Nanostructured Interface Loaded with Chimeric Enzymes for Fluorimetric Quantification of Cyclosporine A and FK506.

Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.

A Universal Multichannel Platform for Assembling Enzyme-Based Boolean Logic Gates.

Concatenated enzyme-based Boolean logic gates activated with 5 chemical input signals were analyzed with a smartphone photo camera. Simultaneous detection of 32 input combinations was conveniently performed using enzyme-modified fiberglass sensing spots generating fluorescence with different intensities for the 0 and 1 binary outputs. The developed technology offers an easy readout method for multi-channel logic systems.

A magneto-controlled biocatalytic cascade with a fluorescent output.

A biocatalytic cascade based on concerted operation of pyruvate kinase and luciferase with a bioluminescent output was switched reversibly between low and high activity by applying an external magnetic field at different positions or removing it. The enzymes participating in the reaction cascade were bound to magnetic nanoparticles to allow their translocation or aggregation/dispersion to be controlled by the magnetic field. The reaction intensity, measured as the bioluminescent output, was dependent on the effective distances between the enzymes transported on the magnetic nanoparticles controlled by the magnets.

Electrochemically stimulated protein release from pH-switchable electrode-immobilized nitroavidin-biotin and avidin-iminobiotin systems.

Bovine serum albumin (BSA), used as a model protein, was immobilized on a buckypaper electrode by formation of covalent bonds with avidin/iminobiotin or nitroavidin/biotin complexes. pH-sensitive affinity interactions between avidin and iminobiotin or between nitroavidin and biotin allowed splitting of the affinity bonds upon pH variation, thus resulting in BSA release. Local (interfacial) pH was changed electrochemically. The pH was decreased upon electrochemical oxidation of ascorbate or increased upon electrochemical reduction of O2. The local pH change resulted in the weakening of the affinity complexes, resulting in BSA release from the avidin/iminobiotin or nitroavidin/biotin systems when the pH was decreased or increased, respectively. Importantly, protein release was only observed when the number of chemical bonds with the affinity systems was decreased by blocking a part (ca. 50%) of the binding sites in avidin/nitroavidin with iminobiotin/biotin molecules missing the possibility of attaching the protein. Without this blocking effect, multiple bond formation with the protein preserved BSA at the electrode surface, by not allowing its release upon electrochemical pH change.

Electrochemically produced local pH changes stimulating (bio)molecule release from pH-switchable electrode-immobilized avidin-biotin systems.

Immobilized avidin-biotin complexes were used to release biotinylated (bio)molecules upon producing local pH changes near an electrode surface by electrochemical reactions. The nitro-avidin complex with biotin was dissociated by increasing local pH with electrochemical O2 reduction. The avidin complex with iminobiotin was split by decreasing local pH with electrochemical oxidation of ascorbate. Both studied systems were releasing molecule cargo species in response to small electrical potentials (-0.4 V or 0.2 V for the O2 reduction or ascorbate oxidation, respectively) applied on the modified electrodes.

Fluorescent sensor based on pyrroloquinoline quinone (PQQ)-glucose dehydrogenase for glucose detection with smartphone-adapted signal analysis.

A new nano-structured platform for fluorescent analysis using PQQ-dependent glucose dehydrogenase (PQQ-GDH) was developed, particularly using a smartphone for transduction and quantification of optical signals. The PQQ-GDH enzyme was immobilized on SiO2 nanoparticles deposited on glass microfiber filter paper, providing a high load of the biocatalytic enzyme. The platform was tested and optimized for glucose determination using a wild type of the PQQ-GDH enzyme. The analysis was based on the fluorescence generated by the reduced form of phenazine methosulfate produced stoichiometrically to the glucose concentration. The fluorescent signals were generated at separate analytical spots on the paper support under wavelength (365 nm) UV excitation. The images of the analytical spots, dependent on the glucose concentration, were obtained using a photo camera of a standard smartphone. Then, the images were processed and quantified using software installed in a smartphone. The developed biocatalytic platform is the first step to assembling a large variety of biosensors using the same platform functionalized with artificial allosteric chimeric PQQ-dependent glucose dehydrogenase activated with different analytes. The future combination of the artificial enzymes, the presently developed analytical platform, and signal processing with a smartphone will lead to novel point-of-care and end-user biosensors applicable to virtually all possible analytes.

Circular Permutated PQQ-Glucose Dehydrogenase as an Ultrasensitive Electrochemical Biosensor.

Protein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ-glucose dehydrogenase (PQQ-GDH). We designed allosterically regulated circular permutated variants of PQQ-GDH that show large (>10-fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N- and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub-nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporine A could be measured in 1 µL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporine A as low as 20 pM and retained functionality in samples containing at least 60 % human serum.

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 H2 O2 as the result of the biocatalytic reactions. The "successful" combination of the input signals leading to the H2 O2 production was different for different logic gates, following the corresponding truth tables of the logic gates. When H2 O2 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.

Implication and Inhibition Boolean Logic Gates Mimicked with Enzyme Reactions.

The enzyme system mimicking Implication (IMPLY) and Inhibition (INHIB) Boolean logic gates has been designed. The same enzyme system was used to operate as the IMPLY or INHIB gate simply by reformulating the input signals. The optical analysis of the logic operation confirmed the output generation as expected for the studied logic gates. The conceptual approach to the IMPLY and INHIB logic gates allows their construction with many other enzymes operating in a similar way.

A Microelectronic Sensor Device Powered by a Small Implantable Biofuel Cell.

Biocatalytic buckypaper electrodes modified with pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and bilirubin oxidase for glucose oxidation and oxygen reduction, respectively, were prepared for their use in a biofuel cell. A small (millimeter-scale; 2×3×2 mm3 ) enzyme-based biofuel cell was tested in a model glucose-containing aqueous solution, in human serum, and as an implanted device in a living gray garden slug (Deroceras reticulatum), producing electrical power in the range of 2-10 µW (depending on the glucose source). A microelectronic temperature-sensing device equipped with a rechargeable supercapacitor, internal data memory and wireless data downloading capability was specifically designed for activation by the biofuel cell. The power management circuit in the device allowed the optimized use of the power provided by the biofuel cell dependent on the sensor operation activity. The whole system (power-producing biofuel cell and power-consuming sensor) operated autonomously by extracting electrical energy from the available environmental source, as exemplified by extracting power from the glucose-containing hemolymph (blood substituting biofluid) in the slug to power the complete temperature sensor system and read out data wirelessly. Other sensor systems operating autonomously in remote locations based on the concept illustrated here are envisaged for monitoring different environmental conditions or can be specially designed for homeland security applications, particularly in detecting bioterrorism threats.

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

The front cover artwork is provided by groups of Prof. Evgeny Katz and Prof. Artem Melman (Clarkson University, NY, USA) as well as Prof. Kirill Alexandrov (Queensland University of Technology, Brisbane, Australia). The image shows activation/inhibition of a chimeric enzyme with biomolecular signals and a corresponding logic network - the artistic vision. Read the full text of the Communication at 10.1002/cphc.201901050.

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.

Biosensors-Recent Advances and Future Challenges.

Biosensors are analytical devices that are able to convert a biological response into an electrical signal [...].

Enzyme-Based Biosensors: Tackling Electron Transfer Issues.

This review summarizes the fundamentals of the phenomenon of electron transfer (ET) reactions occurring in redox enzymes that were widely employed for the development of electroanalytical devices, like biosensors, and enzymatic fuel cells (EFCs). A brief introduction on the ET observed in proteins/enzymes and its paradigms (e.g., classification of ET mechanisms, maximal distance at which is observed direct electron transfer, etc.) are given. Moreover, the theoretical aspects related to direct electron transfer (DET) are resumed as a guideline for newcomers to the field. Snapshots on the ET theory formulated by Rudolph A. Marcus and on the mathematical model used to calculate the ET rate constant formulated by Laviron are provided. Particular attention is devoted to the case of glucose oxidase (GOx) that has been erroneously classified as an enzyme able to transfer electrons directly. Thereafter, all tools available to investigate ET issues are reported addressing the discussions toward the development of new methodology to tackle ET issues. In conclusion, the trends toward upcoming practical applications are suggested as well as some directions in fundamental studies of bioelectrochemistry.

Boolean Logic Gates Realized with Enzyme-catalyzed Reactions - Unusual Look at Usual Chemical Reactions.

Research in the area of molecular computing systems, in the general framework of unconventional computing, has received high attention and resulted in rapid progress in formulating signal-controlled switchable molecules capable to perform Boolean logic operations and basic arithmetic functions. Extension of this research to biomolecular systems allowed sophisticated computational functions much easier than using synthetic molecular and supramolecular species. The advantage of biomolecular systems comparing with synthetic molecular systems is in their complementarity and compatibility allowing easy assembling multi-component systems from various biomolecules, thus increasing their functional complexity. While DNA-based computing systems are promising faster computing than Si-based electronics, at least for solving some combinatorial problems, due to massive parallel operation, enzyme-based logic systems are less promising for computational applications in their narrow definition. However, they offer novel biosensing and bioactuation features operating in binary Yes/No format. The present review article overviews different kinds of enzyme logic gates exemplified with specific enzymatic reactions/cascades. Motivation for this research and its possible applications are discussed. The review will be helpful to researchers working in this specific area to see the comprehensive collection of logic operations performed by the enzyme reactions. The newcomers to the reviewed area will benefit from the example systems representing various logic functions systematically.

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.

Magneto-Controlled Biocatalytic Cascades with Logically Processed Input Signals - Substrate Channeling versus Free Diffusion.

Magnetic nanoparticles (MNPs) functionalized with various enzymes (amyloglucosidase, glucose oxidase and horseradish peroxidase) were used to perform biocatalytic cascades in two different states, solute suspension or aggregated, produced in the absence or presence of an external magnetic field. The biocatalytic reactions proceeded through bulk solution diffusion of intermediate substrates or substrate channeling, when the systems were dispersed or aggregated, respectively. The both pathways have shown very similar kinetics, unless the intermediate substrate was consumed by an additional biocatalytic process called "filter" for brevity. In the presence of the "filter" process, the diffusional process in the bulk solution was significantly inhibited, while the process based on the substrate channeling was still active. The systems were switched reversibly between the inhibited dispersed state and the active aggregated state by removing and applying the external magnetic field, respectively. The signal-controlled biocatalytic cascades were considered as Boolean logic circuits with the inputs consisting of biomolecules and the magnetic field on-off.

Magnetic Field-Activated Sensing of mRNA in Living Cells.

Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers.

Enzyme-Based Logic Gates and Networks with Output Signals Analyzed by Various Methods.

The paper overviews various methods that are used for the analysis of output signals generated by enzyme-based logic systems. The considered methods include optical techniques (optical absorbance, fluorescence spectroscopy, surface plasmon resonance), electrochemical techniques (cyclic voltammetry, potentiometry, impedance spectroscopy, conductivity measurements, use of field effect transistor devices, pH measurements), and various mechanoelectronic methods (using atomic force microscope, quartz crystal microbalance). Although each of the methods is well known for various bioanalytical applications, their use in combination with the biomolecular logic systems is rather new and sometimes not trivial. Many of the discussed methods have been combined with the use of signal-responsive materials to transduce and amplify biomolecular signals generated by the logic operations. Interfacing of biocomputing logic systems with electronics and "smart" signal-responsive materials allows logic operations be extended to actuation functions; for example, stimulating molecular release and switchable features of bioelectronic devices, such as biofuel cells. The purpose of this review article is to emphasize the broad variability of the bioanalytical systems applied for signal transduction in biocomputing processes. All bioanalytical systems discussed in the article are exemplified with specific logic gates and multi-gate networks realized with enzyme-based biocatalytic cascades.

DNA Release from Fe3+ -Cross-Linked Alginate Films Triggered by Logically Processed Biomolecular Signals: Integration of Biomolecular Computing and Actuation.

Signal-controlled release of DNA from Fe3+ -cross-linked alginate hydrogel electrochemically deposited on an electrode surface was studied. The multiple input signals were logically processed with the help of the enzyme-biocatalyzed reactions. Boolean logic gates, OR, AND, INH, were realized with the biocatalytic reactions performed by the enzymes entrapped in the alginate film. Hydrogen peroxide produced by the enzymatic reactions resulted in the degradation of the alginate hydrogel and DNA release. The alginate degradation was facilitated by the formation of free radicals in the Fenton-type reaction catalyzed by iron cations cross-linking the alginate hydrogel. The studied approach is versatile and can be adapted to various chemical signals processed by various enzymes with differently implemented Boolean logic. This work illustrates a novel concept of functional integration of biomolecular computing and actuation.