The electronic changes which occur in chemical or biological molecules when interacting with other molecules provide a sensitive and convenient method of studying molecule/molecule association. For example, technology capable of recording the electronic changes which occur in specific DNA/RNA sequences has been of great importance in numerous applications, such as in medical research and clinical diagnosis.
In general, DNA/RNA array sensors are based on single-stranded oligonucleotide (ODN) probes (of known sequence) immobilised on a physical substrate.
Addition of tagged ODNs in the sample may result in immobilisation of tagged ODNs via Watson-Crick base pairing (or hybridization) if the sequences are complementary. The resulting double stranded ODN is immobile, being anchored by the probe, as well as tagged by prior sample tagging.
The immobilisation of tagged ODNs provides the basis of sample detection and the resulting pattern is detected by (for example) fluorescence or chemiluminescences from the tag. The main disadvantages of such detection methods are cost, complexity and inefficiency of tagging reactions.
A number of alternative ‘label-free’ methodologies have also been developed, such as acoustic (Okahata et al., 1992; Ma, et al., 2002), optical (Piunno et al., 1994; Isola et al., 1998) and electrochemical (XU et al., 2000; Wang et al., 2001, 2003; Hashimoto et al., 1994; Napier et al., 1997; Pividori et al., 2000) methods which seek to detect the hybridization event directly. Electrochemical approaches using metal complexes (Takenaka et al., 2000), organic redox indicators (Millan et al., 1993) or nano particles (Wang et al., 2001, 2003) have also been investigated for their suitability to report hybridization.
Conducting polymers are attractive substrates for nucleotide sensors as they can act as an electronic transducer for charged species binding to their surface, and several methods for the immobilisation of ODN probes onto conducting polymers have been reported. In addition, the rich chemistry associated with fabrication of conducting polymer sensors and electrodes allows the detection of a broad range of chemical or biological materials according to the selection of probe and the capacity of the conducting polymer to bind the probe (Janata & Josowicz, 2003; Saxena & Malhotra, 2003).
Initial approaches to construct conducting polymer ODN sensors included direct adsorption of the ODN probes onto oxidized polypyrrole (PPy) films by electrostatic attractions (Palecek, 2002; Minehan et al., 1994) or incorporation of the ODN probe into the polymer film as a macro-counterion (Wang et al., 1999). However, these methods constrain the orientation of the ODN probe resulting in high steric and kinetic barriers to hybridization, and the possibility of oxidative damage to the ODN probes. The result is that such methods have poor sensory properties.
To overcome steric constraints, several groups have electro copolymerised N-position substituted pyrroles with pyrrole to functionalise the polymer for ODN probe linkage to the substrate (Wang et al., 2000; Livache et al., 1995; Lassalle et al., 2001). However, the resulting poly(N-substituted pyrrole) possessed lower conductivity, possibly due to a lack of ring planarity in the resulting polymer.
Of most interest has been a report that a 3-substituted pyrroles gave rise to a polymer film with higher conductivity (Delabouglise et al., 1989). A probe may be chemically coupled to a polymer by an amino-modified ODN.
U.S. Pat. No. 6,096,825 discloses such a conducting polymer formed from a 3-substituted polypyrrole polymer. The conducting polymer includes a linker unit located in the 3-position of pyrrole monomers. The linker is bound to the pyrrole, at one end, and to the probe at the other end.
The linker connecting the probe to the polymer represents a key feature in electrochemical transduction between the probe and the polymer. Typically short saturated organic chains of 2- to 4-carbon atoms have been used to connect the probe to the polymer (see also H. Peng et al Label-free electrochemical DNA sensor based on functionalised conducting polymer Biosensor and Bioelectronics 20 (2005) 1821-1828).
There is a continuing need to synthesize new conducting polymers, and in particular linkers which enhance or improve electronic transduction between the probe and the polymer backbone. In addition, there is a continuing need to provide conducting polymers which have negligible effect on the chemical or biological sensory properties of probes they are attached to.
Throughout this specification reference is made to “unsaturated” or “conjugated” organic chains. The words “unsaturated” and “conjugate” refer to double or triple bonds between two or more atoms. The words are used interchangeably throughout the specification.
Throughout the specification reference is made to “probes”. The use of the word “probe” in this specification is intended to be interpreted inclusively and including any chemical or biological species which may be tethered via a linker to a polymer backbone. A probe may include a single or double stranded ODN, a virus, a protein, a cell, a peptide nucleic acid (PNA), a polysaccharide, a drug, or an RNA, for example.
Throughout the specification reference is made to the phrase “sensory properties” in relation to a probe interaction with other chemical or biological molecules or species. Sensory properties should be interpreted as the ability of the probe to hybridize and/or otherwise interact through non-covalent bonding or the like with another chemical or biological molecules or species and to be efficiently transduced into a useful signal. Ideally a biological probe, for example, which is bound to a conducting polymer as far as possible, retains its natural sensory properties.