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.

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in a way we control electronic computers. Enzyme-based computational systems can respond to a great variety of small molecule inputs. They have an 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 since the enzymatic and DNA computing systems are independent of each other in composition and complexity.

An electrochemically generated alginate matrix cross-linked with Fe 3+ 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 Fe 3+ and Fe 2+ cations with alginate. The oxidized Fe 3+ cations strongly interact with alginate resulting in its cross-linking and formation of the hydrogel, while the reduced Fe 2+ 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 Fe 3+ -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. À1.0 V) converted the iron cations to the Fe 2+ 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. † Electronic supplementary information (ESI) available: AFM imaging of the Fe 3+ -cross-linked alginate hydrogel thin-film on the electrode surface with the physically entrapped lysozyme. See

A new Sense-and-Act system was realized by the integration of a biocomputing system, performing analytical processes, with a signal-responsive electrode. A drug-mimicking release process was triggered by biomolecular signals processed by different logic networks, including three concatenated AND logic gates or a 3-input OR logic gate. Biocatalytically produced NADH, controlled by various combinations of input signals, was used to activate the electrochemical system. A biocatalytic electrode associated with signal-processing "biocomputing" systems was electrically connected to another electrode coated with a polymer film, which was dissolved upon the formation of negative potential releasing entrapped drug-mimicking species, an enzyme-antibody conjugate, operating as a model for targeted immune-delivery and consequent "prodrug" activation. The system offers great versatility for future applications in controlled drug release and personalized medicine.

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.