This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme-poly(L-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA-enzyme films show great promise in predicting genotoxicity of new drug and chemical product candidates. These devices combine metabolic biocatalysis, reactive metabolite-DNA reactions, and DNA damage detection. Catalytic voltammetry or electrochemiluminescence (ECL) can be used for high throughput arrays utilizing multiple LbL "spots" of DNA, enzyme and metallopolymer. DNA-enzyme films can also be used to produce nucleobase adduct toxicity biomarkers for detection by LC-MS. These approaches provide valuable high throughput tools for drug and chemical product development and toxicity prediction.

The electrical properties of single-walled carbon nanotubes (CNT) are of intense interest due to applications in nanoelectronics. Cyclic voltammetry and chronoamperometry have been used to explore the Ru(bpy)32+ electrocatalytic oxidation of DNA-solubilized carbon nanotubes. Dramatic current enhancements are observed with the addition of a CNT wrapped in an oligonucleotide sequence containing no oxidizable guanines. The current enhancement observed is solely due to the oxidation of the CNT by electrogenerated Ru(III) and subsequent recycling of the metal complex redox reaction. The chronoamperometric (CA) response is biphasic, and rate constants derived from the CA response were used to develop digital simulations of the cyclic voltammograms collected at the same CNT concentrations. Ten successive C' reactions were required to account for all of the observed signal. The oxidation of the CNT is a multielectron process, and this effect arises from the multiple electron donor sites in the carbon nanotube as well as the over oxidation of each site.

Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electro*des monitored via Ru(bpy)32+–mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (KI*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with KI* of 268.2, 142.3 and 204.2 µM, respectively.

This paper describes redox chemistry in semisolid molten salts ionic liquids of DNA in which the counterions of the phosphates are redox-active metal complexes with bipyridine ligands labeled with MW 350 poly-(ethylene glycol) (PEG) "tails", e.g., M(bpy 350 ) 3 DNA (where M ) Co, Ni, and bpy 350 ) 4,4′-(CH 3 (OCH 2 -CH 2 ) 7 OCO) 2 -2,2′-bipyridine). Other redox-active metal complexes are added to the M(bpy 350 ) 3 DNA melt: (a) the PEG-tailed metal bipyridine complexes Fe(bpy 350 ) 3 (ClO 4 ) 2 and Ru(bpy 350 ) 3 (ClO 4 ) 2 and (b) the nontailed complexes Os(bpy) 3 Cl 2 (bpy ) 2,2′-bipyridine) and Os(bpy) 2 dppzCl 2 (dppz ) dipyridophenazine). In example a, electrogeneration of the powerful oxidizers [Fe(bpy 350 ) 3 ] 3+ and [Ru(bpy 350 ) 3 ] 3+ gives microelectrode voltammetry indicative of electrocatalytic oxidation of DNA base sites. Since physical diffusion of the metal complexes is slow in the viscous semisolids (and that of DNA is nil), the rate of electron hopping between the base sites of the DNA becomes a significant contributor to the overall charge transport rate, as deduced from analysis of the voltammetry. DNA base site self-exchange rate constants of 1.1 × 10 6 and 1.8 × 10 6 s -1 are estimated from measurements using Fe(bpy 350 ) 3 3+ and Ru(bpy 350 ) 3 3+ oxidants, respectively. In example b, a complex known to be a DNA intercalator in aqueous solutions is found to not be an intercalator in the DNA molten salt environment, as deduced from measurements showing the physical diffusion coefficients of aqueous nonintercalator Os(bpy) 3 Cl 2 and aqueous intercalator Os(bpy) 2 dppzCl 2 to be indistinguishable in the M(bpy 350 ) 3 DNA melt.

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