Bioelectronic Interface Connecting Reversible Logic Gates Based on Enzyme and DNA Reactions

It is believed that connecting biomolecular computation elements in complex networks of communicating molecules may eventually lead to a biocomputer that can be used for diagnostics and/or the cure of physiological and genetic disorders. Here, a bioelectronic interface based on biomolecule‐modified electrodes has been designed to bridge reversible enzymatic logic gates with reversible DNA‐based logic gates. The enzyme‐based Fredkin gate with three input and three output signals was connected to the DNA‐based Feynman gate with two input and two output signals—both representing logically reversible computing elements. In the reversible Fredkin gate, the routing of two data signals between two output channels was controlled by the control signal (third channel). The two data output signals generated by the Fredkin gate were directed toward two electrochemical flow cells, responding to the output signals by releasing DNA molecules that serve as the input signals for the next Feynman logic gate based on the DNA reacting cascade, producing, in turn, two final output signals. The Feynman gate operated as the controlled NOT gate (CNOT), where one of the input channels controlled a NOT operation on another channel. Both logic gates represented a highly sophisticated combination of input‐controlled signal‐routing logic operations, resulting in redirecting chemical signals in different channels and performing orchestrated computing processes. The biomolecular reaction cascade responsible for the signal processing was realized by moving the solution from one reacting cell to another, including the reacting flow cells and electrochemical flow cells, which were organized in a specific network mimicking electronic computing circuitries. The designed system represents the first example of high complexity biocomputing processes integrating enzyme and DNA reactions and performing logically reversible signal processing.     

Boolean Logic Networks Mimicked with Chimeric Enzymes Activated/Inhibited by Several Input Signals

Reactions catalyzed by artificial allosteric enzymes, chimeric proteins with fused biorecognition and catalytic units, were used to mimic multi-input Boolean logic systems. The catalytic parts of the systems were represented by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). Two biorecognition units, calmodulin or artificial peptide-clamp, were integrated into PQQ-GDH and locked it in the OFF or ON state respectively. The ligand-peptide binding cooperatively with Ca²⁺ cations to a calmodulin bioreceptor resulted in the enzyme activation, while another ligand-peptide bound to a clamp-receptor inhibited the enzyme. The enzyme activation and inhibition originated from peptide-induced allosteric transitions in the receptor units that propagated to the catalytic domain. While most of enzymes used to mimic Boolean logic gates operate with two inputs (substrate and co-substrate), the used chimeric enzymes were controlled by four inputs (glucose – substrate, dichlorophenolindophenol – electron acceptor/co-substrate, Ca²⁺ cations and a peptide – activating/inhibiting signals). The biocatalytic reactions controlled by four input signals were considered as logic networks composed of several concatenated logic gates. The developed approach allows potentially programming complex logic networks operating with various biomolecular inputs representing potential utility for different biomedical applications.     

Biomolecular Release from Alginate‐modified Electrode Triggered by Chemical Inputs Processed through a Biocatalytic Cascade – Integration of Biomolecular Computing and Actuation

Biocatalytic cascades involving more than one or two enzyme‐catalyzed step are inefficient inside alginate hydrogel prepared on an electrode surface. The problem originates from slow diffusion of intermediate products through the hydrogel from one enzyme to another. However, enzyme activity can be improved by surface immobilization. We demonstrate that a complex cascade of four consecutive biocatalytic reactions can be designed, with the enzymes immobilized in an LBL‐assembled polymeric layer at the alginate‐modified electrode surface. The product, hydrogen peroxide, then induces dissolution of iron‐cross‐linked alginate, which results in release process of entrapped biomolecular species, here fluorescently marked oligonucleotides, denoted F‐DNA. The enzymatic cascade can be viewed as a biocomputing network of concatenated AND gates, activated by combinations of four chemical input signals, which trigger the release of F‐DNA. The reactions, and diffusion/release processes were investigated by means of theoretical modeling. A bottleneck reaction step associated with one of the enzymes was observed. The developed system provides a model for biochemical actuation triggered by a biocomputing network of reactions.     

