Shay Mailloux

Bridging the Two Worlds: A Universal Interface between Enzymatic and DNA Computing Systems

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in the way we control electronic computers. Enzyme-based computational systems can respond to a great variety of small molecule inputs. They have the advantage of signal amplification and highly specific recognition. DNA computing systems are most often controlled by oligonucleotide inputs/outputs and are capable of sophisticated computing as well as controlling gene expressions. Here, we developed an interface that enables communication of otherwise incompatible nucleic-acid and enzyme-computational systems. The enzymatic system processes small molecules as inputs and produces NADH as an output. The NADH output triggers electrochemical release of an oligonucleotide, which is accepted by a DNA computational system as an input. This interface is universal because the enzymatic and DNA computing systems are independent of each other in composition and complexity.

Electrochemically stimulated release of lysozyme from an alginate matrix cross-linked with iron cations

An electrochemically generated alginate matrix cross-linked with Fe3+ cations was used to entrap lysozyme and then release it upon application of an electrochemical signal. The switchable behavior of the alginate hydrogel was based on the different interaction of Fe3+ and Fe2+ cations with alginate. The oxidized Fe3+ cations strongly interact with alginate resulting in its cross-linking and formation of the hydrogel, while the reduced Fe2+ cations weakly interact with alginate and do not keep it in the hydrogel state. Thus, the electrochemical oxidation of iron cations at +0.8 V (Ag/AgCl) in the presence of alginate and lysozyme resulted in the Fe3+-cross-linked alginate hydrogel thin-film on the electrode surface with the physically entrapped lysozyme. On the other hand, application of reductive potentials (e.g. −1.0 V) converted the iron cations to the Fe2+ state, thus resulting in dissolution of the alginate thin-film and lysozyme release. The bactericidal effect of the electrochemically released lysozyme was tested on the Gram-positive bacterium Micrococcus luteus demonstrating the same activity as the unadulterated lysozyme commercially supplied by Sigma-Aldrich. The present result represents the first step towards drug delivering systems (exemplified by the lysozyme release) based on alginate hydrogels and activated by electrochemical stimuli.

Majority and Minority Gates Realized in Enzyme-Biocatalyzed Systems Integrated with Logic Networks and Interfaced with Bioelectronic Systems

Biocatalytic reactions operating in parallel and resulting in reduction of NAD(+) or oxidation of NADH were used to mimic 3-input majority and minority logic gates, respectively. The substrates corresponding to the enzyme reactions were used as the input signals. When the input signals were applied at their high concentrations, defined as logic 1 input values, the corresponding biocatalytic reactions were activated, resulting in changes of the NADH concentration defined as the output signal. The NADH concentration changes were dependent on the number of parallel reactions activated by the input signals. The absence of the substrates, meaning their logic 0 input values, kept the reactions mute with no changes in the NADH concentration. In the system mimicking the majority function, the enzyme-biocatalyzed reactions resulted in a higher production of NADH when more than one input signal was applied at the logic 1 value. Another system mimicking the minority function consumed more NADH, thus leaving a smaller residual output signal, when more than one input signal was applied at the logic 1 value. The performance of the majority gate was improved by processing the output signal through a filter system in which another biocatalytic reaction consumed a fraction of the output signal, thus reducing its physical value to zero when the logic 0 value was obtained. The majority gate was integrated with a preceding AND logic gate to illustrate the possibility of complex networks. The output signal, NADH, was also used to activate a process mimicking drug release, thus illustrating the use of the majority gate in decision-making biomedical systems. The 3-input majority gate was also used as a switchable AND/OR gate when one of the input signals was reserved as a command signal, switching the logic operation for processing of the other two inputs. Overall, the designed majority and minority logic gates demonstrate novel functions of biomolecular information processing systems.

Substance Release Triggered by Biomolecular Signals in Bioelectronic Systems

A new approach to bioelectronic Sense-and-Act systems was developed with the use of modified electrodes performing sensing and substance-releasing functions. The sensing electrode was activated by biomolecular/biological signals ranging from small biomolecules to proteins and bacterial cells. The activated sensing electrode generated reductive potential and current, which stimulated dissolution of an Fe(3+)-cross-linked alginate matrix on the second connected electrode resulting in the release of loaded biochemical species with different functionalities. Drug-mimicking species, antibacterial drugs, and enzymes activating a biofuel cell were released and tested for various biomedical and biotechnological applications. The studied systems offer great versatility for future applications in controlled drug release and personalized medicine. Their future applications in implantable devices with autonomous operation are proposed.

Activation of a biocatalytic electrode by removing glucose oxidase from the surface--application to signal triggered drug release.

