Bio-logic analysis of injury biomarker patterns in human serum samples

Digital biosensor systems analyzing biomarkers characteristic of liver injury (LI), soft tissue injury (STI) and abdominal trauma (ABT) were developed and optimized for their performance in serum solutions spiked with injury biomarkers in order to mimic real medical samples. The systems produced ‘Alert’-type optical output signals in the form of “YES-NO” separated by a threshold value. The new approach aims at the reliable detection of injury biomarkers for making autonomous decisions towards timely therapeutic interventions, particularly in conditions when a hospital treatment is not possible. The enzyme-catalyzed reactions performing Boolean AND/NAND logic operations in the presence of different combinations of the injury biomarkers allowed high-fidelity biosensing. Robustness of the systems was confirmed by their operation in serum solutions, representing the first example of chemically performed logic analysis of biological fluids and a step closer towards practical biomedical applications of enzyme-logic bioassays.     

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.     

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.     

Redox-Guest-Induced Multimode Photoluminescence Switch for Sequential Logic Gates in a Photoactive Coordination Cage

Molecular or supramolecular level photoluminescence (PL) modulation combining chemical and photonic input/output signals together in an integrated system can provide potential high-density data memorizing and process functions intended for miniaturized devices and machines. Herein, a PL-responsive supramolecular coordination cage has been demonstrated for complex interactions with redox-active guests. PL signals of the cage can be switched and modulated by adding or retracting Fc derivatives or converting TTF into different oxidation states through chemical or photochemical pathways. As a result, reversible or stepwise PL responses are displayed by these host–guest systems because of the occurrence of photoinduced electron-transfer (PET) or fluorescence resonance energy transfer (FREnT) processes, providing unique nanodevice models bearing off/on logic gates or memristor-like sequential memory and Boolean operation functions.     

分子基逻辑材料*

在适当的条件下分子开关将输入的信息转换为输出信号,利用这一特点,可在分子体系根据二进位布尔逻辑规则实现信号转换。目前,用化学体系进行基本的布尔逻辑功能执行 (PASS、YES、NOT、AND、NAND、OR、NOR、XNOR和INH)都已成为可能。在此基础上,逻辑门的整合与编程,以及更进一步的复杂分子运算开始受到人们的关注。迄今为止,以高灵敏性的荧光输出信号为主,人们在分子水平上设计实现了多种复杂的逻辑电路,包括组合逻辑电路和时序逻辑电路等,并开始涉及信息处理的安全平台设计。本文主要介绍了近年来利用分子荧光开关体系模拟数字逻辑电路过程中所取得的最新进展,对分子逻辑电路研究的热点和问题进行了展望。     

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.     

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.     

Logic Gates Based on Magnetic Nanoparticles Functionalized with a Bioelectrocatalytic System

Magneto‐controlled OR, AND and INHIB logic gates were designed using cobalt ferrite magnetic nanoparticles (CoFe₂O₄, saturated magnetization ca. 70 emu g^?1, 17±2 nm diameter) functionalized with microperoxidase‐11. Tunable magnetic field generated by three external permanent magnets (NdFeB) upon moving them below the electrochemical cell resulted in translocation of the biofunctionalized magnetic nanoparticles between conductive and nonconductive domains of a solid plate. This resulted in electrochemically readable output signals with the Boolean logic controlled by the magnetic input signals. The current corresponding to the reversible redox process of the heme measured at ?0.4 V (vs. SCE) was considered as “1” output signal, while a small background current obtained from the conducting interface in the absence of the magnetic nanoparticles was considered as “0” output signal. Addition of H₂O₂ to the solution resulted in the generation of a cathodic catalytic current when the microperoxidase‐11‐functionalized magnetic nanoparticles are associated with the conductive domain of the support. This resulted in the amplification of “1” output signal and the increased difference between “1” and “0” signals generated by the cell, thus reducing the possibility of errors in the Boolean logic operations.     

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.     

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.     

Biofuel cell controlled by enzyme logic network — Approaching physiologically regulated devices

A “smart” biofuel cell switchable ON and OFF upon application of several chemical signals processed by an enzyme logic network was designed. The biocomputing system performing logic operations on the input signals was composed of four enzymes: alcohol dehydrogenase (ADH), amyloglucosidase (AGS), invertase (INV) and glucose dehydrogenase (GDH). These enzymes were activated by different combinations of chemical input signals: NADH, acetaldehyde, maltose and sucrose. The sequence of biochemical reactions catalyzed by the enzymes models a logic network composed of concatenated AND/OR gates. Upon application of specific “successful” patterns of the chemical input signals, the cascade of biochemical reactions resulted in the formation of gluconic acid, thus producing acidic pH in the solution. This resulted in the activation of a pH-sensitive redox-polymer-modified cathode in the biofuel cell, thus, switching ON the entire cell and dramatically increasing its power output. Application of another chemical signal (urea in the presence of urease) resulted in the return to the initial neutral pH value, when the O₂-reducing cathode and the entire cell are in the mute state. The reversible activation–inactivation of the biofuel cell was controlled by the enzymatic reactions logically processing a number of chemical input signals applied in different combinations. The studied biofuel cell exemplifies a new kind of bioelectronic device where the bioelectronic function is controlled by a biocomputing system. Such devices will provide a new dimension in bioelectronics and biocomputing benefiting from the integration of both concepts.     

