U.S. patent application number 10/514191 was filed with the patent office on 2006-08-10 for polyelectrolyte complex(e.g.zwitterionic polythiophenes) with a receptor (e.g.polynucleotide, antibody etc.) for biosensor applications.
Invention is credited to Peter Asborg, Olle Inganas, Peter Nilsson.
Application Number | 20060175193 10/514191 |
Document ID | / |
Family ID | 20287871 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060175193 |
Kind Code |
A1 |
Inganas; Olle ; et
al. |
August 10, 2006 |
Polyelectrolyte complex(e.g.zwitterionic polythiophenes) with a
receptor (e.g.polynucleotide, antibody etc.) for biosensor
applications
Abstract
A complex between a conjugated polyelectrolyte, and one or more
receptor molecules specific for a target biomolecule analyte, the
polyelectrolyte and the receptor being non-covalently bound to each
other, is usable as a probe for biomolecular interactions. It also
relates to a method of determining selected properties of
biomolecules. Thereby, a complex as above is exposed to a target
biomolecule analyte whereby the analyte and the receptor interact,
and a change of a property of the polyelectrolyte in response to
the interaction between the receptor and the analyte is detected.
The detected change is used to determine the selected property of
the biomolecule.
Inventors: |
Inganas; Olle; (Linkoping,
SE) ; Asborg; Peter; (Linkoping, SE) ;
Nilsson; Peter; (Linkoping, SE) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET
2ND FLOOR
ARLINGTON
VA
22202
US
|
Family ID: |
20287871 |
Appl. No.: |
10/514191 |
Filed: |
May 9, 2003 |
PCT Filed: |
May 9, 2003 |
PCT NO: |
PCT/SE03/00762 |
371 Date: |
February 13, 2006 |
Current U.S.
Class: |
204/242 |
Current CPC
Class: |
G01N 33/531 20130101;
C12Q 1/6825 20130101; C12Q 1/6827 20130101; C12Q 1/6825 20130101;
G01N 33/54366 20130101; C12Q 1/6827 20130101; G01N 33/566 20130101;
C12Q 2563/125 20130101; C12Q 2565/628 20130101 |
Class at
Publication: |
204/242 |
International
Class: |
C25B 9/00 20060101
C25B009/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2002 |
SE |
0201468-6 |
Claims
1. A complex between a conjugated polyelectrolyte, and one or more
receptor molecules specific for a target biomolecule analyte, said
polyelectrolyte and said receptor being non-covalently bound to
each other, and wherein said conjugated polyelectrolyte has one or
more zwitterionic side chain functionalities, said complex being
usable as a probe for biomolecular interactions.
2. The complex as claimed in claim 1, wherein the polyelectrolyte
comprises copolymers or homopolymers of thiophene, pyrrole,
aniline, furan, phenylene, vinylene or their substituted forms.
3. The complex as claimed in claim 2, wherein said zwitterionic
side chain functionalities comprises amino acids, amino acid
derivatives, neurotransmittors, monosaccharides, nucleic acids, or
combinations and chemically modified derivatives thereof.
4. The complex as claimed in claim 2, wherein the zwitterionic
functionalities comprise one or more anionic and cationic side
chain functionalities.
5. The complex as claimed in claim 1, wherein said receptor
molecules are selected from the group consisting of peptides,
carbohydrates, nucleic acids, lipids, pharmaceuticals, antigens,
antibodies, proteins, any organic polymers or combination of these
molecules capable of interacting with said target analyte.
6. The complex as claimed in claim 1, wherein said conjugated
polyelectrolyte is confined, adsorbed or covalently attached to a
solid support.
7. The complex as claimed in claim 1, wherein said conjugated
polyelectrolyte is in solution.
8. The complex as claimed in claim 7, wherein water, organic
solvents, buffer systems or combination thereof are used as a
solvent.
9. The complex as claimed in claim 6, wherein said solid support
comprises silicon wafers, glass, glass slides, glass beads, glass
wafers, silicon rubber, polystyrene, polyethylene, fluorinated
hydrocarbon polymers, silica gel beads, gold, indium tin oxide
coated materials, filter paper made from nylon, cellulose or
nitrocellulose, standard copy paper or variants and separation
media or other chromatographic media.
10. The complex as claimed in claim 1, wherein said conjugated
polyelectrolyte is entrapped inside polymer matrices.
11. The complex as claimed in claim 10, wherein the said polymer
matrices comprises poly [3,4-(ethylenedioxy)
thiophene]/poly(styrenesulfonicacid) (PEDOT/PSS), poly
(diallyldimethylammonium chloride) (PDADMAC), poly-4-vinylpyridine
(PVPy), poly(pyrrole) (PPy), poly(vinylalcohol) (PVA),
poly(aniline) (PANI) or combinations thereof.
12. The complex as claimed in claim 1, wherein said target analytes
are selected from the group consisting of cells, viruses, bacteria,
spores, microorganisms, peptides, carbohydrates, nucleic acids,
lipids, pharmaceuticals, antigens, antibodies, proteins, enzymes,
toxins, any organic polymers or combination of these molecules that
are capable of interacting with said receptors.
13. A biosensor device for determining selected properties of
biomolecules, comprising a complex as claimed in claim 1, and a
receptacle for said complex in which said complex is exposable to
said target analyte.
14. A biosensor device as claimed in claim 13, wherein said
polyelectrolyte is immobilized on a surface of said receptacle.
15. A biosensor device as claimed in claim 13, wherein said
receptacle is a flow cell.
16. A method of determining selected properties of biomolecules,
comprising: exposing a complex as claimed in claim 1, to a target
biomolecule analyte whereby the analyte and the receptor interact,
detecting a change of a property of said polyelectrolyte in
response to the interaction between the receptor and the analyte;
and using the detected change to determine said selected property
of said biomolecule.
17. The method as claimed in claim 16, wherein the change of said
property is detected by measuring fluorescence, Forster resonance
energy transfer (FRET), quenching of emitted light, absorption,
impedance, refraction index, mass, visco-elastic properties,
thickness or other physical properties.
18. A method of manufacturing a biosensor device, wherein a complex
as claimed in claim 1 is attached to a surface in a suitable
receptacle.
19. The method as claimed in claim 18, wherein a conjugated
polyelectrolyte is transferred to said surface by a method selected
from dip coating, spin-coating, contact printing, screen printing,
ink jet technologies, spraying, dispensing and microfluidic
printing by the use of soft lithography, or combinations thereof,
and forming a complex between a suitable receptor and said
polyeletrolyte.
20. The method as claimed in claim 19, wherein the zwitterionic
conjugated polyelectrolytes is immobilized by physical adhesion to
the solid support at elevated temperatures or by entrapment in a
hydrogel matrix.
21. A biosensor device, comprising a plurality of spots, array or
lines of a complex according to claim 1.
22. A biosensor device as claimed in claim 21, wherein the
plurality of spots, array or lines are printed by micro contact
printing using elastomer stamps; by spotting zwitterionic
conjugated polyelectrolyte solutions; or by ink jetting
polyelectrolyte solutions onto said solid support.
23. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for detection of
biomolecular interactions through the detection of alterations of
the intra- and inter-chain processes in materials based on
zwitterionic conjugated polyelectrolytes.
