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Adam Heller, Ernest Cockrell Sr. Chair in Engineering Emeritus of the John J. McKetta Department of Chemical Engineering at The University of Texas at Austin, recalls his childhood in the Holocaust and his contributions to science and technology that earned him the US National Medal of Technology and Innovation in a conversation with Elton J. Cairns, Professor of Chemical and Biomolecular Engineering at the University of California, Berkeley. Dr. Heller, born in 1933, describes the enslavement of his father by Hungarians in 1942; the confiscation of his family's home, business, and all its belongings in 1944; and his incarceration in a brick factory with 18,000 Jews who were shipped by the Hungarians to be gassed by Germans in Auschwitz. Dr. Heller and his immediate family survived the Holocaust and arrived in Israel in 1945. He studied under Ernst David Bergmann at the Hebrew University, and then worked at Bell Laboratories and GTE Laboratories, where he headed Bell Lab's Electronic Materials Research Department. At GTE Laboratories, he built in 1966 the first neodymium liquid lasers and in 1973 with Jim Auborn conceived and engineered the lithium thionyl chloride battery, one of the first to be manufactured lithium batteries, which is still in use. After joining the faculty of engineering of The University of Texas at Austin, he cofounded with his son Ephraim Heller TheraSense, now a major part of Abbott Diabetes Care, which produced a microcoulometer that made the monitoring of glucose painless by accurately measuring the blood glucose concentration in 300 nL of blood. He also describes the electrical wiring of enzymes, the basis for Abbott's state-of-the-art continuous glucose monitoring system. He discusses his perspective of reducing the risk of catastrophic global warming in a wealth-accumulating, more-energy-consuming world and provides advice for students entering careers in science or engineering.

Membranes formed of thermodynamically stable cubic phase lyotropic liquid crystals (LLCs) could replace the presently used polymeric membranes, applied to reduce the flux of glucose in semicontinuous, subcutaneously implanted, user-replaced, miniature, amperometric glucose sensors, assisting in the management of diabetes. LLC-forming amphiphilic compounds set and toughen spontaneously after mixing with water, without undergoing chemical change. When applied by doctor-blading, they form membranes having three-dimensionally interconnected water channels of uniform diameter, with reproducible glucose transport-characteristics. We find that the best studied cubic phase LLCs, which are formed of monoolein and water, are not useful in their intended application because they are hydrolyzed by serum lipases. Those formed of phytantrioi, a liquid at ambient temperature, and water, are not hydrolyzed but change their shape and size in a dehydration and rehydration cycle. Because glucose sensors are sterilized and stored in a sealed package in a dry atmosphere, drying and rehydralion must not change the transport characteristics. A third, novel, LLC-forming, amphiphile 1-O-[beta]-(3,7,11,15-tetramethylhexadecyl)-D-ribopyranoside, I, was synthesized, and its phase diagram was tailored by adding Vitamin E acetate, to form a cubic phase. The phase was stable through the 20 [degrees]C-90 [degrees]C temperature range in excess of water and had the desired glucose-transport characteristics. A preferred LLC, II, was formed of water and I containing 7 wt % of Vitamin E acetate. When II was applied to a wired glucose oxidase bioelectrocatalyst, sensors of reproducible glucose-sensitivity were formed. At a 0.1 mm thickness of II, the membrane reduced the glucose flux 5-fold and increased the 90% response-time by less than 2 rain. The membrane was mechanically rugged, withstanding the ~ 1 N [m.sup.-2] maximal shear stress at 5 mm diameter electrodes rotating at 4000 rpm. The activation energy for glucose permeation through II was reduced to 15.6 kJ/mol, making the sensors's current less temperature-dependent than that of the polymericmembrane overcoated implantable glucose sensors.