Switchable electrode controlled by Boolean logic gates using enzymes as input signals

Application of Boolean logic operations performed by enzymes to control electrochemical systems is presented. Indium–tin oxide (ITO) electrodes with the surface modified with poly-4-vinyl pyridine (P4VP) brush were synthesized and used as switchable electrochemical systems. The switch ON and OFF of the electrode activity were achieved by pH changes generated in situ by biocatalytic reactions in the presence of enzymes used as input signals. Two logic gates operating as AND/OR Boolean functions were designed using invertase and glucose oxidase or esterase and glucose oxidase as input signals, respectively. The electrode surface coated with a shrunk P4VP polymer at neutral pH values was not electrochemically active because of the blocking effect of the polymer film. The positive outputs of the logic operations yielded a pH drop to acidic conditions, resulting in the protonation and swelling of the P4VP polymer allowing penetration of a soluble redox probe to the conducting support, thus switching the electrode activity ON. The electrode interface was reset to the initial OFF state, with the inhibited electrochemical reaction, upon in situ pH increase generated by another enzymatic reaction in the presence of urease. Logically processed biochemical inputs of various enzymes allowed reversible activation–inactivation of the electrochemical reaction.     

Alert-type biological dosimeter based on enzyme logic system

A cooperative effect of two biomarkers, α-amylase and lactate dehydrogenase, was used to analyze radiation-caused tissue damage in vitro in model solutions of human serum. The analytical system was based on the recently emerged biocomputing concept applying biocatalytic cascades for logic processing of biochemical input signals. The studied system resembled a Boolean NAND logic gate in which the change of the optical output signal from a high level (logic value 1) to a low level (logic value 0) confirmed the presence of both biomarkers at pathological concentrations (1,1 input signals), thus yielding the conclusion about radiation tissue damage. The system operates in a digital YES/NO format as an alert-type biosensor with a built-in Boolean logic.     

Digital biosensors with built-in logic for biomedical applications—biosensors based on a biocomputing concept

This article reviews biomolecular logic systems for bioanalytical applications, specifically concentrating on the prospects and fundamental and practical challenges of designing digitally operating biosensors logically processing multiple biochemical signals. Such digitally processed information produces a final output in the form of a yes/no response through Boolean logic networks composed of biomolecular systems, and hence leads to a high-fidelity biosensing compared with traditional single (or parallel) sensing devices. It also allows direct coupling of the signal processing with chemical actuators to produce integrated “smart” “sense/act” (biosensor-bioactuator) systems. Unlike common biosensing devices based on a single input (analyte), devices based on biochemical logic systems require a fundamentally new approach for the sensor design and operation and careful attention to the interface of biocomputing systems and electronic transducers. As common in conventional biosensors, the success of the enzyme logic biosensor would depend, in part, on the immobilization of the biocomputing reagent layer. Such surface confinement provides a contact between the biocomputing layer and the transducing surface and combines efficiently the individual logic-gate elements. Particular attention should thus be given to the composition, preparation, and immobilization of the biocomputing surface layer, to the role of the system scalability, and to the efficient transduction of the output signals. By processing complex patterns of multiple physiological markers, such multisignal digital biosensors should have a profound impact upon the rapid diagnosis and treatment of diseases, and particularly upon the timely detection and alert of medical emergencies (along with immediate therapeutic intervention). Other fields ranging from biotechnology to homeland security would benefit from these advances in new biocomputing biosensors and the corresponding closed-loop “add/act” operation.     

Concentric glucose/O2 biofuel cell

A concentric glucose/O₂ biofuel cell has been developed. The device is constituted of two carbon tubular electrodes, one in the other, and combines glucose electrooxidation at the anode and oxygen electroreduction at the cathode. The anodic catalyst is glucose oxidase co-immobilized with the mediator 8-hydroxyquinoline-5-sulfonic acid hydrate, and the cathodic catalyst is bilirubin oxidase co-immobilized with the mediator 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt. Both enzymes and mediators are entrapped at the surface of the tubular electrodes by an electrogenerated polypyrrole polymer. The originality of the concentric configuration is to compartmentalize the anode and cathode electrodes and to supply dissolved oxygen separate from the electrolyte in order to avoid secondary reactions. The dissolved oxygen circulates through the inside of the cathode tube and diffuses from the inner to the external surface of the tube to react directly with the immobilized bilirubin oxidase. The assembled biofuel cell is studied at 37 °C in phosphate buffer pH 7.4. We show the influence of the thickness of the polypyrrole polymer on the electrochemical activity of the biocathodes. We also demonstrate the effect of the chemical reticulation of the enzymes by glutaraldehyde within the polymer on the performances of the bioelectrodes. The maximum power density delivered by the assembled glucose/O₂ biofuel cell reaches 42 μW cm⁻², evaluated from the geometric area of the electrodes, at a cell voltage of 0.30 V with 10 mM glucose. The results demonstrate that the concentric design of the BFC based on compartmented electrodes is a promising architecture for further development of micro electronic devices.     

DNAzyme logic-controlled biofuel cells for self-powered biosensors

The integration of a biosensor employing a DNAzyme logic system within a biofuel cell is presented. The self-powered DNAzyme logic biosensor conforms with INH logic operation and generates power output in accordance with a truth table. The new concept of logic-activated DNAzyme by the input signals has wide-ranging implications in the self-powered diagnostics domain.     

Single‐Enzyme Biofuel Cells

Herein, we demonstrate that the intramolecular electron transfer within a single enzyme molecule is an important alternative pathway that can be harnessed to generate electricity. By decoupling the redox reactions within a single type of enzyme (for example, Trametes versicolor laccase), we harvested electricity efficiently from unconventional fuels including recalcitrant pollutants (for example, bisphenol A and hydroquinone) in a single‐laccase biofuel cell. The intramolecular electron‐harnessing concept was further demonstrated with other enzymes, including power generation during CO₂ bioconversion to formate catalyzed by formate dehydrogenase from Candida boidinii . The novel single‐enzyme biofuel cell is shown to have potential for utilizing wastewater as a fuel as well as for generating energy while driving bioconversion of chemical feedstock from CO₂.     

A Biofuel Cell Based on Biocatalytic Reactions of Lactate on Both Anode and Cathode Electrodes – Extracting Electrical Power from Human Sweat

Electrodes composed of carbon fibers were modified with graphene nano‐sheets in order to increase their surface area and facilitate electrochemical reactions. Electrocatalytic species, such as Meldola's blue (MB) and hemin were immobilized on the graphene surface due to their π‐π stacking and then used for electrocatalytic oxidation of NADH and reduction of H₂O₂, respectively. Further modification of these electrodes with enzymes producing NADH and H₂O₂ in situ (lactate dehydrogenase, LDH, and lactate oxidase, LOx, respectively), allowed assembling of a biofuel cell operating in the presence of lactate, oxygen and NAD⁺. The cathode of the biofuel cell required lactate and O₂ for its operation, while the anode operated in the presence of lactate and NAD⁺. Notably, both bioelectrocatalytic electrodes operated in the presence of lactate, one producing H₂O₂ in the reaction catalyzed by LOx in the presence of O₂, second producing NADH in the reaction catalyzed by LDH in the presence of NAD⁺. Both reactions were performed in the biofuel cell without separation of the cathodic and anodic solutions and with no need of a membrane. The biofuel cell was tested in solutions mimicking human sweat and then in real human sweat samples, demonstrating substantial power release being able to activate electronic devices.     

Controlled Logic Gates—Switch Gate and Fredkin Gate Based on Enzyme‐Biocatalyzed Reactions Realized in Flow Cells

Controlled logic gates, where the logic operations on the Data inputs are performed in the way determined by the Control signal, were designed in a chemical fashion. Specifically, the systems where the Data output signals directed to various output channels depending on the logic value of the Control input signal have been designed based on enzyme biocatalyzed reactions performed in a multi‐cell flow system. In the Switch gate one Data signal was directed to one of two possible output channels depending on the logic value of the Control input signal. In the reversible Fredkin gate the routing of two Data signals between two output channels is controlled by the third Control signal. The flow devices were created using a network of flow cells, each modified with one enzyme that biocatalyzed one chemical reaction. The enzymatic cascade was realized by moving the solution from one reacting cell to another which were organized in a specific network. The modular design of the enzyme‐based systems realized in the flow device allowed easy reconfiguration of the logic system, thus allowing simple extension of the logic operation from the 2‐input/3‐output channels in the Switch gate to the 3‐input/3‐output channels in the Fredkin gate. Further increase of the system complexity for realization of various logic processes is feasible with the use of the flow cell modular design.     

Biomolecule Release from Alginate Composite Hydrogels Triggered by Logically Processed Signals

Alginate composite hydrogels that exhibit highly sensitive stimuli-responsive behavior were used for signal-stimulated release of pre-loaded insulin. The alginate pores, particularly located at the periphery, were blocked by interpenetration of polyvinyl alcohol (PVA) cross-linked with 1,3-benzenediboronic acid (IPN), thus, significantly reducing uncontrolled leakage of the entrapped biomolecules. The beads were loaded with insulin and various enzymes mimicking different Boolean logic gates (AND, OR, NOR, IMP, INHIB). The enzymes were activated with biologically relevant input signals applied in four logic combinations: 0 , 0 ; 1 , 0 ; 0 , 1 ; 1 , 1 , having the production of H₂O₂ as the result of the biocatalytic reactions. The “successful” combination of the input signals leading to the H₂O₂ production was different for different logic gates, following the corresponding truth tables of the logic gates. When H₂O₂ was produced, boronate ester bonds were oxidized and the IPN was irreversibly degraded, thus re-opening the original pores of the hydrogel. This process allowed release of insulin from the alginate beads. The smart soft material that we have developed tackled well-known limitations of these systems and it may prove valuable in future medical diagnostics or treatments.     

Membrane-bound dehydrogenases from Gluconobacter sp.: Interfacial electrochemistry and direct bioelectrocatalysis

Although membrane-bound dehydrogenases isolated from Gluconobacter sp. (mainly PQQ-dependent alcohol and fructose dehydrogenase) have been used for preparing diverse forms of bioelectronic interfaces for almost 2 decades, it is not an easy task to interpret an electrochemical behaviour correctly. Recent discoveries regarding redox properties of membrane-bound dehydrogenases along with extensive investigations of direct electron transfer (DET) or direct bioelectrocatalysis with these enzymes are summarized in this review. The main aim of this review is to draw general conclusions about possible electronic coupling paths of these enzymes on various interfaces via direct electron transfer or direct bioelectrocatalysis. A short overview of the metabolism and respiration chain in Gluconobacter relevant to interfacial electrochemistry is given. Biosensor devices based on DET or direct bioelectrocatalysis using membrane-bound dehydrogenases from Gluconobacter sp. are described briefly with the emphasis given on practical applications of preparing enzymatic biofuel cells. Moreover, interfacial electrochemistry of Gluconobacter oxydans related to the construction of microbial biofuel cells is also discussed.     

Enzyme-regulated the changes of pH values for assembling a colorimetric and multistage interconnection logic network with multiple readouts

Based on enzymatic reactions-triggered changes of pH values and biocomputing, a novel and multistage interconnection biological network with multiple easy-detectable signal outputs has been developed. Compared with traditional chemical computing, the enzyme-based biological system could overcome the interference between reactions or the incompatibility of individual computing gates and offer a unique opportunity to assemble multicomponent/multifunctional logic circuitries. Our system included four enzyme inputs: β-galactosidase (β-gal), glucose oxidase (GOx), esterase (Est) and urease (Ur). With the assistance of two signal transducers (gold nanoparticles and acid–base indicators) or pH meter, the outputs of the biological network could be conveniently read by the naked eyes. In contrast to current methods, the approach present here could realize cost-effective, label-free and colorimetric logic operations without complicated instrument. By designing a series of Boolean logic operations, we could logically make judgment of the compositions of the samples on the basis of visual output signals. Our work offered a promising paradigm for future biological computing technology and might be highly useful in future intelligent diagnostics, prodrug activation, smart drug delivery, process control, and electronic applications.     

Not-XOR (NXOR) Logic Gate Realized with Enzyme-Catalyzed Reactions: Optical and Electrochemical Signal Transduction

The studied enzyme-based biocatalytic system mimics NXOR Boolean logic gate, which is a logical operator that corresponds to equality in Boolean algebra. It gives the functional value true ( 1 ) if both functional arguments (input signals) have the same logical value ( 0 , 0 or 1 , 1 ), and false ( 0 ) if they are different ( 0 , 1 or 1 , 0 ). The output signal producing reaction is catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), which is inhibited at acidic and basic pH values. Two other reactions catalyzed by esterase and urease produce acetic acid and ammonium hydroxide, respectively, shifting solution pH from the optimum pH for PQQ-GDH to acidic and basic values ( 1 , 0 and 0 , 1 input combinations, respectively), thus switching the enzyme activity off (output 0 ). When the input signals are not applied ( 0 , 0 combination) or both applied compensating each other ( 1 , 1 combination) the optimum pH is preserved, thus keeping PQQ-GDH running at the high rate (output 1 ). The biocatalytic cascade mimicking the NXOR gate was characterized optically and electrochemically. In the electrochemical experiments the PQQ-GDH enzyme communicated electronically with a conducting electrode support, thus resulting in the electrocatalytic current when signal combinations 0 , 0 and 1 , 1 were applied. The logic gate operation, when it was realized electrochemically, was also extended to the biomolecular release controlled by the gate. The release system included two electrodes, one performing the NXOR gate and another one activated for the release upon electrochemically stimulated alginate hydrogel dissolution. The studied system represents a general approach to the biocatalytic realization of the NXOR logic gate, which can be included in different catalytic cascades mimicking operation of concatenated gates in sophisticated logic circuitries.     