BACKGROUND
[0002] The development of materials that are capable of selectively
detecting biomolecular interactions have come under increasing
attention, owing to their large potential for molecular electronics
and biosensors. In this regard, conjugated polymers (CPs) such as
poly(thiophene) and poly(pyrrole) can be used to couple
analyte/receptor interactions, as well as non-specific
interactions, into observable responses. CPs based sensors are
sensitive to very minor perturbations, due to amplification by a
collective system response and therefore offer a key advantage
compared to small molecules based sensors. The possibility to use
CPs as detecting elements for biological molecules requires that
polymers are compatible with aqueous environment. This has been
accomplished by making conjugated (and sometimes luminescent)
polyelectrolytes, as recently used to detect biomolecules through
their impact on the conditions for photoinduced charge or
excitation transfer. Conjugated polyelectrolytes offer
possibilities for very sensitive measurements, and may become
ubiquitous for genomics and proteomics in the future, if the
optical or electronic processes in these materials can be used to
track biospecific interactions.
[0003] The physical and chemical properties of conjugated polymers
can be modified by the introduction of suitable side chains in the
3-position. Polythiophene derivatives that exhibit biotin and
different carbohydrates has been synthesized and shown to undergo
colorimetric transitions in response to binding of streptavidin and
different types of bacteria and viruses, respectively. The
presently demonstrated systems use covalent attachment of a
receptor to the side chains of the conjugated polymer. Therefore,
methods without the need of covalent attachment of the receptor
would be desirable, and such systems have been developed, see
Boissinot, M., Leclerc, M, Ho, H-A. Patent Appl. WO02081735, 2002.
However, these methods, which use polyanionic or polycationic
conjugated polyelectrolytes, based on interactions mainly dominated
by electrostatic forces, sometimes requires labelling of the
analyte. Methods without any labelling of the analyte or any
covalent attachment of the receptor would be attractive.
SUMMARY OF THE INVENTION
[0004] Thus, there remains a need for simpler and more sensitive
methods for detection of molecular interactions. Methods based on
conjugated polyelectrolytes that can create versatile interactions
with molecules and detect molecular interactions, without any
labelling of the analytes or any covalent attachment of the
receptors, would therefore be desirable.
[0005] The object of the present invention is therefore to provide
means and methods that meet these and other needs.
[0006] This object is in a first aspect achieved with a complex
between a conjugated polyelectrolyte, and one or more receptor
molecules specific for a target biomolecule analyte, said
polyelectrolyte and said receptor being non-covalently bound to
each other, usable as a probe for biomolecular interactions,
defined in claim 1.
[0007] For the purpose of this invention, the term "probe" shall be
taken to mean any form of a complex as defined in claim 1, capable
of responding to biomolecular interactions occurring between a
receptor in the complex and another species, such as molecules,
cells, viruses, bacteria, spores, microorganisms, peptides,
carbohydrates, nucleic acids, lipids, pharmaceuticals, antigens,
antibodies, proteins, enzymes, toxins, any organic polymers or
combination of these molecules that interacts with receptors of
interest, by changing at least one property of the complex that can
be detected by external means.
[0008] Suitably the polyelectrolyte comprises copolymers or
homopolymers of thiophene, pyrrole, aniline, furan, phenylene,
vinylene or their substituted forms, and preferably the conjugated
polyelectrolyte has one or more zwitterionic side chain
functionalities.
[0009] In a further aspect of the invention, there is provided a
biosensor device for determining selected properties of
biomolecules, comprising a complex of the kind identified above,
and a substrate for said complex in which said complex is exposable
to said target analyte. The biosensor device is defined in claim
14. In still another aspect of the invention there is provided a
method of determining selected properties of biomolecules,
comprising exposing a complex as defined above, to a target
biomolecule analyte whereby the analyte and the receptor interact,
detecting a change of a property of said polyelectrolyte in
response to the interaction between the receptor and the analyte;
and using the detected change to determine said selected property
of said biomolecule. The method is defined in claim 17.
[0010] The multiplicity of biomolecular interactions that one may
wish to identify also implies that the invention in a still further
aspect, can be implemented in the form of a microarray, and which
calls for anchoring and patterning of the detecting system on a
surface, defined in claim 22.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the chemical structure of
poly(3-[(S)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylene
hydrochloride) (POWT), a zwitterionic polythiophene derivative.
[0012] FIG. 2 schematically illustrates the method according to the
invention.
[0013] FIG. 3 shows the absorptionspectra of 1.16 .mu.mol POWT (on
a monomer basis) and 0 mol (.quadrature.), 6.4 nmol (.diamond.) of
an oligonucleotide (5'-CAT GAT TGA ACC ATC CAC CA-3') after 5
minutes of incubation in 10 mM Na-phosphate buffer pH 7.5, or in
the same buffer system with 6.4 nmol of a complementary
oligonucleotide (.DELTA.).
[0014] FIG. 4 shows the emission spectra of 23.1 nmol POWT (on a
monomer basis) and 0 mol (.quadrature.), 1.28 nmol (.diamond.), and
2.56 nmol (x) of an oligonucleotide (5'-CAT GAT TGA ACC ATC CAC
CA-3') after 5 minutes of incubation in 10 mM Na-phosphate buffer
pH 7.5, or in the same buffer system with 1.28 nmol of a
complementary oligonucleotide (.DELTA.). All of the emission
spectra were recorded with excitation at 400 nm.
[0015] FIG. 5 shows the emission spectra of 100 nmol POWT (on a
monomer basis) (x) with 1.0 equivalent (on a monomer basis) of a
positively charged peptide, JR2K (.diamond.), 1.0 equivalent of a
negatively charged peptide, JR2E (.DELTA.), 0.5 equivalents JR2E
plus an addition of 0.5 equivalents JR2K (.quadrature.), 0.5
equivalents JR2K plus an addition of 0.5 equivalents JR2E
(.box-solid.), 2.0 equivalent JR2K (.diamond-solid.), and 2.0
equivalent JR2E (.tangle-solidup.) after 10 min of incubation in a
20 mM Na-phosphate buffer pH 7.4. All of the emission spectra were
recorded with excitation at 400 nm.
[0016] FIG. 6 shows the Emission spectra of 26.1 nmol POWT in 20 mM
Na-phosphate pH 7.5, upon addition of 4.9 .mu.M of a synthetic
peptide, with a receptor site for carbonic anhydrase (thin line),
and after addition of 13 .mu.M of carbonic anhydrase (bold
line).
[0017] FIG. 7 shows the fluorescence images of POWT/DNA complexes.
Hydrogels of POWT and single stranded DNA after binding of
complementary DNA (bottom left) and non-complementary DNA (bottom
right). Cross points (100.times.100 .mu.m) of POWT and single
stranded DNA after binding of complementary DNA (top left) or
non-complementary DNA (top right). The fluorescence was recorded
with an epifluorescence microscope (Zeiss Axiovert inverted
microscope A200 Mot) equipped with a CCD camera (Axiocam HR).
[0018] FIG. 8 shows the DNA-hybridisation event on a POWT/gold chip
monitored with a BiacoreX instrument Injection and wash out of (in
order): ssDNA1 (characterization, 1540 RU), ssDNA1
(non-complementary, 30 RU), ssDNA2 (complementary, 860 RU). 0.15 M
PBS buffer was used.
[0019] FIG. 9 shows the microcontact printing of POWT. A square net
of POWT on plasma etched polystyrene, with lines 25 .mu.m wide
surrounding the polystyrene squares of 100.times.100 .mu.m. Optical
microscopy in reflected light.
[0020] Table 1 shows the difference in ratio of emission intensity
at the wavelengths 540 nm/585 nm and 540 nm/670 nm upon addition of
1.28 nmol of different oligonucleotides to a mixture of 23.1 nmol
POWT and 1.28 nmol of a single stranded oligonucleotide.
[0021] Table 2 shows the absorption maximum and the ratio of the
intensity of the emitted light at 540 nm/610 nm for POWT and
POWT/peptide complexes after 10 min incubation in 20 mM
Na-phosphate pH 7.4
DETAILED DESCRIPTION OF THE INVENTION
[0022] In general terms, the present invention relates to a novel
complex between zwitterionic conjugated polyelectrolytes and a
receptor, the polyelectrolyte acting as a carrier for said
receptor, without the requirement to label the analytes or to
covalently attach the receptors to the carrier. The complex is used
as a probe for responding to biomolecular interactions. It also
relates to a biosensor device comprising such complex and a method
for detection of molecular interactions.
[0023] The invention is based on zwitterionic polyelectrolyte
forming a complex with one or more receptor molecules. This complex
is formed without covalent bonding and is based on hydrogen
bonding, electrostatic- and non-polar interactions between the
zwitterionic conjugated polymers and the receptor molecules, herein
referred to as non-covalent bonding, which further includes any
type of bonding that is not covalent in its nature.
[0024] The present invention utilizes changes of the zwitterionic
conjugated polyelectrolyte/receptor molecules complex or
alterations of the net charge of the receptor molecules, which
induce conformational transitions of the backbone of the
zwitterionic conjugated polyelectrolyte, separation or aggregation
of zwitterionic conjugated polyelectrolyte chains. Furthermore,
conformational transitions of the backbone of the zwitterionic
conjugated polyelectrolyte, separation or aggregation of
zwitterionic conjugad polyelectrolyte chains, alter the intra- and
inter-chain processes of the zwitterionic conjugated
polyelectrolytes. These changes can be detected in solution or on a
surface.
[0025] In particular the present invention allows, but is not
limited to, detection of biospecific recognition through DNA (base
pairing), proteins (antigen/antibody), glycoproteins or shorter
purpose designed peptides.
[0026] The novel complex is suitably implemented as an active part
of a biosensor device, e.g. by immobilizing the polyelectrolyte on
a substrate in a biosensor cell. Suitably the biosensor device
comprises a suitable receptacle for said substrate, and a complex
between polyelectrolyte and receptor is formed on the
substrate.
[0027] However, other configurations are possible, e.g the complex
can be provided in solution and passed through a flow cell while an
analyte solution is mixed with the flow of complex solution. The
interaction can be monitored by various analytical techniques.
[0028] As an example of polymers exhibiting the above discussed
characteristics
poly(3-[(S)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylene
hydrochloride) (POWT, see FIG. 1) can be mentioned. Studies of this
polymer (see Andersson, M.; Ekeblad, P. O.; Hjertberg, T.;
Wennerstrom, O.; Inganas, O. Polymer Commun. 1991, 32, 546-548;
Berggren, M.; Bergman, P.; Fagerstrom, J.; Inganas, O.; Andersson,
M.; Weman, H.; Granstrom, M.; Stafstrom, S.; Wennerstrom, O.;
Hjertberg, T. Chem. Phys. Lett. 1999, 304, 84-90. Nilsson, K. P.
R.; Andersson, M. R.; Inganas, O. Journal of Physics: Condensed
Matter 2002, 14, 10011-10020), which is the first polythiophene
carrying a zwitterionic side chain, have shown interesting optical
and electronic processes due to different electrostatic
interactions and hydrogen bonding patterns within a single polymer
chain and between adjacent polymer chains. The interactions, due to
the zwitterionic side chains, forces the polymer backbone to adopt
alternative conformations, separation or aggregation of polymer
chains. Especially the separation and aggregation of polymer chains
induce novel intra- and inter chain processes. The intra-chain
processes are related to optical and electronic processes within a
polymer chain and the inter-chain processes are related to optical
and electronic processes between adjacent polymer chains. This
cause novel optical absorption and emission properties, due to the
novel intra- and inter chain processes, that have not been seen for
polycationic or polyanionic conjugated polyelectrolytes.
[0029] The functional groups of the zwitterionic side chain,
charged anionic or cationic at different pH, make this
polythiophene derivative suitable for forming polyelectrolyte
complexes with negatively or positively charged oligomers and
polymers. In addition, the zwitterionic groups create versatile
hydrogen bonding patterns with different molecules.
[0030] The detailed description of the invention that follows will
deal separately with the zwitterionic conjugated polyelectrolytes,
receptor molecules, analytes, methods of detection, immobilization
of conjugated polyelectrolytes and receptors, and arrays and lines.
The invention is finally exemplified with a number of experiments
demonstrating the utility thereof.
I Zwitterionic Conjugated Polymers
[0031] The present invention relates to a variety of conjugated
polyelectrolytes, with a minimum of 5 mers, consisting of mers
derived from the monomers thiophene, pyrrole, aniline, furan,
phenylene, vinylene or their substituted forms, forming
homopolymers and copolymers there from. Furthermore, monomers with
anionic-, cationic or zwitterionic side chain functionalities are
included within the scope of the invention. The side chain
functionalities is derived from, but not limited to, amino acids,
amino acid derivatives, neurotransmittors, monosaccharides, nucleic
acids, or combinations and chemically modified derivatives thereof.
The conjugated polyelectrolytes of the present invention may
contain a single side chain functionality or may comprise two or
more different side chain functionalities. The functional groups of
the zwitterionic conjugated polyelectrolytes, charged anionic or
cationic at different pHs, make these polyelectrolyte derivatives
suitable for forming strong polyelectrolyte complexes with
negatively or positively charged oligomers and polymers. In
addition, the zwitterionic groups create versatile hydrogen bonding
patterns with different molecules.
II Receptor Molecules
[0032] The zwitterionic polyelectrolytes of the present invention
form a complex with one or more receptor molecules (FIG. 2). This
complex is formed without covalent bonding and based on hydrogen
bonding, electrostatic- and non-polar interactions between the
zwitterionic conjugated polymers and the receptor molecules. The
receptor molecules will act as the recognition site for analytes or
as anchors for performing enzymatic reactions, such as
phosphorylation. A wide variety of receptor molecules can be used
and the choice of molecule is only limited by the affinity to the
conjugated polymers and the recognition properties of desirable
analytes. Appropriate receptor molecules include, but are not
limited to, peptides, carbohydrates, nucleic acids, lipids,
pharmaceuticals, antigens, antibodies, proteins, any organic
polymers or combination of these molecules that are capable of
interacting with analytes of interest.
[0033] The receptor molecules can be chemically modified to
interact with the conjugated polymers of interest. Methods of
derivatizing a diverse range of compounds (e.g. carbohydrates,
proteins, nucleic acids and other chemical groups) are well known.
For example, amino acid side chains can easily be modified to
contain polar and non-polar groups, or groups with hydrogen bonding
abilities.
III Analytes
[0034] Upon binding of or exposure to one or more analytes, the
conjugated polyelectrolyte/receptor molecules complex is subject to
changes or alterations of the net charge of the receptor
molecules.
[0035] The changes of the conjugated polyelectrolyte/receptor
molecules complex or the alterations of the net charge of the
receptor molecules will induce conformational transitions of the
backbone of the zwitterionic conjugated polyelectrolyte, separation
or aggregation of polyelectrolyte chains, that leads to altered
intra- and intra chain processes of the zwitterionic conjugated
polyelectrolyte.
[0036] Appropriate analytes include, but are not limited to, cells,
viruses, bacteria, spores, microorganisms, peptides, carbohydrates,
nucleic acids, lipids, pharmaceuticals, antigens, antibodies,
proteins, enzymes, toxins, any organic polymers or combination of
these molecules that interacts with receptors of interest.
[0037] The analytes can be chemically modified to interact with the
receptor molecules of interest. Methods of derivatizing a diverse
range of compounds (e. g. carbohydrates, proteins, nucleic acids
and other chemical groups) are well known. For example, amino acid
side chains can easily be modified to contain polar and non-polar
groups, or groups with hydrogen bonding abilities.
IV Methods of Detection
[0038] As already indicated, the present invention is based on the
utilization of alterations of intra and inter chain processes of
zwitterionic conjugated polyelectrolytes. These alterations can be
observed by fluorescence, Forster resonance energy transfer (FRET),
quenching of emitted light, absorption, impedance, refraction
index, change in mass, visco-elastic properties, change in
thickness or other physical properties. The conformational
transitions of the backbone of the zwitterionic conjugated
polyelectrolyte, separation or aggregation of polyelectrolyte
chains will alter the intra- and inter-chain processes of the
zwitterionic conjugated polyelectrolyte and can for example be
detected as a change in the ratio of the intensities of the emitted
light at two or more different wavelengths (see example 3). The
emission intensities can be recorded by a fluorometer and
enhancement of the photon flow in the detector can increase the
sensitivity. This can be achieved using a laser as the excitation
source.
[0039] The fluorometric change can also be detected by the use of a
fluorescence microscope or a confocal microscope. A combination of
excitation or emission filter can be used and the picture can be
recorded by a CCD-camera (see example 7 and 9), video camera,
regular camera or by a Polaroid camera. The pictures can then be
analyzed by image processing software on a computer, Image
correlation spectroscopy (ICS) or by other means.
[0040] Changes in impedance can be detected by using the method of
impedance spectroscopy. According to the invention the zwitterionic
conjugated polymers can be immobilized inside a conducting polymer
hydrogel matrix for example poly
[3,4-(ethylenedioxy)thiophene]/poly(styrenesulfonicacid)
(PEDOT/PSS). Changes in resistance, capacitance and inductance can
then be tracked with the zwitterionic conjugated polyelectrolytes,
receptor or analyte molecules in an aqueous environment.
[0041] Surface plasmon resonance (SPR) enables detection of minute
changes in refraction index. A change in refraction index occurs
when the intra- and inter-chain processes of the zwitterionic
conjugated polyelectrolytes are altered by the interactions between
receptor and analyte molecules or alteration of the net charge of
the receptor molecules. These interactions can also lead to
aggregation of the polyelectrolyte chains and is thus detected as a
change in refraction index.
[0042] Quartz crystal microbalance and dissipation (QCM-D) is a
sensitive and versatile technique to measure both adsorbed mass and
visco-elastic properties of adsorbed layers of molecules in liquid.
Alteration of the intra- and inter-chain processes of the
zwitter-ionic conjugated polyelectrolyte, due to interaction
between receptor and analyte molecules or change in the net charge
of the receptor molecules, can lead to changes in mass or
visco-elastic properties and thus be detected by QCM-D or other
techniques.
[0043] When the analyte molecules interact with the receptor
molecules, complexed with the zwitterionic conjugated
polyelectrolyte adsorbed to a solid support changes in thickness
may occur. Ellipsometry, imaging or null ellipsometry, is an
optical technique that uses polarised light to sense the dielectric
properties of a sample and can be used to detect these changes in
thickness on a sub-angstrom level. These techniques can thus be
used for measuring alteration in intra- and inter chain processes
of the zwitterionic conjugated polyelectrolytes.
[0044] The interaction of receptor and analyte molecules with
zwitterionic conjugated polyelectrolyte can also be detected by
electrical and electrochemical methods. A gel or network of the
zwitterionic conjugated polyelectrolyte can be formed, and thus a
three dimensional object is obtained where each polymer chain is in
(indirect) contact with all chains in the network. If the
zwitterionic conjugated polyelectrolyte is in a semiconducting
state--such as when the luminescence properties is used--it will
exhibit a rather low conductivity, which is somewhat difficult to
easily distinguish from the ionic conductivity of the aqueous
medium bathing the gel. It is therefore desirable to form highly
conducting gels of the sensitive macromolecule that allow
electrical conduction in the network. A difficulty is that the
doping of the conjugated chains, which gives a metallic polymer and
a high conductivity, will not only turn on conductivity but also
change the mechanical properties and geometry of the chains,
thereby hindering the mechanism at work in the case of luminescence
detection. A solution to that problem is the use of two component
polymer gels, where one component A gives the high conductivity and
another component B the biospecific interactions. If these two
compounds are combined in a suitable manner, the changes of
geometry of the gel due to said interactions can be made to detect
the interaction between component B and biomolecules. Component A
can be an aqueous dispersion of a highly doped polymer and
component B, the zwitterionic conjugated polyelectrolyte can be
combined, to make gels. By measuring the DC or AC conductivity of
these gels with two point and four point probe methods or by
impedance spectroscopy, the change of conductance upon binding or
exposure to analytes or net charge alteration of the receptors can
be followed.
[0045] The intra- and inter-chain processes of the zwitterionic
conjugated polyelectrolytes are altered by the interactions between
receptor and analyte molecules or alteration of the net charge of
the receptor molecule, and leads to changes of the electrochemical
properties of the resulting complex, which can then be used to
build electrochemical detectors for biomolecules. A change of the
redox potential of the hydrogel formed in the presence of a
biomolecule can be used to detect the presence of a complementary
biomolecule. Similar devices, using conjugated polymers with a
covalently attached receptor, have been studied by Korri-Youssoufi,
H., et al in "Toward bioelectronics: Specific DNA recognition based
on an oligonucleotide-functionalized polypyrrole", J. Am. Chem.
Soc. 1997, 119, 7388-7389.
[0046] The above described methods can also be implemented in the
form of microarrays, to give an "image" of the composition of a
biological sample.
V Immobilization of Conjugated Polymers and Receptors
[0047] The zwitterionic conjugated polyelectrolytes, the
zwitterionic conjugated polyelectrolyte/receptor molecules complex
or the receptor molecules of the present invention can be
immobilized on a variety of solid supports, including, but not
limited to silicon wafers, glass (e.g. glass slides, glass beads,
glass wafers etc.), silicon rubber, polystyrene, polyethylene,
Teflon, silica gel beads, gold, indium tin oxide (ITO coated
materials, e.g. glass or plastics), filter paper (e.g. nylon,
cellulose and nitrocellulose), standard copy paper or variants and
separation media or other chromatographic media. Transfer of the
zwitterionic zwitterionic conjugated polyelectrolyte to the solid
support can be achieved by using i.a. but not limited to, dip
coating, spin-coating, contact printing, screen printing, ink jet
technologies, spraying, dispensing and microfluidic printing by the
use of soft lithography or the Biacore.TM. (Biacore, Uppsala,
Sweden) system. Immobilization of the zwitterionic conjugated
polyelectrolytes is achieved by physical adhesion to the solid
support at elevated temperatures or by entrapment in a hydrogel
matrix.
[0048] Immobilization of the zwitterionic conjugated
polyelectrolytes of the present invention may be desired to improve
their ease of use, assembly into devices (e.g. arrays or parallel
lines), stability, robustness, fluorescent response, to fit into
the process of high-throughput-screening (HTS) using micro titer
plates and other desired properties.
[0049] The receptor molecules of the present invention can be
immobilized together with the zwitterionic conjugated
polyelectrolyte (i.e. mixed with the polyelectrolyte solution).
Another way to immobilize the receptor molecules is to place them
underneath or on top of the zwitterionic conjugated
polyelectrolyte. Transfer of the receptor molecules mixed together
with zwitterionic conjugated polyelectrolyte to the solid support
can be achieved by, but not limited to, using dip coating,
spin-coating, contact printing, screen printing, ink jet
technologies, spraying, dispensing and microfluidic printing (see
example 9) by the use of soft lithography (see example 10) or the
Biacore.sup.TM system (see example 8). If the receptor molecules is
to be placed underneath the zwitterionic zwitterionic conjugated
polyelectrolyte it has to be transferred to the solid support in
the same way as it would have been mixed together with the
polyelectrolyte as mentioned above. Placing the receptor molecules
on top of the zwitterionic conjugated polyelectrolyte is done in
the same way but after the polyelectrolyte has been immobilized to
the solid support. The receptor molecules will act as the
recognition site for analytes or as anchors for performing
enzymatic reactions, such as phosphorylation.
[0050] Solvents for the zwitterionic conjugated polyelectrolytes of
the present invention and the receptor molecules during the
immobilization to the solid support can be, but are not limited to,
water, buffered water solutions, methanol, ethanol and combinations
thereof. Supporting polymers of other kinds can also be added in
this step.
[0051] When the receptor molecules are immobilized on the solid
support underneath, on top of or together with the zwitterionic
conjugated polyelectrolyte of the present invention they form a
complex with the polyelectrolyte through non-covalent interactions
(FIG. 2). This complex is formed without covalent chemistry and is
based on hydrogen bonding, electrostatic- and non-polar
interactions between the zwitterionic conjugated polyelectrolyte
and the receptor molecule. Immobilization of the receptors to the
zwitterionic conjugated polyelectrolytes of the present invention
may be desired to improve their ease of use, assembly into devices
(e.g. arrays or parallel lines), stability, robustness, fluorescent
response, to fit into the process of high-throughput-screening
(HTS) using micro titer plates and other desired properties. While
receptor molecules have been immobilized onto cationic or anionic
conjugated polymers for detection of analytes [15], prior to the
the present invention, immobilization without covalent chemistry
and based on hydrogen bonding, electrostatic- and non-polar
interactions between the zwitterionic conjugated polyelectrolytes
and the receptor molecules had not been realized.
[0052] The zwitterionic conjugated polyelectrolyte and receptor
molecules can be entrapped inside polymer matrices on top of a
solid support or free floating in solution. A gel or network of the
zwitterionic conjugated polymers can be formed, where each
zwitterionic conjugated polyelectrolyte chain of the present
invention is in (indirect) contact with all chains in the network.
Realization of these polymer matrices can be done by mixing c
zwitterionic conjugated polyelectrolyte with other polymers such
as, but not limited to, poly [3,4-(ethylenedioxy)
thiophene]/poly(styrenesulfonicacid) (PEDOT/PSS), poly
(diallyldimethylammonium chloride) (PDADMAC), poly-4-vinylpyridine
(PVPy), poly(pyrrole) (PPy), poly(vinylalcohol) (PVA),
poly(aniline) (PANI) or combinations thereof. By swelling these
polymer blends in water a hydrogel is realized, which can be of
interest when using receptor and analyte molecules of biological
origin. The zwitterionic conjugated polyelectrolytes of the present
invention can be mixed together with these polymers before
immobilization to the solid support or transferred afterwards.
Receptor molecules of interest can be transferred together with the
zwitterionic conjugated polyelectrolyte or in a subsequent step. A
microarray or parallel line format can be used if desired,
necessary or for other reasons. In certain embodiments of the
present invention this network or hydrogel approach can be used to
detect conformational changes and aggregation of the zwitterionic
conjugated polyelectrolyte chains due to interaction between
receptor and analyte molecules or change in the net charge of the
receptor molecules. These alterations can then be detected by
measuring absorption, fluorescence, electrical properties,
impedance or by other means.
VI Arrays or Lines
[0053] According to the present invention the generation of large
arrays or parallel lines of the zwitterionic conjugated
polyelectrolytes with the same or different receptor molecules in
each spot or line can overcome shortcomings of a single sensor or a
solution based approach. The array or parallel line approach opens
up the parallel analysis of one or different analytes to one or
different receptors in an easy way. The main purpose of using
arrays or lines is to increase ease of use, portability,
quantification, selectivity among other qualities and
characteristics. With this approach we can explore the ability to
measure multicomponent samples and to use partially selective
sensor spots. This gives the opportunity to analyse two or more
samples of interest at the same time, to do on-chip concentration
determinations and to study the background. By immobilizing the
zwitterionic conjugated polyelectrolyte and/or the receptor
molecules on solid supports of any size and in any chosen patterns
(such as arrays, lines, spots, posts) small, portable, easily read
and interpretable devices can be constructed.
[0054] The use of multiple arrays requires that detection can be
done for a great number of biomolecules, more or less
simultaneously. This is often done in the form of a microarray,
where many individual detector elements (or probes) are integrated
on a small surface area, to allow for massive parallelism in the
detection. As we can construct each individual detector by the
simple blending of the zwitterionic conjugated polyelectrolyte and
biomolecules, we have removed the necessity of covalent chemistry
for making each one of many thousands of detectors in a detector
array (microarray). We have shown that the zwitterionic conjugated
polyelectrolyte and zwitterionic conjugated
polyelectrolyte/biomolecule complexes can be printed by micro
contact printing using elastomer stamps (FIG. 9). Transfer onto a
microarray surface may also be done by spotting zwitterionic
conjugated polyelectrolyte solutions, or by ink jetting
polyelectrolyte solutions or by the other methods mentioned above.
These steps are essential to prepare a multipixel microarray.
EXPERIMANTAL
EXAMPLE 1
Optical Detection of DNA-hybridisation in Solution
[0055] A stock solution containing 0.5 mg ml.sup.-1 POWT in
de-ionised water was prepared and incubated for 30 minutes. 50
.mu.l of the polymer solution was mixed with 64 .mu.l of
DNA-solution (100 nmol ml.sup.-1, 5'-CAT GAT TGA ACC ATC CAC CA-3',
purchased from SGSDNA, Koping, Sweden). After 15 minutes of
incubation, the samples were diluted with de-ionised water, a stock
buffer solution (Na-phosphate pH 7.5 and a 1.0 equivalent amount of
the respective nucleotide (5'-TGG TGG ATG GTT CAA TCA TG-3,
purchased from SGSDNA, Koping, Sweden) to a final volume of 1500
.mu.l containing 10 mM Na-phosphate. The samples were incubated for
5 minutes and the absorption spectra were recorded with a
Perkin-Elmer Lambda 9 UV/VIS/NIR spectrophotometer.
DNA-hybridisation is detected by a shift of the absorption maximum
to shorter wavelengths and a decrease of the shoulder at longer
wavelengths (FIG. 3). The shift of absorption maximum is due to
conformational changes of the POWT backbone and the decrease of the
shoulder at longer wavelengths is due to separation of the POWT
chains.
EXAMPLE 2
Fluorescent Detection of DNA-hybridisation in Solution
[0056] A stock solution containing 0.5 mg ml.sup.-1 POWT in
de-ionised water was prepared and incubated for 30 minutes. 10
.mu.l of the polymer solution was mixed with 12.8 .mu.l of
DNA-solution (100 nmol ml.sup.-1, 5'-CAT GAT TGA ACC ATC CAC CA-3',
purchased from SGSDNA, Koping, Sweden). After 15 minutes of
incubation, the samples were diluted with de-ionised water, a stock
buffer solution (Na-phosphate pH 7.5 and a 1.0 equivalent amount of
the respective nucleotide (5'-TGG TGG ATG GTT CAA TCA TG-3,
purchased from SGSDNA, Koping, Sweden) to a final volume of 1500
.mu.l containing 10 mM Na-phosphate. The samples were incubated for
5 minutes and the emission spectra were recorded with a ISA
Jobin-Yvon spex FluoroMax-2 apparatus. DNA-hybridisation is
detected by an increase of the emitted light and a shift of the
emission maximum to a shorter wavelength (FIG. 4). The emitted
light at 540 nm (intra-chain process) is increased and the emitted
light at 670 nm (inter-chain process) is decreased as formation of
double stranded DNA occurs.
EXAMPLE 3
Fluorescent Detection of Single Nucleotide Polymorphism (SNP)in
Solution
[0057] A stock solution containing 0.5 mg ml.sup.-1 POWT in
de-ionised water was prepared and incubated for 30 minutes. 10
.mu.l of the polymer solution was mixed with 12.8 .mu.l of
DNA-solution (100 nmol ml.sup.-1, 5'-CAT GAT TGA ACC ATC CAC CA-3',
purchased from SGSDNA, Koping, Sweden). After 15 minutes of
incubation, the samples were diluted with de-ionised water, a stock
buffer solution (Na-phosphate pH 7.5 and a 1.0 equivalent amount of
the respective nucleotide (5'-TGG TGG ATG GTT CAA TCA TG-3', 5'-TGG
TGG ATG CTT CAA TCA TG -3', 5'-TGG TGG AAC GTT CAA TCA TG-3',
5'-TGG TGG AAC CTT CAA TCA TG -3' or 5'-CAT GAT TGA ACC ATC CAC CA
-3', purchased from SGSDNA, Koping, Sweden) to a final volume of
1500 .mu.l containing 10 mM Na-phosphate. The samples were
incubated for 5 minutes and the emission spectra were recorded with
a ISA Jobin-Yvon spex FluoroMax-2 apparatus. The difference in
ratio of emission intensity at the wavelengths 540 nm/585 nm and
540 nm/670 nm were calculated. The emitted light at 540 nm and 585
nm is due to intra-chain processes and the emitted light at 670 nm
is due to an inter-chain process (aggregation of POWT chains).
Nucleotides with one, two or three mismatches can easily be
detected, as the difference in ratio of the emission intensity at
the wavelengths 540 nm/585 nm and 540 nm/670 nm are influenced by
the degree of mismatch between the DNA strands (Table 1).
TABLE-US-00001 TABLE 1 Difference in Difference in Ratio Ratio
Sequence 540 nm/585 nm.sup.a 540 nm/670 nm.sup.a 5'-CAT GAT TGA ACC
ATC CAC CA-3' 0.000.sup.b .+-. 0.000 0.000.sup.b .+-. 0.000 3'-TGA
CTA ACT TGG TAG GTG GT-5' 5'-CAT GAT TGA ACC ATC CAC CA-3'
0.041.sup.b .+-. 0.003.sup.c 0.133.sup.b .+-. 0.013.sup.c 3'-TGA
CTA ACT TCG TAG GTG GT-5' 5'-CAT GAT TGA ACC ATC CAC CA-3'
0.052.sup.b .+-. 0.004.sup.c 0.219.sup.b .+-. 0.021.sup.c 3'-TGA
CTA ACT TGC AAG GTG GT-5' 5'-CAT GAT TGA ACC ATC CAC CA-3'
0.074.sup.b .+-. 0.007.sup.c 0.355.sup.b .+-. 0.034.sup.c 3'-TGA
CTA ACT TCC AAG GTG GT-5' .sup.aThe ratio of the intensity of the
emitted light at 540 nm and 585 nm or 540 nm and 670 nm. .sup.bThe
difference in ratio is calculated from the following formula
Ratio.sub.complementary - Ratios.sub.x, where x denotes the double
stranded DNA sequence of interest. .sup.cThe mean value and the
standard deviation for 10 independently performed experiments.
EXAMPLE 4
Optical Detection of Self-assembly of Synthetic Peptides in
Solution
[0058] A stock solution containing 3.7 mg ml.sup.-1 POWT in
de-ionised water was prepared and incubated for 30 minutes. 10
.mu.l of the polymer solution was mixed with 10 .mu.l or 20 .mu.l
of a negatively charged peptide
(NH2-N-A-A-D-L-E-K-A-l-E-A-L-E-K-H-L-E-A-K-G-P-V-D-A-A-Q-L-E-K-Q--
L-E-Q-A-F-E-A-F-E-R-A-G-COOH) or the positively charged peptide
(NH2-N-A-A-D-L-K-K-A-I-K-A-L-K-K-H-L-K-A-K-G-P-V-D-A-A-Q-L-K-K-Q-L-K-Q-A--
F-K-A-F-K-R-A-G-COOH) solution (2.2 mg ml.sup.-1), respectively and
diluted with de-ionised water to a final volume of 300 .mu.l. After
15 minutes of incubation, the samples were diluted with a stock
buffer solution (Na-phosphate pH 7.4) and 10 .mu.l de-ionised water
or 10 .mu.l of the positive/negative peptide solution (2.2 mg
ml.sup.-1) to a final volume of 2000 .mu.l containing 20 mM
Na-phosphate. The samples were incubated for 10 minutes in room
temperature and the absorption spectra was recorded with a
Perkin-Elmer Lambda 9 UV/VIS/NIR spectrophotometer Addition of JR2K
will shift the absorption maximum to shorter wavelengths,
indicative of a non-planar POWT backbone and separation of POWT
chains, and addition of JR2E will shift the absorption maximum to
longer wavelengths, indicative of a planar POWT backbone and
aggregation of POWT chain (Table 2). JR2E and JR2K has been tailor
made to form a four-helix bundle and the formation of this
structure can be detected by a change of the absorption maximum for
POWT (Table 2). TABLE-US-00002 TABLE 2 Ratio of the Ratio of the
intensity of intensity of Absorption the emitted the emitted
maximum light at light at (nm) 540 nm/610 nm 540 nm/670 nm POWT 438
0.72 1.63 POWT + JR2E 451 0.24 0.44 POWT + JR2K 419 1.08 2.88 POWT
+ JR2E + 440 0.49 0.97 JR2K
EXAMPLE 5
Fluorescent Detection of Self-assembly of Synthetic Peptides in
Solution
[0059] A stock solution containing 3.7 mg ml.sup.-1 POWT in
de-ionised water was prepared and incubated for 30 minutes. 10
.mu.l of the polymer solution was mixed with 10 .mu.l or 20 .mu.l
of a negatively charged peptide
(NH2-N-A-A-D-L-E-K-A-I-E-A-L-E-K-H-L-E-A-K-G-P-V-D-A-A-Q-L-E-K-Q--
L-E-Q-A-F-E-A-F-E-R-A-G-COOH) or a positively charged peptide
(NH2-N-A-A-D-L-K-K-A-I-K-A-L-K-K-H-L-K-A-K-G-P-V-D-A-A-Q-L-K-K-Q-L-K-Q-A--
F-K-A-F-K-R-A-G-COOH) solution (2.2 mg ml.sup.-1), respectively and
diluted with de-ionised water to a final volume of 300 .mu.l. After
15 minutes of incubation, the samples were diluted with a stock
buffer solution (Na-phosphate pH 7.4) and 10 .mu.l de-ionised water
or 10 .mu.l of the positive/negative peptide solution (2.2 mg
ml.sup.-1) to a final volume of 2000 .mu.l containing 20 mM
Na-phosphate. The samples were incubated for 10 minutes in room
temperature and the emission spectra (FIG. 5, Table 2) were
recorded with an ISA Jobin-Yvon spex FluoroMax-2 apparatus.
Addition of JR2K will shift the emission maximum to shorter
wavelengths and increase the intensity of the emitted light,
indicative of a non-planar POWT backbone and separation of POWT
chains, and addition of JR2E will shift the emission maximum to
longer wavelengths and decrease the intensity of emitted light,
indicative of a planar POWT backbone and aggregation of POWT chain
(FIG. 5). JR2E and JR2K has been tailor made to form a four-helix
bundle and the formation of this structure can be detected by a
change of the emission maximum and the intensity of the emitted
light from POWT (FIG. 5). The emitted light at 540 nm and 610 nm is
due to intra-chain processes and the emitted light at 670 nm is due
to an inter-chain process (aggregation of polymer chains). The
difference in ratio of the emission intensity at the wavelengths
540 nm/610 nm and 540 nm/670 nm are influenced, as the different
complexes between POWT and the different peptides are formed (Table
2). For instance, the intra- and inter-chain processes of the
POWT/JR2E (receptor) are clearly altered upon addition of JR2K
(analyte).
EXAMPLE 6
Fluorescent Detection of Carbonic Anhydrase in Solution
[0060] A stock solution containing 0.5 mg ml.sup.-1 POWT in
de-ionised water was prepared and incubated for 30 minutes. 10
.mu.l of the polymer solution was mixed with 5 .mu.l of a
negatively charged peptide solution
(NH2-N-A-A-D-L-E-K-A-I-E-A-L-E-K-H-L-E-A-K-G-P-V-D-A-A-Q-L-E-K-Q-L-E-Q-A--
F-E-A-F-E-R-A-G-COOH, modified with a receptor for carbonic
anhydrase) (2.2 mg ml.sup.-1), and diluted with de-ionised water to
a final volume of 100 .mu.l. After 15 minutes of incubation, the
samples were diluted with a stock buffer solution (Na-phosphate pH
7.4) and 40 .mu.l deionised water or 40 .mu.l of a carbonic
anhydrase solution (6.4 mg ml.sup.-1) to a final volume of 1000
.mu.l containing 20 mM Na-phosphate. The samples were incubated for
5 minutes in room temperature and the emission spectra were
recorded with an ISA Jobin-Yvon spex FluoroMax-2 apparatus. The
intensity of the emitted light is increased when carbonic anhydrase
binds to the peptide (FIG. 6), clearly illustrating that the intra-
and inter-chain processes, especially separation/aggregation, of
the polymer chains are altered as carbonic anhydrase (analyte)
binds to the peptide (receptor).
EXAMPLE 7
Fluorescent Detection of DNA-hybridisation in Hydrogel Spots on a
Surface
[0061] 0.5 .mu.l droplets of POWT (0.5 mg ml.sup.-1) were placed on
a polystyrene surface and left to dry for 10 min. The polymer
droplets were cross-linked with 0.5 .mu.l DNA solution containing
0.5 equivalents on a monomer basis of 5'-AGA TTG GCG CAT TAC GAG
GTT AGA-3' or 5'-TCT AAC CTC GTA ATG CGC CAA TCT-3' (purchased from
SGSDNA, Koping, Sweden), respectively. After drying the
polymer/single stranded DNA hydrogel spots were incubated in a
buffer solution (10 mM Na-phosphate pH 7.5) with 10 nmol 5'-AGA TTG
GCG CAT TAC GAG GTT AGA-3' (purchased from SGSDNA, Koping, Sweden)
for 2 h. The fluorescence from the spots was recorded with an
epifluorescence microscope (Zeiss Axiovert inverted microscope A200
Mot) equipped with a CCD camera (Axiocam HR), using a 405/30 nm
bandpass filter (LP450, exposure time: 1500 ms), a 470/40 nm
bandpass filter (LP515, exposure time: 1500 ms) and a 546/12 nm
bandpass filter (LP590, exposure time: 500 ms). The alterations of
the intra- and interchain processes of POWT, due to
DNA-hybridisation, are seen as a change of the colour and the
intensity of the emitted light from POWT (FIG. 7).
EXAMPLE 8
Detection of DNA-hybridisation on a Surface by Surface Plasmon
Resonance (SRP)
[0062] A bare gold sensor chip was spin casted (1000 rpm, 30 s)
with a 5 mg/ml solution of POWT in milliQ water. The film were
annealed by heating the chip at 75.degree. C. for 5 min. Finally,
the chip was assembled on the sensor chip support by using glue or
adhesive strips. Generally an injection sequence consisting of
three injections were performed. The first injection aims to
characterize the polymer with ssDNA(5'-AGA TTG GCG CAT TAC GAG GTT
AGA-3', purchased from SGSDNA, Koping, Sweden), the second to
verify that no unspecific binding occurs and the final injection
aims to prove specific binding in the form of DNA hybridisation
using 5'-AGA TTG GCG CAT TAC GAG GTT AGA-3' or 5'-TCT AAC CTC GTA
ATG CGC CAA TCT-3' (purchased from SGSDNA, Koping, Sweden),
respectively. The polymer films were first swollen in degassed
milliQ water and then equilibrated in degassed 20 mM phosphate pH
7,4 buffer (PBS) with salt concentrations (NaCl) ranging from 0 to
1 M. The injected DNA was solved in the same buffer as the running
buffer and the concentration was usually around 1 .mu.M. The
temperature was set to 25.degree. C. during all experiments. The
hybridisation event was monitored with a BiacoreX instrument from
Biacore AB (Uppsala, Sweden). The instrument has two flow channels
with the approximate size of 0.5.times.2.5 mm. Manual loading is
required and the maximal injection volume is 100 .mu.l. As shown in
FIG. 8, a huge increase of the response unit (RU) is detected after
injection of a DNA strand complementary to the target strand
(receptor). The response unit is just slightly altered by the
injection of non-complementary DNA, clearly showing that
DNA-hybridisation is detected.