When implanted in animals, enzyme-containing battery electrodes, biofuel cell electrodes, and biosensors are often damaged by components of the biological environment. An [O.sub.2] cathode, superior to the classical platinum cathode, which would be implanted, as part of a caseless physiological pH miniature Zn-[O.sub.2] battery or as part of a caseless and membraneless miniature glucose-[O.sub.2] biofuel cell, is rapidly damaged by serum urate at its operating potential. The cathode is made by electrically connecting, or wiring, reaction centers of bilirubin oxidase to carbon with an electron-conducting redox hydrogel. In the physiological pH 7.3 electrolyte battery or biofuel cell, the [O.sub.2] cathode should operate at, or positive of, 0.3 V (Ag/AgCl), where the urate anion, a common serum component, is electrooxidized. Because an unidentified urate electrooxidation intermediate, formed in the presence of [O.sub.2], damages the wired bilirubin oxidase electrocatalyst, urate must be excluded from the cathode. Unlike [O.sub.2], which permeates through both the lipid and the aqueous interconnected networks of cubic-phase lyotropic liquid crystals, urate permeates only through their continuous three-dimensional aqueous channel networks. The aqueous channels have well-defined diameters of ~5 nm in the monoolein/water cubic-phase liquid crystal. Through tailoring the wall charge of the aqueous channels, the anion/cation permeability ratio can be modulated. Thus, doping the monoolein of the monoolein/water liquid crystal with 1,2-dioleoyl-sn-glycero-3-phosphate makes the aqueous channel walls anionic and reduces the urate permeation in the liquid crystal. As a result, the ratio of the urate electrooxidation current to the [O.sub.2] electroreduction current is reduced from 1:3 to 1:100 for 5-mm [O.sub.2] cathodes rotating at 1000 rpm. Doping with 1,2-dioleoylsn-glycero-3-phosphate also increases the shear strength of the cubic-phase monoolein/water lyotropic liquid crystal. While the undoped liquid crystal is promptly damaged at the 0.1 N [m.sup.-2] average shear stress generated by rotating the 5-mm-diameter disk cathode at 1000 rpm in a physiological aqueous solution, the 10 mol % 1,2- dioleoyl-sn-glycero-3-phosphate-doped film remains intact. The mechanical strengthening of the lyotropic liquid crystal by the two-tailed 1,2-dioleoyl-sn-glycero-3-phosphate is attributed to cross-linking hydrophobic bonds (i.e., bonds resulting of the increase in entropy upon the freeing of the translation and rotation of multiple water molecules), which is analogous to the strengthening of polymer-based plastic materials by cross-linking through covalent bonds.

We describe a simple, potentially low-cost, amperometric, enzyme-amplified, sandwich-type immunoassay, monitoring IgG at a concentration as low as ~7 pg/mL with a dynamic range of [10.sup.4]. The assay utilizes a screen-printed carbon electrode on which a redox hydrogel and avidin are co-electrodeposited. To neutralize nonspecifically binding positively charged microdomains of the avidin, two polyanions, poly(acrylic acid-co-maleic acid) and poly(acrylic acid), are applied. These polyanions bind to the film not only electrostatically but also by Michael addition reaction to cysteine, lysine, or arginine functions of the avidin. The electrode is then made specific for the analyte, for which rabbit IgG was chosen, by conjugating the film-bound avidin to biotin-labeled anti-rabbit IgG. After exposure to the tested solution and capture of rabbit IgG, the sandwich is completed by conjugation of horseradish-peroxidase (HRP)-labeled anti-rabbit IgG. Electrical contact between the HRP and the electrode-bound hydrogel results in the formation of an electrocatalyst for the electroreduction of [H.sub.2][O.sub.2] to water. The application of the poly(acrylic acid-co-maleic acid) and the poly(acrylic acid) reduces the nonspecific adsorption-associated noise, lowers the detection limit from 3 ng/mL (~20 pM analyte antibody concentration) to ~7 pg/mL (~40 fM analyte antibody concentration), and also expands the dynamic range to [10.sup.4].

We report the amperometric detection of glucose at 2 fM concentration in a physiological buffer solution at 1 atm 02 pressure. The sensitive assay is based on the close to absolute electroreductive stripping of 02 from the solution near the glucose electrooxidizing anode. The glucose was detected by its electrooxidation on a stationary glassy carbon disk surrounded by an also stationary platinum ring. The disk was coated with a film of glucose oxidase (GOx), electrically 'wired' with PVP-[[Os[(N,N'-dimethyl-2,2'-biimidazole).sub.3]].sup.2+/3+] (polymer I), having a redox potential of -0.19 V versus Ag/AgCl. The ring was coated with bilirubin oxidase (BOD) 'wired' with PAA-PVI-[Os[(4,4'-dichloro-2,2'-bipyridine).sub.2][Cl].sup.+/2+] (polymer II), having a redox potential of + 0.36 V versus Ag/AgCl. The ring-disk electrode was held facing up, and a 30-[micro]L drop was placed on it for the assay, with the ring poised at -0.3 V/AgAgCl and the disk poised at -0.1 V/Ag/AgCl. Even though the atmosphere over the drop was Ou at 1 atm pressure, the wired BOD disk scavenged the [O.sub.2] so effectively that the glucose-reduced FAD[H.sub.2] of GOx was not oxidized by [O.sub.2], the natural cosubstrate of the enzyme.

The electrochemical sandwich-type, enzyme-amplified assay of Zhang, Kim, and Heller (Anal. Chem. 2003, 75, 3267-3269) was simplified by replacing the amplifying horseradish peroxidase with bilirubin oxidase (BOD). BOD catalyzes the reduction of ambient [O.sub.2] to water and obviates the need for adding [H.sub.2][O.sub.2]. Femtomolar ([10.sup.-15] M) concentrations of DNA were detected at a 10-[micro]m-diameter tip of a carbon fiber electrode. Correspondingly, a few thousand copies of DNA were detected in ~5-[micro]L samples. The sandwich is formed in an electron-conducting redox hydrogel, to the polymer of which a DNA capture sequence is bound. Capture of the analyte DNA and its hybridization with a BOD-labeled complementary DNA sequence, electrically connects the BOD label to the electron-conducting redox polymer, which is in electrical contact with the electrode. Placing the BOD in contact with the redox polymer thus converts the noncatalytic base layer into a catalyst for the electroreduction of [O.sub.2] to water at +0.12 V (vs Ag/AgCl) (Figure 1). In an exemplary assay, ~3000 copies of the iron transporting sequence of the sit gene of Shigella flexneri were detected without PCR amplification.

Laccase, a copper enzyme catalyzing the four-electron reduction of [O.sub.2] to water, has been shown by others to be a useful label in enzyme-linked immunoassays, in which the substrate is ambient [O.sub.2] instead of an added chemical, such as hydrogen peroxide, or a phosphate ester of a phenol. Laccase-catalyzed [O.sub.2] reduction is, however, inhibited by halides, which complex the enzyme's copper ions. Replacement of laccase by bilirubin oxidase, a copper enzyme retaining its maximal activity at high chloride concentrations and at pH 7.2, allows enzyme-amplified affinity assays with [O.sub.2] as the substrate in neutral-pH chloride solutions, exemplified here by the assay of DNA, the duplexes of which are unstable at low ionic strength but are stable in strong NaCl solutions.

A redox hydrogel with an apparent electron diffusion coefficient (D(sub app)) is described. The order of magnitude increase in D(sub app) relative to redox hydrogels results from the tethering of redox centers to the backbone of the cross-linked redox polymer backbone through 13 atom spacer arms.

A large fraction of the presently mass-manufactured (> [10.sup.8] units/year) electrochemical biosensors, used mostly by diabetic people to monitor their blood glucose levels, have screen-printed carbon working electrodes. An earlier study (Campbell, C. N., et al. Anal. Chem. 2002, 74, 158-162) showed that nucleic acids can be assayed at 1 nM concentrations by a sandwich-type amperometric method. The assay was performed with vitreous carbon working electrodes on which an electron-conducting polycationic redox polymer and avidin were coelectrodeposited. Because the rate of the electrodeposition increases with the surface density of the polycationic redox polymer, its practicality depends on pretreatment of the surface, which adds anionic functions. (Gao, Z., et al. Angew. Chem. Int. Ed. 2002, 41, 810-813). Here it is shown that the required conducting redox polymer films can be electrodeposited on potentially mass manufacturable electrodes made by screen-printing hydrophilic carbon inks on polyester sheets. The modified electrodes are made in two steps. First a polycationic electron-conducting redox polymer is cross-linked and electrodeposited by applying a negative potential. Next, an amine-terminated 20-base single-stranded oligonucleotide is electrodeposited by ligand-exchange. Both steps involve exchange of a labile inner sphere chloride ligand of the polymer-bound osmium-complex: Cross-linking and electrodeposition of the redox polymer result when innersphere chloride anions of the osmium complexes are exchanged by imidazole functions of neighboring chains. Incorporation of the oligonucleotide in the redox polymer results in the formation of a coordinative bond between the terminal amine (attached through a spacer to the oligonucleotide) and the osmium complex. In testing for the presence of a 38-base oligonucleotide, the analyte, in a 15- or 25-[micro]L droplet of hybridization solution, is hybridized with and captured by the 20-base electrode-bound sequence; then it is hybridized with an 18-base horseradish peroxidase labeled sequence. When the HRP label electrically contacts the redox polymer, the film becomes an electrocatalyst for the reduction of [H.sub.2][O.sub.2] to water at 0.10 V (Ag/AgCl). Flow of the [H.sub.2][O.sub.2]-reduction current indicates the presence of the assayed sequence.

