Synthesis of conducto-magnetic polymers as nano-transducers in biosensor design

This application claims benefit of Provisional Application No. 60/720,601, filed Sep. 26, 2005, which is incorporated herein by reference in its entirety.

This invention was funded in part by the Department of Homeland Security through the National Center for Food Protection and Defense, Homeland Security Grant No. R9106007104. The U.S. Government has certain rights to this invention.

Not Applicable.

(1) Field of the Invention

The present invention relates to novel polymers having conductive polyaniline, tetracyanoquinodimethane and a transferrin family member that can be used in conductimetric biosensors. One member of the transferrin family provided in the polymer is lactoferrin.

(2) Description of Related Art

Polymers, once used for insulating purposes, have gained prominence for electrical conductivity, among other novel traits such as magnetism and biodegradability. The appellation “synthetic metals” has been duly given to these polymers that exemplify the conductive/magnetic properties of the metallic inorganics (e.g. iron), as well as the flexibility and lightness of plastics.

The importance of conductive polymers alone is perhaps best demonstrated by the awarding of the 2000 Nobel Prize in chemistry for effort in the field. Conducting polymers can be utilized in diverse areas ranging from corrosion protection to microwave shielding (M. Gerard, A. Chaubey, and B. D. Malhotra. Application of conducting polymers to biosensors. Biosensors & Bioelectronics (2002), 17, 345). Analytical chemistry and chemical/biological sensors are significant areas of applications as well (Gerard et al. Ibid.). The conductive properties of the synthetic metals arise from the π-electron backbone and the single/double bonds of the n-conjugated system alternating down the polymer chain (Gerard et al. Ibid.). Some conducting polymeric structures include polyaniline (PANi), polypyrrole, polyacetylene, and polyphenylene (Gerard et al. Ibid.). Polyaniline, in particular, has been studied thoroughly because of its stability in fluid form, conductive properties, and strong bio-molecular interactions (Z. Muhammad-Tahir, and E. C. Alocilja. A disposable biosensor for pathogen detection in fresh produce samples. Biosystems Engineering (2004), 88, 145).

Fully organic polymers displaying magnetism are in high demand for their potentially tremendous applications in varying fields: electronic, magnetic, and photonic/photronic devices including information storage, magnetic imaging, static and low frequency magnetic shielding, and magnetic induction (A. J. Epstein, and J. S. Miller. Molecule- and polymer-based magnets, a new frontier. Synthetic Metals (1996), 80, 231). Indeed, organic magnets represent a completely new class of materials. So far, however, limited work has been done on the synthesis of conducting-magnetic polymers (F. Yan, G. Xue, J. Chen, and Y. Lu. Preparation of a conducting polymer/ferromagnet composite film by anodic-oxidation method. Synth Met (2001), 123, 17). Much of the work has involved the simple mixing of inorganic ferromagnetic powder with conducting polymer powder. This approach raises the issue of incompatibility with inorganic and organic phases as well as the difficulty in working with such a blend. A promising organic magnet derived from PANi, however, has been reported recently (N. A. Zaidi, S. R. Giblin, I. Terry, and A. P. Monkman. Room temperature magnetic order in an organic magnet derived from polyaniline. Polymer (2004), 45, 5683). This polymer, named PANiCNQ, is a result of the synthesis from PANi and tetracyanoquinodimethane (TCNQ), an acceptor molecule. Room temperature magnetization was observed in PANiCNQ only after a three month period.

Besides being electrically conductive and/or magnetic, organic polymers with biodegradable attributes can be appreciably employed in tissue engineering by regulating cellular activities such as cell migration, cell adhesion, DNA synthesis, and protein secretion (G. Shi, M. Rouabhia, Z. Wang, L. H. Dao, and Z. Zhang. A novel electrically conductive and biodegradable composite made of polypyrrole nanoparticles and polylactide. Biomaterials (2004), 25, 2477). Implantable biosensors may require biodegradability and biocompatibility as well. One promising biodegradable polymer is β-carotene, a source of vitamin A. It has large, delocalized π-electron systems over a chain of roughly 20 carbon atoms connected by bonds (G. Leatherman, E. N. Durantini, D. Gust, T. A. Moore, A. L. Moore, S. Stone, Z. Zhou, P. Rez, Y. Z. Liu, and S. M. Lindsay. Carotene as a molecular wire: Conducting atomic force microscopy. J. Phys. Chem. B (1999), 103, 4006). Furthermore, the molecule can be oxidized electrochemically, thus implying β-carotene may have conductive properties (Leatherman et al. Ibid.). As of yet unexplored polymeric composites with β-carotene and other polymers represent areas of investigation for feasible biodegradable and biocompatible materials with conductive properties.

