Field of the Invention

The invention relates to an electrically conductive. DNA polymer hybrid; in particular it relates to a DNA polymer hybrid nanowire. The invention also relates to a method of manufacturing an electrically conductive DNA polymer h brid conductive nanowire, a sensing device and an integrated circuit comprising the DNA polymer hybrid.

Background to the Invention

Techniques such as chemical vapour deposition and atomic layer deposition are well developed and useful for fabricating nanoscale layers of inorganic materials. However, the. synthesis of one- dimensional nanostructures, or nanowkes, is a less developed area of technology and presents many m re chall e nge s .

Conducting polymer nanowkes bring the advantage of significantly enhanced sensitivity over electrode/polymer film systems. The improvement in sensitivity in nanowire devices over bulk hlms employed as chemical sensors anses because a nanowire has a much large rs surface to volume ratio. This factor means that the conductance of the nanowire is much more strongly affected by chemical, species interacting with its surface than would be a bulk him of the same material. The result is the percentage change in current flowing through the nanowire is much larger and therefore nanowire devices can be used to measure smaller quantities or chemical species. Nanowkes are also flexible and their conductivity can be chemically controlled.

One attractive approach towards preparing one-dimensional nanostructures is to integrate bio- molecules, often with additional synthetic functionality, into bio-hybrid materials.

In particular, natural double-stranded DNA, a well defined biopolymer of a single dimension, has been a promising bio-building block. DNA is extremely adaptable for use in the formation of nanomateriais, due to its relative stability and programmable length. Structurally, DNA can be ret ot micrometres long but only several nanometres in diameter. It has therefore gained much intere as a template for conductive material.

It is possible to combine DNA with a polymer to form a DMA polymer hybrid comprising an anionic DNA strand and a cationic polymer strand.

Several groups have recently reported the. use of conducting polymers, such as polyamline and. polyp yrrole to form nanowires using DNA templates.

Although the surface modification of semiconductor and metallic substrates, such as silicon and gold, is well understood, there are significantly fewer reports in the literature that demonstrate the selective functionali ation of polymer nanowires.

There has been growing recent: interest in the utilisation of one -dimensional nanosmictures in nanoseale devices, such as nhotodetecfors, waveguides and chemical sensors.

It would be desirable to provide an improved electrically conductive D A polymer hybrid.

Statement of lavetmon

The invention provides an electrically conductive DNA polymer hybrid as claimed in Claim 1. The invention farther provides an integrated circuit as claimed in Claim 8. The invention further provides a sensor as claimed in Claim 9.

Another aspect of the invention provides a method of making an electrically conductive DNA polymer hybrid as claimed in Claim 10.

Another aspect of the invention provides a method of making an electrically conductive D A polymer hybrid as claimed in Claim 1 1.

The inventio further provides a method of making a molecule as claimed in Claim 15. Another aspect of the invention provides a monomer as claimed in Claim 16. Another aspect of the invention, provides a polymer as claimed in Claim 20.

Preferred aspects f the invention are specified in the claims dependent on Claims 1, 10 and 11 and the description that follows:

The invention provides an electrically conductive DNA polymer hybrid comprising a polymer having a first functional group that is complementary to a second functional group on a molecule, wherein the said functional groups are reactable by a coupling reaction to bind the molecule to the polymer.

In a preferred embodiment, the coupling reaction is azide-alkyne cyci.oaddition.

The first functional group may be an atkyne group and the second functional group may be an azide group.

The first functional group may be an azide group and the second functional group may be an alkyne group.

Preferably, the polymer is derived from -pentynyl-2- (2-thienyl)-pyrrole.

The molecule may be an antibody, single stranded DNA, double stranded DNA, RNA, peptide nucleic acid, an ap tamer, protein, supramolecular ion cage sensor, polymer, metal or semiconductor nanoparticle.

Advantageously, the DNA polymer hybrid is a nanowire.

The invention further provides an integrated circuit comprising the electrically conductive DNA polymer hybrid.

The invention further provides a sensor comprising the electrically conductive DNA polymer hybrid.

The invention further provides a method of making an electrically conductive DNA polymer hybrid comprising the steps of

(i) functionaiising a first molecule with a first functional group

(ii) reacting the iirst molecule with D A in the presence oi an oxidant. The invention further provides a method of making an electrically conductive DNA polymer hybrid comprising the step:; of

(i) functionalising a first molecule with a first iunctional group

(a) functionalising a second molecule with a second functional group complementary to the first functional group of the first molecule

(iii) reacting the first and second functional groups fay a coupling reaction.

Preferably, the first molecule is functionalised by performing N-alkylatk of 2-(2-Thienyl)-pyrroJe. in one embodiment, the first molecule is a monomer and die method farther comprises the step of polymerisation of the monomer.

The method may further comprise the step of reacting at least one functional group on the first molecule with a complementary functional group on the second molecule by azide-alkyne cyc!oadditior..

The invention further provides a method of making a molecule for use in 2 DNA polymer hybrid comprising the step of performing N-alkylation of 2-(2-Thienyi)-pyrro;e with 5-chloro-l -pent ne.