A biocatalytic electrode activated by pH signals was prepared with a multilayered nanostructured interface including PQQ-dependent glucose dehydrogenase (PQQ-GDH) directly associated with the conducting support and glucose oxidase (GOx) located on the external interface. GOx was immobilized through a pH-signal-cleavable linker composed of an iminobiotin/avidin complex. In the presence of GOx, glucose was intercepted at the external interface and biocatalytically oxidized without current generation, thus keeping the electrode in its nonactive state. When the pH value was lowered from pH 7.5 to 4.5 the iminobiotin/avidin complex was cleaved and GOx was removed from the interface allowing glucose penetration to the electrode surface where it was oxidized by PQQ-GDH yielding a bioelectrocatalytic current, thus switching the electrode to its active state. This process was used to trigger a drug-mimicking release process from another connected electrode. Furthermore, the pH-switchable electrode can be activated by biochemical signals logically processed by biocatalytic systems mimicking various Boolean gates. Therefore, the developed switchable electrode can interface biomolecular computing/sensing systems with drug-release processes.

Enzymatic filter for improved separation of output signals in enzyme logic systems towards ‘sense and treat’ medicine

The major challenge for the application of autonomous medical sensing systems is the noise produced by non-zero physiological concentrations of the sensed target. If the level of noise is high, then a real signal indicating abnormal changes in the physiological levels of the analytes might be hindered. Inevitably, this could lead to wrong diagnostics and treatment, and would have a negative impact on human health. Here, we report the realization of a filter system implemented to improve both the fidelity of sensing and the accuracy of consequent drug release. A new filtering method was tested in the sensing system for the diagnosis of liver injury. This sensing system used the enzymes alanine transaminase (ALT) and aspartate transaminase (AST) as the inputs. Furthermore, the output of the sensing system was designed to trigger drug release, and therefore, the role of the filter in drug release was also investigated. The drug release system consists of beads with an iron-cross-linked alginate core coated with different numbers of layers of poly-l-lysine. Dissolution of the beads by the output signals of the sensing system in the presence and absence of the filter was monitored by the release of rhodamine-6G dye encapsulated in the beads, mimicking the release of a real drug. The obtained results offer a new view of the problem of noise reduction for systems intended to be part of sense and treat medical devices.

Model system for targeted drug release triggered by immune-specific signals

AbstractA new sense-and-act system was realized by integrating a biocatalytic/bioaffinity electrode responding to immune signals represented by an antibody and a polymer-modified electrode loaded with drug-mimicking species. The release of the drug-mimicking species was achieved specifically in response to a signal antibody, thus demonstrating for the first time an immune-induced drug-releasing process. The present approach promises new options for future applications in controlled drug release and personalized medicine. FigureElectrochemical immune-sensing system was integrated with the substance-releasing modified electrode to demonstrate the immune-triggered drug release process

Enzymatic analysis of α-ketoglutaramate—A biomarker for hyperammonemia

Two enzymatic assays were developed for the analysis of α-ketoglutaramate (KGM)-an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to α-ketoglutarate (KTG) via a reaction catalyzed by ω-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (λ=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids.

Biocomputing, Biosensing and Bioactuation Based on Enzyme Biocatalyzed Reactions

Abstract The focus of this review paper is on the design and implementation of smart ‘Sense-and-Treat’ systems using enzyme-biocatalytic systems. These systems were used to perform biomolecular computing and they were functionally integrated with signal responsive materials aiming towards their biomedical use. Electrode interfaces, functionalized with signal-responsive materials, find applications in biocomputing, biosensing, and, specifically, triggered release of bioactive substances. ‘Sense-and-Treat’ systems require multiple components working together, including biosensors, actuators, and filters, in order to achieve closed-loop and autonomous operation. In general, biochemical logic networks were developed to process single biochemical or chemical inputs as well as multiple inputs, responding to nonphysiological (for concept demonstration purposes) and physiological signals (for injury detection or diagnosis). Actuation of drug-mimicking release was performed using the responsive material iron-cross-linked alginate with entrapped biomolecular species, responding to physical, chemical or biochemical signals.

Role of biomolecular logic systems in biosensors and bioactuators

Abstract. An overview of recent advances in biosensors and bioactuators based on biocomputing systems is presented. Biosensors digitally process multiple biochemical signals through Boolean logic networks of coupled biomolecular reactions and produce an output in the form of a YES/NO response. Compared to traditional single-analyte sensing devices, the biocomputing approach enables high-fidelity multianalyte biosensing, which is particularly beneficial for biomedical applications. Multisignal digital biosensors thus promise advances in rapid diagnosis and treatment of diseases by processing complex patterns of physiological biomarkers. Specifically, they can provide timely detection and alert medical personnel of medical emergencies together with immediate therapeutic intervention. Application of the biocomputing concept has been successfully demonstrated for systems performing logic analysis of biomarkers corresponding to different injuries, particularly as exemplified for liver injury. Wide-ranging applications of multianalyte digital biosensors in medicine, environmental monitoring, and homeland security are anticipated. “Smart” bioactuators, for signal-triggered drug release, for example, were designed by interfacing switchable electrodes with biocomputing systems. Integration of biosensing and bioactuating systems with biomolecular information processing systems advances the potential for further scientific innovations and various practical applications.