Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6825—Nucleic acid detection involving sensors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

• Polyelectrolyte complex e.g. zwitterionic polythiophenes
• a receptor e.g. polynucleotide, antibody etc.
• 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.
• conjugated polymers 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.
• conjugated 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.
• 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.
• the object of the present invention is therefore to provide means and methods that meet these and other needs.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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..
• Figure 1 shows the chemical structure of poly(3-[(S)-5-amino-5-carboxyl-3- oxapentyl]-2,5-thiophenylene hydrochloride) (POWT), a zwitterionic poly thiophene derivative.
• FIG. 2 schematically illustrates the method according to the invention.
• Figure 3 shows the absorptionspectra of 1. 16 ⁇ mol POWT (on a monomer basis) and 0 mol ( ⁇ ), 6.4 nmol (0) 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 ( ⁇ ).
• Figure 4 shows the emission spectra of 23.1 nmol POWT (on a monomer basis) and 0 mol ( ⁇ ), 1.28 nmol (0), 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 ( ⁇ ). All of the emission spectra were recorded with excitation at 400 nm.
• Figure 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 (0), 1.0 equivalent of a negatively charged peptide, JR2E ( ⁇ ), 0.5 equivalents JR2E plus an addition of 0.5 equivalents JR2K ( ⁇ ), 0.5 equivalents JR2K plus an addition of 0.5 equivalents JR2E ( ⁇ ), 2.0 equivalent JR2K ( ⁇ ), and 2.0 equivalent JR2E (A) 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.
• Figure 6 shows the Emission spectra of 26.1 nmol POWT in 20 mM Na- phosphate pH 7.5, upon addition of 4.9 ⁇ M of a synthetic peptide, with a receptor site for carbonic anhydrase (thin line), and after addition of 13 ⁇ M of carbonic anhydrase (bold line).
• Table 1 shows the difference in ratio of emission intensity at the wavelengths 540nm/585nm and 540nm/670nm 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.
• Table 2 shows the absorption maximum and the ratio of the intensity of the emitted light at 540nm/610nm for POWT and POWT/peptide complexes after 10 min incubation in 20 mM Na-phosphate pH7.4
• 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.
• 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.
• 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.
• 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.
• 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.
• the biosensor device comprises a suitable receptacle for said substrate, and a complex between polyelectrolyte and receptor is formed on the substrate.
• 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.
• poly(3-[(S)-5-amino-5-carboxyl-3-oxapentyl]-2,5-thiophenylene hydrochloride) (POWT, see Figure 1) can be mentioned.
• Studies of this polymer see Andersson, M.; Ekeblad, P. O.; Hjertberg, T.; Wennerstr ⁇ m, O.; Inganas, O. Polymer Commun.
• 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.
• the zwitterionic groups create versatile hydrogen bonding patterns with different molecules.
• 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.
• 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, neuro transmitters, 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 zwitterionic groups create versatile hydrogen bonding patterns with different molecules.
• the zwitterionic polyelectrolytes of the present invention form a complex with one or more receptor molecules ( Figure 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.
• 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
• amino acid side chains can easily be modified to contain polar and non-polar groups, or groups with hydrogen bonding abilities.
• the conjugated polyelectrolyte/ receptor molecules complex 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.
• 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.
• 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
• amino acid side chains can easily be modified to contain polar and non-polar groups, or groups with hydrogen bonding abilities.
• 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, F ⁇ rster 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.
• FRET F ⁇ rster resonance energy transfer
• 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.
• 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.
• ICS Image correlation spectroscopy
• 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.
• 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.
• SPR Surface plasmon resonance
• Quartz crystal microbalance and dissipation 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.
• Ellipsometry imaging or null 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.
• 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.
• 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.
• the above described methods can also be implemented in the form of microarrays, to give an "image" of the composition of a biological sample.
• 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.
• 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
• 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 micro fluidic printing by the use of soft lithography or the BiacoreTM (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.
• 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.
• HTS high-throughput-screening
• 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 BiacoreTM system (see example 8).
• the receptor molecules 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.
• 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.
• the receptor molecules 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 ( Figure 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.
• HTS high-throughput- screening
• 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.
• 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.
• PDAMAC poly(diallyldimethylammonium chloride)
• PVPy poly-4-vinylpyridine
• PVPy poly (pyrrole) (PPy)
• PVA poly(vinylalcohol)
• PANI poly (aniline)
• 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.
• 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.
• 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.
• 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.
• 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).
• zwitterionic conjugated polyelectrolyte and zwitterionic conjugated polyelectrolyte/ biomolecule complexes can be printed by micro contact printing using elastomer stamps ( Figure 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.
• Example 1 Optical detection of DNA-hybridisation in solution.
• a stock solution containing 0.5 mg ml- 1 POWT in de-ionised water was prepared and incubated for 30 minutes.
• 50 ⁇ l of the polymer solution was mixed with 64 ⁇ l of DNA-solution (100 nmol mH, 5 ' - CAT GAT TGA ACC ATC CAC CA -3 ' , purchased from SGSDNA, K ⁇ ping, Sweden).
• Example 2 Fluorescent detection of DNA-hybridisation in solution.
• a stock solution containing 0.5 mg ml" 1 POWT in de-ionised water was prepared and incubated for 30 minutes. 10 ⁇ l of the polymer solution was mixed with 12.8 ⁇ l of DNA-solution (100 nmol mH, 5 ' - CAT GAT TGA ACC ATG CAC CA -3 ' , purchased from SGSDNA, K ⁇ ping, Sweden).
• Example 3 Fluorescent detection of single nucleotide polymorphism (SNP) in solution.
• a stock solution containing 0.5 mg ml" 1 POWT in de-ionised water was prepared and incubated for 30 minutes. 10 ⁇ l of the polymer solution was mixed with 12.8 ⁇ l of DNA-solution (100 nmol ml" 1 , 5 ' - CAT GAT TGA ACC ATC CAC CA -3 ' , purchased from SGSDNA, K ⁇ ping, Sweden).
• 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, K ⁇ ping, Sweden) to a final volume of 1500 ⁇ l containing 10 mM Na-phosphate.
• a stock buffer solution Na- phosphate pH 7.5 and a 1.0 equivalent amount of the respective nucleotide
• 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 540nm/585nm and 540nm/670nm 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 540nm/585nm and 540nm/670nm are influenced by the degree of mismatch between the DNA strands (Table 1).
• Example 4 Optical detection of self-assembly of synthetic peptides in solution.
• a stock solution containing 3.7 mg ml 1 POWT in de-ionised water was prepared and incubated for 30 minutes. 10 ⁇ l of the polymer solution was mixed with 10 ⁇ l or 20 ⁇ 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 the positively charged peptide (NH2-N-A-A-D-L-K-K-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 mH
• the samples were diluted with a stock buffer solution (Na-phosphate pH 7.4) and 10 ⁇ l de-ionised water or 10 ⁇ l of the positive /negative peptide solution (2.2 mg ml" 1 ) to a final volume of 2000 ⁇ l containing 20 mM Na-phosphate.
• a stock buffer solution Na-phosphate pH 7.4
• 10 ⁇ l de-ionised water or 10 ⁇ l of the positive /negative peptide solution (2.2 mg ml" 1 ) to a final volume of 2000 ⁇ l containing 20 mM Na-phosphate.
• Example 5 Fluorescent detection of self-assembly of synthetic peptides in solution.
• a stock solution containing 3.7 mg ml- 1 POWT in de-ionised water was prepared and incubated for 30 minutes.
• 10 ⁇ l of the polymer solution was mixed with 10 ⁇ l or 20 ⁇ 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-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-
• the samples were diluted with a stock buffer solution (Na-phosphate pH 7.4) and 10 ⁇ l de-ionised water or 10 ⁇ l of the positive /negative peptide solution (2.2 mg ml- 1 ) to a final volume of 2000 ⁇ l containing 20 mM Na-phosphate.
• the samples were incubated for 10 minutes in room temperature and the emission spectra (Figure 5, Table 2) were recorded with an ISA Jobin-Yvon spex FluoroMax-2 apparatus.
• 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
• 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 (figure 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 (figure 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 interchain process (aggregation of polymer chains).
• the difference in ratio of the emission intensity at the wavelengths 540nm/610nm and 540nm/670nm are influenced, as the different complexes between POWT and the different peptides are formed (Table 2).
• the intra- and inter-chain processes of the POWT/JR2E (receptor) are clearly altered upon addition of JR2K (analyte).
• a stock solution containing 0.5 mg ml 1 POWT in de-ionised water was prepared and incubated for 30 minutes. 10 ⁇ l of the polymer solution was mixed with 5 ⁇ 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- 1 ), and diluted with de- ionised water to a final volume of 100 ⁇ l.
• 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-
• the samples were diluted with a stock buffer solution (Na-phosphate pH 7.4) and 40 ⁇ l deionised water or 40 ⁇ l of a carbonic anhydrase solution (6.4 mg ml 1 ) to a final volume of 1000 ⁇ l containing 20 mM Na-phosphate.
• a stock buffer solution Na-phosphate pH 7.4
• 40 ⁇ l deionised water or 40 ⁇ l of a carbonic anhydrase solution 6.4 mg ml 1
• 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 (figure 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.
• 0.5 ⁇ l droplets of POWT 0.5 mg ml" 1
• the polymer droplets were cross-linked with 0.5 ⁇ 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, K ⁇ ping, Sweden), respectively.
• 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 470/40nm bandpass filter (LP515, exposure time: 1500 ms) and a 546/ 12 nm bandpass filter (LP590, exposure time: 500 ms) .
• LP450 405/30 nm bandpass filter
• LP590 exposure time: 500 ms
• Example 8 Detection of DNA-hybridisation on a surface by surface plasmon resonance (SRP).
• SRP surface plasmon resonance
• 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 °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, K ⁇ ping, 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, K ⁇ ping, 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.
• PBS phosphate pH 7,4 buffer
• NaCl salt concentrations
• the injected DNA was solved in the same buffer as the running buffer and the concentration was usually around 1 ⁇ M.
• the temperature was set to 25 °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 0.5x2.5 mm. Manual loading is required and the maximal injection volume is 100 ⁇ l.
• Example 9 Fluorescent detection of DNA-hyridisation in an array prepared by microfluidic channels.
• Sylgard 184 (Dow Corning, UK), a two component silicone rubber (poly (dime thylsiloxane), 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, MA, USA) as the structural element on top of silicon wafers. Curing is accomplished by heating to 130°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 H2O2 (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.
• silanization (dimethyl-dicholorosilane) was done to obtain the proper surface energy of the SU-8 template.
• a solution of POWT (10 mg ml- 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.
• a PDMS stamp with 100 ⁇ 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.
• Example 10 Microcontact printing of POWT.
• 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, MA, USA) as the structural element on top of silicon wafers. Curing is accomplished by heating to 130°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 H2O2 (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.
• silanization (dimethyl-dicholorosilane) was done to obtain the proper surface energy of the SU-8 template.
• the PDMS stamps were plasma treated for - 10 sec before being dip-coated in a water-based solution of POWT (5 mg ml 1 ). The polymer was dried on the top of the stamp with N 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 figure 9.
• 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.

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