EXAMPLE 9
Fluorescent Detection of DNA-hyridisation in an Array Prepared by
Microfluidic Channels
[0063] Sylgard 184 (Dow Corning, UK), a two component silicone
rubber (poly(dimethylsiloxane), PDMS), was used for preparing
elastomer stamps used for transferring POWT to solid surfaces. The
prepolymer and the curing agent is mixed according to the
instructions provided by the manufacturer. This is then poured on
templates prepared by photolithography using the negative
photoresist SU-8 (Micro Chem Inc., Newton, Mass., USA) as the
structural element on top of silicon wafers. Curing is accomplished
by heating to 130.degree. C. for at least 20 min. The height of
structures was 18 micrometer, and the substrate was a Si wafer
cleaned in a boiling aqueous solution containing 5% each of ammonia
and H.sub.2O.sub.2 (TL-1 wash). The geometry for templates was
designed in CleWin Version 2.51 (WieWeb Software), and transferred
to a Cr mask, which was used in the photolithography step. After
developing the SU-8 structures on the silicon wafer, the template,
silanization (dimethyl-dicholorosilane) was done to obtain the
proper surface energy of the SU-8 template. A solution of POWT (10
mg ml.sup.-1 in methanol) was spin coated (2600 rpm) on to a glass
surface previously cleaned by a TL-1 wash and modified by a 10 sec
oxygen plasma treatment. The films were annealed for 5 min at
75.degree. C. A PDMS stamp with 100 .mu.m wide channels was
modified by 10 sec oxygen plasma treatment and then placed onto the
polymer film. The channels were filled with the desired nucleotide
solution (20 nmol 5'-AGA TTG GCG CAT TAC GAG GTT AGA-3 ' or 5'-TCT
AAC CTC GTA ATG CGC CAA TCT -3' in deionised water) and then left
to dry in room temperature before the stamp was removed. A second
PDMS stamp, modified in the same way as the first one, was placed
onto the polymer film with the channels perpendicular to the first
one. These channels were filled with a buffer solution (10 mM
Na-phosphate pH 7.5) containing 20 nmol 5'-AGA TTG GCG CAT TAC GAG
GTT AGA-3' and left to dry in room temperature. The PDMS stamp was
removed and the fluorescence from the cross points (100.times.100
.mu.m) was recorded with an epifluorescence microscope (Zeiss
Axiovert inverted microscope A200 Mot) equipped with a CCD camera
(Axiocam HR), using a 405/30 nm bandpass filter (LP450, exposure
time: 750 ms), a 470/40 nm bandpass filter (LP515, exposure time:
1500 ms) and a 546/12 nm bandpass filter (LP590, exposure time:
3500 ms). The alterations of the intra- and interchain processes of
POWT, due to. DNA-hybridisation, are seen as a change of the colour
and the intensity of the emitted light from POWT (FIG. 7).
EXAMPLE 10
Microcontact Printing of POWT
[0064] Sylgard 184 (Dow Corning, UK), a two component silicone
rubber (poly(dimethylsiloxane), PDMS), was used for preparing
elastomer stamps used for transferring POWT to solid surfaces. The
prepolymer and the curing agent is mixed according to the
instructions provided by the manufacturer. This is then poured on
templates prepared by photolithography using the negative
photoresist SU-8 (Micro Chem Inc., Newton, Mass., USA) as the
structural element on top of silicon wafers. Curing is accomplished
by heating to 130.degree. C. for at least 20 min. The height of
structures was 18 micrometer, and the substrate was a Si wafer
cleaned in a boiling aqueous solution containing 5% each of ammonia
and H.sub.2O.sub.2(TL-1 wash). The geometry for templates was
designed in CleWin Version 2.51 (WieWeb Software), and transferred
to a Cr mask, which was used in the photolithography step. After
developing the SU-8 structures on the silicon wafer, the template,
silanization (dimethyl-dicholorosilane) was done to obtain the
proper surface energy of the SU-8 template. The PDMS stamps were
plasma treated for .about.10 sec before being dip-coated in a
water-based solution of POWT (5 mg ml.sup.-1). The polymer was
dried on the top of the stamp with N.sub.2. The stamp was put face
down for 20-25 minutes, on a glass substrate previously cleaned
with a TL-1 wash, or a polystyrene surface modified by a 10 sec
oxygen plasma treatment. Both substrates were moistened before
stamp contact. After removal of the stamp, POWT had partly
transferred to the glass as shown in FIG. 9.
EXAMPLE 11
Electrical Detection of Hydrogels
[0065] When preparing the zwitterionic conjugated polymers with
single stranded oligonucleotide molecules (ssDNA), the detection
capability of recognizing another DNA molecule is utilized. The
conjugated zwitterionic polymer is mixed together with a 0.1
equivalent amount (on a monomer basis) of a single stranded
oligonucleotide in deionized water. Gold electrodes, which can be
patterned in any way if desired, on a glass support is cleaned with
ethanol. On top of these electrodes is a dispersion of a conducting
polymer (PEDOT-PSS, commercial name Baytron from Bayer AG) is
deposited. The zwitterionic polymer/oligonucleotide complex is
transferred on to the polymer surface by solution casting, contact
printing, ink-jet printing or in other ways. The resulting layer is
analyzed using 2- or 4-point resistance measurement, by
electrochemical methods or by impedance spectroscopy.
Sequence CWU 1
1
9 1 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 catgattgaa ccatccacca 20 2 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 2 tggtggatgg ttcaatcatg 20 3 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 3 tggtggatgc ttcaatcatg 20 4 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 4 tggtggaacg ttcaatcatg 20 5 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 5 tggtggaacc ttcaatcatg 20 6 42 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 6 Asn
Ala Ala Asp Leu Glu Lys Ala Ile Glu Ala Leu Glu Lys His Leu 1 5 10
15 Glu Ala Lys Gly Pro Val Asp Ala Ala Gln Leu Glu Lys Gln Leu Glu
20 25 30 Gln Ala Phe Glu Ala Phe Glu Arg Ala Gly 35 40 7 42 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 7 Asn Ala Ala Asp Leu Lys Lys Ala Ile Lys Ala Leu Lys Lys
His Leu 1 5 10 15 Lys Ala Lys Gly Pro Val Asp Ala Ala Gln Leu Lys
Lys Gln Leu Lys 20 25 30 Gln Ala Phe Lys Ala Phe Lys Arg Ala Gly 35
40 8 24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 8 agattggcgc attacgaggt taga 24 9 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 9 tctaacctcg taatgcgcca atct 24
* * * * *