sprecher, et al., Biopolymers (1979), 18: 1009; Bokma, et
al., Biopolymers (1987), 26: 893, the teachings of which are
incorporated herein by reference in their entirety), it was
concluded that the formation of polyaniline caused a change similar
to a `B` to `C` polymorphic transition. As a control, a DNA
solution of same concentration was treated with 6 molar ammonium
fluoride. The shape of UV region of CD spectra of the DNA solution
containing 6 molar ammonium fluoride resembled that of
DNA-polyaniline (FIG. 11). However, in the case of DNA-polyaniline,
the concentration of aniline used in the reaction was limited to a
few millimoles. Yet the changes in the CD spectrum were
significant. This confirmed the formation of a polyelectrolyte
complex of DNA-polyaniline.
[0094] The visible region of the CD spectrum provided interesting
information on the secondary structure of the polyaniline. The
increase of the 367 nm and 444 nm CD bands until 80 minutes
indicated the development of chirality in the polyaniline
concomitant with the increase of molecular weight.
Chirality/optically activity has been observed in chemically
(Majidi, M. R., et al., Polymer (1995), 36: 3597, the teachings of
which are incorporated herein by reference in their entirety) and
electrochemically (Majidi, M. R., et al., Polymer (1994), 35: 3113,
the teachings of which are incorporated herein by reference in
their entirety) synthesized complex and colloids (Barisci, J. N.,
et al., Synth. Met. (1997), 84: 181, the teachings of which are
incorporated herein by reference in their entirety) of polyaniline
and (IR)-(-)10-Camphorsulfonic acid. The observed macroasymmetry of
the polyaniline salts formed in the presence of (+) or
(-)-camphorsulfonic acid has been rationalized in terms of the
polyaniline chain adopting a preferred one-sense helical screw
maintained by the dopant anions via electrostatic and H-bonding. It
is therefore probable that the electrostatic interactions between
the DNA double helix and polyaniline, induced a macroasymmetry in
the polyaniline.
[0095] The enzyme catalyzed synthesis described here, can be
extended to the polymerization of functional monomers (substituted
phenols/anilines) with interesting optical and electrical
properties. This method can also be extended to other ionic
biological polyelectrolytes such as collagen. Chiral organization
of polyaniline around DNA may enhancement in the electrical
conductivity of polyaniline. In addition, the chirality and
electrical properties of polyaniline combined with the selectivity
of DNA may be useful in the design of highly specific
biosensors.
Example 4
[0096] A. Changes in CD Spectra During Oxidation or Reduction of
Polyaniline
[0097] The secondary structure of DNA was readily controlled by the
changing the extent of oxidation of polyaniline. The pH of the
DNA-polyaniline solution was changed from 4-10 by adding 1 M NaOH
and the CD spectra was obtained (FIG. 12A). It was evident that the
CD spectra of DNA changed rapidly, and at pH 6, the DNA reverted
back to its loosely wound state (`B` form). The neutralization of
polyaniline minimized the electrostatic interaction between the DNA
and polyaniline resulting in the uncoiling of DNA, back to its
native state.
[0098] Evidence that the observed conformational changes were due
to reduction of the polyaniline was obtained from the UV-Vis
spectra (FIG. 13A). A decrease and subsequent disappearance of the
polaron bands at 404 and 755 nm as the pH of the solution increased
was observed. Simultaneously, the exciton transition of the quinoid
rings at 564 nm and the .pi.-.pi.* transition of the benzenoid
rings at 320 nm emerged. The solution went through a series of
color changes, from green to blue to purple indicating the
transition of the polyaniline to the emeraldine base form. Two
isobestic points at 354 nm and 458 nm were observed. The changes
observed in the UV-Vis and the CD spectra under increasingly basic
conditions were consistent with partial unwinding of the DNA duplex
as consequence of the reduction of polyaniline.
[0099] When DNA-polyaniline was reoxidized using HCl, it was
observed that the CD spectrum approaches the original CD spectra of
oxidized DNA-polyaniline at pH 4 (FIG. 12B). The evidence for
reoxidation of polyaniline was observed in the UV-Vis spectra (FIG.
13B) as indicated by the recovery of polaron bands and the decrease
of the exciton transition band. The average recovery of the CD
bands at 290=nm, 285 nm, 275 nm and 245 nm was greater than 50%.
The same solution was reduced and reoxidized (taken to pH 10 and
back to pH 4). CD spectra obtained during this process indicated a
significantly better recovery (75%) in the DNA region. Therefore,
it was possible to reversibly change the conformation of the DNA
(over-wind and unwind) by controlling the degree of oxidation of
polyaniline.
[0100] B. Control Experiments
[0101] Control experiments were performed by mixing a molecular
complex of polystyrene sulfonic acid and polyaniline, with Calf
thymus DNA. There was no observable change in the conformation of
DNA. This experiment provides unambiguous evidence for existence of
polyaniline, closely bound to the DNA, only if the synthesis of
polyaniline is performed in the presence of DNA.
[0102] The present study has demonstrated the use of DNA as a
substrate for the synthesis of polymers with unique electrical and
optical properties. The conducting polymer (polyaniline) bound to
the DNA can be used as a "tool" to manipulate the conformation of
the DNA. In principle, the doping, dedoping and redoping process
and the conformational switching can also be accomplished
electrochemically. This can enhance the speed and ease of
doping/dedoping process. The remarkable specificity in the
recognition capabilities of DNA can be combined with the doping
dependent electrical properties of polyaniline to develop methods
for highly selective DNA detection and biosensing.
[0103] Equivalents
[0104] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *