Jan Halámek

From “cyborg” lobsters to a pacemaker powered by implantable biofuel cells

Enzyme-based biofuel cells implanted into living lobsters or designed as fluidic systems mimicking human blood circulation were used for powering electronic devices. Two lobsters with implanted biofuel cells connected in series were able to generate open circuit voltage (Voc) up to 1.2 V and an electrical watch, selected as a model electronic device, was activated by the power extracted from the “living battery”. The fluidic system composed of five cells filled with human serum solution connected in series generated Voc of ca. 3 V and was able to power a pacemaker. Sustainable operation of the pacemaker was achieved with the system closely mimicking human physiological conditions characteristic of normal and pathophysiological glucose concentrations with the fluidic rate typical for a blood circulation upon resting or performing physical exercises. While the “cyborg” lobsters demonstrate a model system with future possible military, homeland security and environmental monitoring applications, the system activating a pacemaker presents practicality for biomedical applications. The first demonstration of the pacemaker activated by the physiologically produced electrical energy shows promise for future electronic implantable medical devices powered by electricity harvested from the human body.

Living battery - biofuel cells operating in vivo in clams.

Biofuel cells implanted in living clams and producing sustainable electrical power in vivo were integrated in batteries. The “electrified” clams, being biotechnological living “devices”, were able to generate electrical power using physiologically produced glucose as the fuel. The activity of the living batteries was dependent on the environmental conditions which are affecting physiological processes in clams. The living batteries generated open circuitry voltage (Voc), short circuitry current (Isc) and maximum power (Pmax) of ca. 800 mV, 25 μA, 5.2 μW and ca. 360 mV, 300 μA, 37 μW for the serial and parallel connections of 3 “electrified” clams, respectively. A clam-battery was connected to a capacitor which was charged up to 240 mV providing accumulation of electrical energy up to 28.8 mJ. Discharging the capacitor on an electrical motor resulted in the motor rotation. The “electrified” clams integrated in batteries demonstrated the possibility of activating electrical/electronic devices using energy produced in vivo.

Multianalyte digital enzyme biosensors with built-in Boolean logic.

Novel biosensors based on the biocomputing concept digitally process multiple biochemical signals through Boolean logic networks of coupled biomolecular reactions and produce output in the form of a YES/NO response. Compared to traditional single-analyte sensing devices, biocomputing approach enables a high-fidelity multianalyte biosensing, particularly beneficial for biomedical applications.

Electrochemically controlled drug-mimicking protein release from iron-alginate thin-films associated with an electrode.

Novel biocompatible hybrid-material composed of iron-ion-cross-linked alginate with embedded protein molecules has been designed for the signal-triggered drug release. Electrochemically controlled oxidation of Fe(2+) ions in the presence of soluble natural alginate polymer and drug-mimicking protein (bovine serum albumin, BSA) results in the formation of an alginate-based thin-film cross-linked by Fe(3+) ions at the electrode interface with the entrapped protein. The electrochemically generated composite thin-film was characterized by electrochemistry and atomic force microscopy (AFM). Preliminary experiments demonstrated that the electrochemically controlled deposition of the protein-containing thin-film can be performed at microscale using scanning electrochemical microscopy (SECM) as the deposition tool producing polymer-patterned spots potentially containing various entrapped drugs. Application of reductive potentials on the modified electrode produced Fe(2+) cations which do not keep complexation with alginate, thus resulting in the electrochemically triggered thin-film dissolution and the protein release. Different experimental parameters, such as the film-deposition time, concentrations of compounds and applied potentials, were varied in order to demonstrate that the electrodepositon and electrodissolution of the alginate composite film can be tuned to the optimum performance. A statistical modeling technique was applied to find optimal conditions for the formation of the composite thin-film for the maximal encapsulation and release of the drug-mimicking protein at the lowest possible potential.

Enzyme logic gates for the digital analysis of physiological level upon injury.

A biocomputing system composed of a combination of AND/IDENTITY logic gates based on the concerted operation of three enzymes: lactate oxidase, horseradish peroxidase and glucose dehydrogenase was designed to process biochemical information related to pathophysiological conditions originating from various injuries. Three biochemical markers: lactate, norepinephrine and glucose were applied as input signals to activate the enzyme logic system. Physiologically normal concentrations of the markers were selected as logic 0 values of the input signals, while their abnormally increased concentrations, indicative of various injury conditions were defined as logic 1 input. Biochemical processing of different patterns of the biomarkers resulted in the formation of norepiquinone and NADH defined as the output signals. Optical and electrochemical means were used to follow the formation of the output signals for eight different combinations of three input signals. The enzymatically processed biochemical information presented in the form of a logic truth table allowed distinguishing the difference between normal physiological conditions, pathophysiological conditions corresponding to traumatic brain injury and hemorrhagic shock, and abnormal situations (not corresponding to injury). The developed system represents a biocomputing logic system applied for the analysis of biomedical conditions related to various injuries. We anticipate that such biochemical logic gates will facilitate decision-making in connection to an integrated therapeutic feedback-loop system and hence will revolutionize the monitoring and treatment of injured civilians and soldiers.

Enzyme-Based NAND and NOR Logic Gates with Modular Design

The logic gates NAND/NOR were mimicked by enzyme biocatalyzed reactions activated by sucrose, maltose and phosphate. The subunits performing AND/OR Boolean logic operations were designed using maltose phosphorylase and cooperative work of invertase/amyloglucosidase, respectively. Glucose produced as the output signal from the AND/OR subunits was applied as the input signal for the INVERTER gate composed of alcohol dehydrogenase, glucose oxidase, microperoxidase-11, ethanol and NAD(+), which generated the final output in the form of NADH inverting the logic signal from 0 to 1 or from 1 to 0. The final output signal was amplified by a self-promoting biocatalytic system. In order to fulfill the Boolean properties of associativity and commutativity in logic networks, the final NADH output signal was converted to the initial signals of maltose and phosphate, thus allowing assembling of the same standard units in concatenated sequences. The designed modular approach, signal amplification and conversion processes open the way toward complex logic networks composed of standard elements resembling electronic integrated circuitries.

Responsive Interface Switchable by Logically Processed Physiological Signals: Toward “Smart” Actuators for Signal Amplification and Drug Delivery

Biomarkers characteristic of liver injury, alanine transaminase and lactate dehydrogenase, were processed by an enzyme-based system functioning as a logic AND gate. The NAD+ output signal produced by the system upon its activation in the presence of both biomarkers was then biocatalytically converted to a decrease in pH. The acidic pH value biocatalytically produced by the system as a response to the biomarkers triggered the restructuring of a polymer-modified electrode interface. This allowed a soluble redox species to approach the electrode surface, thus switching the electrochemical reaction ON. The redox transformations activated by the biochemical signals resulted in an amplification of signals. This system represents the first example of an integrated sensing-actuating chemical device with the implemented AND Boolean logic for processing natural biomarkers at their physiologically relevant concentrations.

Enzymatic AND Logic Gates Operated Under Conditions Characteristic of Biomedical Applications

Experimental and theoretical analyses of the lactate dehydrogenase and glutathione reductase based enzymatic AND logic gates in which the enzymes and their substrates serve as logic inputs are performed. These two systems are examples of the novel, previously unexplored class of biochemical logic gates that illustrate potential biomedical applications of biochemical logic. They are characterized by input concentrations at logic 0 and 1 states corresponding to normal and pathophysiological conditions. Our analysis shows that the logic gates under investigation have similar noise characteristics. Both significantly amplify random noise present in inputs; however, we establish that for realistic widths of the input noise distributions, it is still possible to differentiate between the logic 0 and 1 states of the output. This indicates that reliable detection of pathophysiological conditions is indeed possible with such enzyme logic systems.

Electrode interfaces switchable by physical and chemical signals for biosensing, biofuel, and biocomputing applications

AbstractThis review outlines advances in designing modified electrodes with switchable properties controlled by various physical and chemical signals. Irradiation of the modified electrode surfaces with various light signals, changing the temperature of the electrolyte solution, application of a magnetic field or electrical potentials, changing the pH of the solutions, and addition of chemical/biochemical substrates were used to change reversibly the electrode activity. The increasing complexity in the signal processing was achieved by integration of the switchable electrode interfaces with biomolecular information processing systems mimicking Boolean logic operations, thus allowing activation and inhibition of electrochemical processes on demand by complex combinations of biochemical signals. The systems reviewed range from simple chemical compositions to complex mixtures modeling biological fluids, where the signal substrates were added at normal physiological and elevated pathological concentrations. The switchable electrode interfaces are considered for future biomedical applications where the electrode properties will be modulated by the biomarker concentrations reflecting physiological conditions. FigureModified electrodes were reversibly switched between active and inactive states by various physical and chemical signals.

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.