Enzyme-free and DNA-based universal platform for the construction of various logic devices based on graphene oxide and G-quadruplex

In the fields of biocomputing and biomolecular, DNA molecules are applicable to be regarded as data of logical computing platform that uses elaborate logic gates to perform a variety of tasks. Graphene oxide (GO) is a type of novel nanomaterial, which brings new research focus to materials science and biosensors due to its special selectivity and excellent quenching ability. G-quadruplex as a unique DNA structure stimulates the intelligent application of DNA assembly on the strength of its exceptional binding activity. In this paper, we report a universal logic device assisted with GO and G-quadruplex under an enzyme-free condition. Integrated with the quenching ability of GO to the TAMRA (fluorophore, Carboxytetramethylrhodamine) and the enhancement of fluorescence intensity produced by the peculiar binding of G-quadruplex to the NMM (N-methylmesoporphyrin IX), a series of basic binary logic gates (AND. OR. INHIBIT. XOR) have been designed and verified through biological experiments. Given the modularity and programmability of this strategy, two advanced logic gates (half adder and half subtractor) were realized on the basis of the same work platform. The fluorescence signals generated from different input combinations possessed satisfactory results, which provided proof of feasibility. We believe that the proposed universal logical platform that operates at the nanoscale is expected to be utilized for future applications in molecular computing as well as disease diagnosis.     

Implanted biofuel cell operating in a living snail

Implantable biofuel cells have been suggested as sustainable micropower sources operating in living organisms, but such bioelectronic systems are still exotic and very challenging to design. Very few examples of abiotic and enzyme-based biofuel cells operating in animals in vivo have been reported. Implantation of biocatalytic electrodes and extraction of electrical power from small living creatures is even more difficult and has not been achieved to date. Here we report on the first implanted biofuel cell continuously operating in a snail and producing electrical power over a long period of time using physiologically produced glucose as a fuel. The "electrified" snail, being a biotechnological living "device", was able to regenerate glucose consumed by biocatalytic electrodes, upon appropriate feeding and relaxing, and then produce a new "portion" of electrical energy. The snail with the implanted biofuel cell will be able to operate in a natural environment, producing sustainable electrical micropower for activating various bioelectronic devices.     

DNA Computing Systems Activated by Electrochemically‐triggered DNA Release from a Polymer‐brush‐modified Electrode Array

An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA‐based Identity, AND and XOR logic gates. Single‐stranded DNA molecules were loaded on the mixed poly(N ,N ‐dimethylaminoethyl methacrylate) (PDMAEMA)/poly(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAEMA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals.     

Reversible Logic Gates Based on Enzyme‐Biocatalyzed Reactions and Realized in Flow Cells: A Modular Approach

Reversible logic gates, such as the double Feynman gate, Toffoli gate and Peres gate, with 3‐input/3‐output channels are realized using reactions biocatalyzed with enzymes and performed in flow systems. The flow devices are constructed using a modular approach, where each flow cell is modified with one enzyme that biocatalyzes one chemical reaction. The multi‐step processes mimicking the reversible logic gates are organized by combining the biocatalytic cells in different networks. This work emphasizes logical but not physical reversibility of the constructed systems. Their advantages and disadvantages are discussed and potential use in biosensing systems, rather than in computing devices, is suggested.     

Biofuel Cell Operating in Vivo in Rat

Biocatalytic electrodes made of buckypaper were modified with PQQ‐dependent glucose dehydrogenase on the anode and with laccase on the cathode. The enzyme modified electrodes were assembled in a biofuel cell which was first characterized in human serum solution and then the electrodes were placed onto exposed rat cremaster tissue. Glucose and oxygen dissolved in blood were used as the fuel and oxidizer, respectively, for the implanted biofuel cell operation. The steady‐state open circuitry voltage of 140±30 mV and short circuitry current of 10±3 µA (current density ca. 5 µA cm^?2 based on the geometrical electrode area of 2 cm²) were achieved in the in vivo operating biofuel cell. Future applications of implanted biofuel cells for powering of biomedical and sensor devices are discussed.