In recent years, industrial applications for immobilized enzymes have grown dramatically, for example, in the diverse fields of fine pharmaceutical synthesis, clinical analysis, and in the production of bulk chemicals. However, the majority of industrial enzymes perform one of only three reactions: isomerization, hydrolysis or oxidation. None of these industrial enzymatic processes involves biological endothermic synthesis, and thus the utility of biological catalysts has been greatly limited. Attempts have been made to overcome this limitation by finding a way to artificially generate high-energy, nonphysiological and biological electron sources, including enzyme cofactor electron sources. Energy requiring enzymes such as formate dehydrogenase, hydrogenase, or ntrogenase could then be used in the synthesis of fuel or other important chemicals and in highly specific electrochemical sensors.
In order to catalyze a biochemical reaction involving one or more biochemical substrates, an enzyme must be capable of effecting electron transfer to or from the substrates. However, the amino acids which form the protein moieties of enzymes (the apoenzymes) cannot undergo changes in redox state. Therefore, all redox active enzymes require nonprotein, redox active organic, metal or metal-organic cofactors in order to perform these biological electron transfers. There exist several types of cofactors to accomplish a variety of electron transfer processes, each apoenzyme requiring a specific cofactor for activation.
In nature, electron transfer processes occur either between two protein-bound cofactors or between a protein bound cofactor and some small metabolic compound. Among the most versatile of the redox active cofactors are the flavin nucleotides which are involved both in transferring charge between protein-bound cofactors and in catalyzing metabolic redox changes. The flavin cofactors are unique because they can transfer either single or pairs of electrons. Flavin cofactors form an integral part of the redox active sites of many different enzymes. In these enzymes the apoenzyme confers specificity to the reaction, permitting only specific chemicals to arrive at the active site. Thus, a given cofactor can perform several different processes depending on the protein environment at a specific active site. These redox active flavin cofactors are derived from flavin compounds having the formula ##STR1## in which R.sub.1 is a ribose derivative; e.g. riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). In natural systems FMN and FAD allow the required electron transfer between substrates.
Attempts have been made to provide electrochemical control of such biochemical reactions and regeneration of the flavin cofactor's redox state by coupling the natural system (e.g. the cofactor, apoenzyme and substrate) with an electronic assembly (e.g. an electrode and a current source). Many of these methods, however, have had the disadvantages of less than optimum electron transfer efficiency, diffusional resistance, chemical instability of the components, steric and electronic interference, and/or adherence problems.
As in designing any electrocatalytic system, a method must be found for rapidly and efficiently transporting charges between the electrocatalytic species (in this case the enzyme cofactor) and the electrode surface. Additionally, the biochemical substrate must be free to interact with the electrocatalytic species without steric or electronic interference. Thus, a need exists for a means of achieving a more direct electronic linkage between the electrode and the active site of the enzyme in order to effect efficient energy transfer for the catalysis of many biochemical reactions.
The orientation of the cofactor in the enzyme has been found to be critical for the proper functioning of the enzyme. For example, it is usually found that the flavin cofactors must be oriented with the benzenoid end of the flavin cofactor projecting into the solution, while the other, heterocyclic end is surrounded by the apoenzyme. Attempts have been made to avoid steric and electronic interference by coupling the flavin cofactor to the electrode surface at the flavin 8-alpha methyl position. However, prior to the present invention, most such electrodes have required strongly basic coupling conditions, have exhibited a low biological activity level, and have been subject to breakdown of the chemical bond at the 8-alpha methyl position of the flavin moiety when exposed to even weakly basic environments.
The successful formation of an efficient, biologically active, chemically stable, electrochemically coupled flavin cofactor modified electrode would provide the basis for the development of a new class of bioelectronic detectors and catalysts. Further, the use of an appropriate means of linking the electrode surface and the cofactor would allow large active surface coverages of the cofactor-mediators and encourage effective electron transfer from the electrode to the enzyme by presenting a low energy barrier to such transfer due to the short, low resistance distance (less than 10 angstroms) between the electrode and the enzyme. Monitoring the current flow from an electrode with a protein renatured on its surface or a flavoprotein or holoenzyme in solution would allow specific quantitation (due to enzymatic specificity) of analytes by convenient electronic methods. Further, using the electrode to maintain the biological cofactor at a specific potential would permit biocatalytic reactions to be driven by electrochemical energy without the need for replenishing of the expensive biological energy sources, e.g. redox active cofactors.
Commonly owned, copending U.S. application Ser. No. 862,951, filed May 14, 1986 and issued Nov. 3, 1987 as Pat. No. 4,744,193, disclosing a cofactor modified electrode in which a flavin cofactor moiety is bonded to an electrode surface via an imidazole moiety and a suitable linking group, presents to the art one such modified electrode, as well as methods for its preparation and use.