The invention further provides a monomer having the chemical structure:

The invention further provides a polymer having the chemical structure:

Brief Description of the Drawings

Figure 1 is a schematic representation of die formation of a conductive aikynyi polymer/ DNA nanowiie and modification by a "click" coupling reaction.

Figure 2 is a schematic representation of the formation of a conductive aikynyi polymer/DNA nanowire and modification by a "click" coupling reaction.

Figure 3 sbov/s resuirs of Fourier Transform infra Red (FUR) spectroscopic studies.

Detailed Description, of the Preferred Embodiments 2-f2-ThienyS)-pyrrale (1) is a known monomer having the following structure:

Synthesis of 2-(2-Thienyi) -pyrrole (1) has been decribed by Vautrm er al (FiecUOchemistry

Communications 1 (1999) 233-237).

According to the present invention, 2-(2-Thienyl)-pyrrole (1) was synthesised in three steps described by Vautrin et ai.

N-alkylation of ( 1) with 5-chloro- 1 -pentyne results in the co-monomer N-pentynyl-2-(2.-thicnyl)- pyrrole (2) as shown below:

Unlike polypyrrole or polytliiophene, it is difficult 1:0 produce rcgiorcgular polymers from a monomer having the structure or (2). The invention provides a DNA-po!ymer hybrid nanowire comprising a polymer based on (2), which is shown below (where n is any number). The polymer (2') is formed in situ by reacting the monomer (2) (30mM), DNA (~80μ§ ,uL ^" ') as a template, and FeCl, (lmM) as an oxidant, in a water/ DMF (dimethylformamide) medium.

A DNA/polymer hybrid based on the polymer (2'} is formed in situ by reacting the monomer (2), DNA, and FeCi ₃ , in a water/DMF medium and incubating for two hours. T ^' his forms a nanowire structure, which is a hybrid of D A and the polymer (2'). The process is shown in more detail, in Figure 1.

Surprisingly, 2' retains its conductivity when polymerised in die form of a wire.

Furthermore, it is possible to perform further reactions on the wire to functionatise the polymer, while retaining conductivity. This is surprising since aikynyl groups themselves are known to polymerise and hence become unavailable for further reactions.

Reactions of the type known as "click" chemistry are well known and understood in the field of chemistry. One such "click" reaction is copper catalysed azide-alkyne Huisgen cycioaddirion. This reaction can be performed to modify the (2')/DNA hybrid. In this way. any desired molecule having an azide group can be "clicked" onto a nanowire at the aikynyl group.

The aikynyl groups of the polymer 2' remain intact after polymerisation and the resulting polymer/ DNA hybrid nanowire. has exposed aikynyl groups onto which a desired molecule may be

"clicked".

S The product (3) oi the reaction is shown below, Here the alkyne group is coupled to an azide group of a desired molecule or receptor R, where R is any desired group, molecule or receptor that is "clicked" onto the alkynyl group vis copper catalysed azide-alkyne cydoaddirion.

Surprisingly, the polymer (3) also retains conductivity, despite the potential for loss of planarity of the polymer system, and hence electronic conjugation, due to steric interference from bulky R groups.

Referring now to Figure 2, in the formation of the DN A/polymer nanowire, (2) is oxidised in the presence of D A. Oxidation results iu (2) becoming positively charged and attracted to the negatively charged phosphate groups 4 of the D A 5, The alkynyl groups 6 are free to bond -with the aaide groups of desired receptors R. in a preferred embodiment the DNA that is used as template is λ-DNA. However, calf- thymus (CT) or other DNA could be used.

The resulting nanowire preferably has a diameter in the range of 5-10nni.

The conductive wire of the invention may be used as a sensing element for a wide range of applications. The a¾ido derivative of a receptor is simply "clicked" onto the nanowire.

Changes in the conductivity of the polymer/DNA nanowire hybrid, caused for example, by binding of a substrate to the receptor site, are then used to detect the presence of that particular substrate. DNA-polymer hybrid tranow ^' ires based on polymer 2' were formed in situ by reacting the monomer (2) (30mM), DNA (~ 80pg uL ^! ) as a template, and Fed-, (ImM) as an oxidant, in a ater/DMF medium.

A iter incubation for two hours, nanowire formation was confirmed by atomic force microscopy (AFM) by deposition and molecular combining of droplets of these solutions across an n-<l l l >-Si wafer. Tapping mode AFM of the poly2'A-DNA hybrid indicated the formation of wire-like structures aligned on the surface.

In an alternative embodiment of the invention, the functionalisation or "click" reaction may be performed on the monomer (2) or polymer (2 ^* ) before it is bonded ro DNA. in particular, the "click" reaction may be performed on the monomer (2) and the resulting "click" conjugate is then polymerised in the presence of DNA.

Figure 3 shows the .results of FTJR spectroscopic studies, which were performed to ascertain that the nanowire hybrids imaged by AFM were in fact the polymer 2 ^* emplated onto DNA .

In order to establish th t the terminal alkyne group of (2) remains available for further chemistry following polymerisation, polymer (2') and bundles of polymer (2')/CT-D hybrids were analysed by FTiR spectroscopy.