A significant approach to test novel polymers with conductive, magnetic, biodegradable, and/or biocompatible traits is the biosensor, an analytical device capable of pathogen detection. Organic conductive polymers act as electrochemical transducers to transform biological signals to electric signals. Conductive polymers that also demonstrate magnetic properties are advantageous in biosensors. It is possible to guide biosensors within a body or organism by applying external magnets (e.g. glucose biosensors in diabetics). Furthermore, the polymers may be concentrated by drawing the sensors together with magnets, thereby increasing the yield of polymers conjugated with antibodies, and strengthening the output electrical signal.

Statistics has shown that pathogens result in an estimated 14 million illnesses, 60,000 hospitalizations, 1,800 deaths, and cost approximately $2.9-$6.7 billion in the United States each year due to food-borne diseases (P. S. Mead, L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. Food-related illness and death in the United States. Emerg Infect Dis (1999), 5, 607; J. C. Buzby, T. Roberts, C. T. J. Lin, and J. M. MacDonald. Bacterial foodborne disease: Medical costs and productivity losses. Agricultural Economics Report No. 741:100. USDA Economic Research Service, Washington, D.C., USA (1996)). Furthermore, possible bioterror threats after Sep. 11, 2001 has presented urgent needs of biosensors for surveillance of foods to prevent the contamination of food supplies (World Health Organization (WHO), Terrorist threats to food: Guidance for establishing and strengthening prevention . . . . WHO Food Safety Dept., Geneva, Switzerland (2002)). Of these numerous food-borne pathogens, Bacillus cereus has garnered notice as bacteria that can cause two types of food poisoning: a diarrheal type, and an emetic type (P. E. Granum, and T. Lund. Bacillus cereus and its food poisoning toxins. FEMS Microbiology Letters (1997), 157, 223). The former leads to diarrhea while the latter results in vomiting (Granum et al. Ibid.). The ubiquitous nature of the B. cereus pathogen is demonstrated by its status as a common soil saprophyte and association with foods, primarily plants, but also meats, eggs, and dairy products (Granum et al. Ibid.). It was implicated in a third of all cases of food poisoning in Norway (1988-1993), 47% in Iceland (1985-1992), and 22% in Finland (1992) (Granum et al. Ibid.). Furthermore, recent research has concluded that Bacillus anthracis and Bacillus cereus are of the same species (E. Helgason, O. A. Okstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and A. Koisto. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One species on the basis of genetic evidence. Applied and Environmental Microbiology (2000), 66, 2627). B. anthracis is responsible for the lethal disease anthrax, an agent in biological terrorism/warfare (Helgason et al. Ibid.). Thus, detection and defense using biosensors against B. cereus may accurately model and lead to heightened security with respect to B. anthracis.

Identification of pathogens by conventional methods, however, necessitates manual work and anywhere from 24 to 48 hours of incubation time (Z. Muhammad-Tahir, and E. C. Alocilja. Fabrication of a disposable biosensor Escherichia coli O157:H7 Detection. IEEE Sensors Journal (2003), 3, 345). Biosensors that are rapid, portable, accurate, sensitive and easy to use are crucial for optimal detection of pathogens, including B. cereus. Electrochemical immunosensors, one type of biosensor, utilize antibodies as a biological sensing element connected to an electrochemical transducer (the organic conductive polymer) (Z. Muhammad-Tahir, and E. C. Alocilja. A disposable biosensor for pathogen detection in fresh produce samples. Biosystems Engineering (2004), 88, 145). An advantage of utilizing antibodies is that the employment of antibodies specific to other pathogens can result in biosensors effective for said pathogens (e.g. E. coli O157:H7). Thus, the biosensor is not limited in effectiveness to just B. cereus.

The present invention provides a polymer which comprises an electrically conductive reaction product of an emeraldine polyaniline (PANi), tetracyanoquinodimethane (TCNQ) and a transferrin family member. In further embodiments, the transferrin family member is lactoferrin. In further embodiments, the polymer has magnetic properties. In still further embodiments, the polyaniline is provided as an emeraldine base or an emeraldine salt.