A study reported the characteristics of a novel cathode on which O2 is electroreduced to water under physiological conditions. The cathode is made by electrically connecting (wiring) redox centers of bilirubin oxidase (BOD) from Trachyderma tsunodae (Tt) to carbon electrodes through a redox polymer.

Scanning electrochemical microscopy (SECM) was used to study horseradish peroxidase (HRP) immobilized with copolymer on insulating substrates (glass slide or polycarbonate membrane filter). Two methods were used to immobilize HRP: In the first, HRP was coimmobilized by cross-linking on a glass slide with a copolymer swelled in water to form a hydrogel; in the second, the same copolymer and avidin were coimmobilized on the glass slide and biotin-labeled HRP was conjugated to the avidin of the film. SECM was then used to detect the presence of the bound enzyme by observing the feedback current in a solution of benzoquinone and hydrogen peroxide, when hydroquinone was generated at the tip. A detection limit less than 7 x [10.sup.5] HRP molecules within a ~7-[micro]m-diameter area was demonstrated.

The electroreduction of O2 to water under physiological conditions at a currently density of 5 mA cm(super -2) and at a potential only 0.18 V reducing versus that of the reversible O2/H2O electrode at pH 7.4 is investigated. The potential application of this reaction carried out on immobilized electrode is suggested for use in flow systems and in bioinert films.

A one-step enzyme-amplified amperometric sandwich hybridization test for RNA and DNA is described. The test utilizes a carbon electrode, modified with a film of co-electrodeposited avidin and redox polymer; the redox polymer electrically 'wiring' horseradish peroxidase (HRP) reaction centers upon contact. The film is made specific for the particular RNA or DNA sequence tested by conjugating its avidin with a biotinylated oligonucleotide, complementary to the assayed sequence. This oligonucleotide-modified redox polymer film, prepared prior to the test, forms the base of the sandwich. The center layer of the sandwich, added in the test, is the analyte RNA or DNA; its top is a second complemetary oligonucleotide, which is HRP-labeled, and is cohybridized in the test. The test consists of mixing the analyte DNA or RNA solution, the HRP-labeled oligonucleotide solution, and a hydrogen peroxide solution, immersing the base-layer carrying electrode applying a potential of 0 V versus Ag/AgCl, and measuring the [H.sub.2O.sub.2] electroreduction current. Completion of the sandwich brings the HRP label into electrical contact with the redox polymer, convening the nonelectrocatalytic base layer into an electrocatalyst for the electroreduction of [H.sub.2O.sub.2] to water. Flow of [H.sub.2O.sub.2] electroreduction current when the electrode is poised near Ag/AgCl potential indicates the presence of the analyte RNA or DNA. The current density for the maximally sandwich-covered electrode was 250 [micro] A [cm.sup.-2], exceeding more than a 100-fold the current density flowing upon nonspecific binding of the HRP-labeled oligonucleotide. High concentrations of irrelevant DNA and diluted serum did not interfere with the assay. When the electrodes were rotated in order to make the solution-phase mass transport rapid, the test was completed in ~30 min. The test was applied in probing for the presence of a 60-base E. coli mRNA sequence.