The polymer (2') was prepared, by the oxidative polymerisation of (2) by using FeC!3 in a water/DMF (2: 1) mixture and analysed by FTIR after drying onto a native silicon oxide wafer.

The expected bands due. to die CSC bond, (2120 cm ^' ), alkyne C-H (3300cm ^! ) and aikane C-Fl (2879cm ^'1 ) stretches are observed in the spectra of (2') and (2')/ DNA hybrids (shown as C in Figure 2) and correlate well with drat of the monomer (2) (shown, as A in Figure 3).

Therefore upon polymerisation, the alkynyl group of (2) remains intact and the polymer/ DN A nanowires exhibit alkynyl groups at which a "click" reaction can be performed. Example i - Click reaction with 3-azido propanol

The standard click procedure involved the treatment of monomer 2 of polymer 27DNA with a "click' ^" solution of water/ rert- butyl alcohol (2: 1) containing sodium ascorbate (Stri ), copper (11) sulphate (ImM and 3-azido propanol (98mM). The mixture was stirred gently in darkness for 8 hours before washing with excess of nanopure water and drying n vacuum oven at 25"C.

As shown in Figure 3, FTIR spectra of (2'}/DNA wires, before and after treatment , showed that the alkynyl-hyfarid materia! undergoes molecular modification via "click chemistry". in Figure 3, line A shows the FTIR. spectrum of (2). Line B shows the FTIR spectrum of (2) after a click reaction with 3-azido propanol.

Line C in Figure 3 shows the FTIR spectrum of ( )/ΌΝΑ and line D shows the FTIR spectrum of (2')/DNA after click reaction with 3-azido propanol.

The molecular modification of the D A/ lkynyl hybrid following the click reaction is evident: primarily through the disappearance of the C≡C (2100cm ^' ) and die aikyne C-H (3300cm ') bands. In addition the terminal hydroxy! group of the propanol moiety appears in the spectra as a typically broad 0--H stretching band at 100··3όΟΟ<:ηϊ '. The characteristic bands corresponding to the C~0 stretching in the pyrimidine bases of DNA (1723 cm ^"1 and 1674cm ^{' 1} ) remain unaffected in the spectrum of polymer 2V calf thymus DNA before and after the reaction, although they are shifted to a higher wavenumber in comparison to natural DNA, 1705cm ' and 1658cm ¹ . The reason for this shift is thought to be due to electrostatic interaction of the cationic polymer 2' with the anionic DN phosphate backbone.

This successfully demonstrates that the "click" reaction can be performed at alkytiyi groups exposed along the polymer 2' /DNA nanowires.

Example 2— CHck reaction with dansyl azide

Atomic Force Microscopy (AFM) images of polymer 2'/D A hybrid nanowires after

functionalisation with dansyl azide were also recorded.

Individual wires were visualised and were similar in height (5-15nm) to the non-modified polymer 2 ^* /DHA iiybrid materials. The coupling reaction has no discernible detrimental effect on the one dimensional structure of the polymer/DNA hybrid material and therefore new functionalities or sensing oxoum mav be incorporated, into a conductive nanowire via this method.

The electronic conductance of the nanowires was characterised by Electrostatic Force Microscopy (EFM) phase imaging ("scanned conductance microscopy").

Polymer 2\/DN A wires were combed onto a silaftised silicon oxide chip and then imaged. The polymer 2'A -DNA nanowires were immobilised on die silicon chip and were dien incubated for 16 hours with a "click" solution containing dansyl azide instead of 3-azido propanol.

After "click" modification with the dansyl azide fluorophore the nanowires were re-imaged by EFM. The nanowires remained structurally intact and conductive after the addition of the triazole-coupied fluorophore. Fluorescence micrographs of the silicon chip showed the fiinctionalised conductive nanowires were clearly visible under excitation at 365am.

"Click" type reactions do nor require high temperature or aggressive reactions. They occur under simple reaction conditions and usually create no by-products. Therefore the reaction for modification of the polymer/DNA hybrid may be performed at room temperature and be tolerant of the chemical character of the desired receptor eg an antibody.

Functionalisation of the polymer may be perfomed by an alternative synthetic organic coupling reaction.

R may be any desired molecule or biomolecule such as an antibody, single stranded UNA, double stranded DNA, R A, peptide nucleic acid, an aptamer, protein, suprarnolecular ion cage sensor, polymer, metal or semiconductor nanoparticle. In fact R may be any entity that has a free azide group. In an alternative embodiment, the polymer may be funcrionalised with an azide group and the receptor to be bound thereto is functionalised with a complementary alkyne. In this embodiment R is therefore any entity with a free alkyne group. in an alternative embodiment, the carbon chain length between the .ting system and the alkyne group of the monomer 2 can be varied and hence the polymer 2' structure may be varied. For example the chain could include i, 2, or 3 carbon atoms or even a number of CH2 groups long. The number of rings could also be changed.

Some examples of alternative structures of the monomer 2 axe shown below:

Thus a generic structure of the monomer 2 a :an be written as follows:

1 , 2, 3