Modular multi-level circuits from immobilized DNA-based logic gates

One of the fundamental goals of molecular computing is to reproduce the tenets of digital logic, such as component modularity and hierarchical circuit design. An important step toward this goal is the creation of molecular logic gates that can be rationally wired into multi-level circuits. Here we report the design and functional characterization of a complete set of modular DNA-based Boolean logic gates (AND, OR, and AND-NOT) and further demonstrate their wiring into a three-level circuit that exhibits Boolean XOR (exclusive OR) function. The approach is based on solid-supported DNA logic gates that are designed to operate with single-stranded DNA inputs and outputs. Since the solution-phase serves as the communication medium between gates, circuit wiring can be achieved by designating the DNA output of one gate as the input to another. Solid-supported logic gates provide enhanced gate modularity versus solution-phase systems by significantly simplifying the task of choosing appropriate DNA input and output sequences used in the construction of multi-level circuits. The molecular logic gates and circuits reported here were characterized by coupling DNA outputs to a single-input REPORT gate and monitoring the resulting fluorescent output signals.     

DNA-Based Adaptive Plasmonic Logic Gates

Self-assembled plasmonic logic gates that read DNA molecules as input and return plasmonic chiroptical signals as outputs are reported. Such logic gates are achieved on a DNA-based platform that logically regulate the conformation of a chiral plasmonic nanostructure, upon specific input DNA strands and internal computing units. With systematical designs, a complete set of Boolean logical gates are realized. Intriguingly, the logic gates could be endowed with adaptiveness, so they can autonomously alter their logics when the environment changes. As a demonstration, a logic gate that performs AND function at body temperature while OR function at cold storage temperature is constructed. In addition, the plasmonic chiroptical output has three distinctive states, which makes a three-state molecular logic gate readily achievable on this platform. Such DNA-based plasmonic logic gates are envisioned to execute more complex tasks giving these unique characteristics.     

DNA‐Based Adaptive Plasmonic Logic Gates

Self‐assembled plasmonic logic gates that read DNA molecules as input and return plasmonic chiroptical signals as outputs are reported. Such logic gates are achieved on a DNA‐based platform that logically regulate the conformation of a chiral plasmonic nanostructure, upon specific input DNA strands and internal computing units. With systematical designs, a complete set of Boolean logical gates are realized. Intriguingly, the logic gates could be endowed with adaptiveness, so they can autonomously alter their logics when the environment changes. As a demonstration, a logic gate that performs AND function at body temperature while OR function at cold storage temperature is constructed. In addition, the plasmonic chiroptical output has three distinctive states, which makes a three‐state molecular logic gate readily achievable on this platform. Such DNA‐based plasmonic logic gates are envisioned to execute more complex tasks giving these unique characteristics.     

Microchip extraction of catecholamines using a boronic acid functional affinity monolith

We report on the development of a rapid enzyme logic gate-based electrochemical assay for the assessment of traumatic brain injury (TBI). The concept harnesses a biocatalytic cascade that emulates the functionality of a Boolean NAND gate in order to process relevant physiological parameters in the biochemical domain. The enzymatic backbone ensures that a high-fidelity diagnosis of traumatic brain injury can be tendered in a rapid fashion when the concentrations of key serum-based biomarkers reach pathological levels. The excitatory neurotransmitter glutamate and the enzyme lactate dehydrogenase were used here as clinically-relevant input TBI biomarkers, in connection to the low-potential detection of the NADH product in the presence of methylene green at a glassy carbon electrode. A systematic optimization of the gate and the entire protocol has resulted in the effective discrimination between the physiological and pathological logic levels. Owing to its robust design, the enzyme-based logic gate mitigates potential interferences from both physiological and electroactive sources and is able to perform direct measurements in human serum samples. Granted further detailed clinical validation, this proof-of-concept study demonstrates the potential of the electrochemical assay to aid in the rapid and decentralized diagnosis of TBI.     

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.     

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.     

含氧化还原活性基团的荧光分子开关构建与门

报道了一个基于光致电子转移(PET)机理的双稳态荧光分子开关,其中具有氧化还原活性的二茂铁基团作为荧光团蒽PET过程的氧化还原控制单元,通过双Schiff碱C=N键与蒽相连。研究了不同条件下蒽的荧光发射行为。以不同的化学试剂作为体系的输入信号,以不同的荧光发射强度作为输出信号,利用荧光分子开关的质子化反应和氧化反应实现了一个分子水平的逻辑功能。输入信号及其相应的输出结果在分子水平上符合数字逻辑与门的真值表。     

DNA logic gates

A conceptually new logic gate based on DNA has been devised. Methoxybenzodeazaadenine ((MD)A), an artificial nucleobase which we recently developed for efficient hole transport through DNA, formed stable base pairs with T and C. However, a reasonable hole-transport efficiency was observed in the reaction for the duplex containing an (MD)A/T base pair, whereas the hole transport was strongly suppressed in the reaction using a duplex where the base opposite (MD)A was replaced by C. The influence of complementary pyrimidines on the efficiency of hole transport through (MD)A was quite contrary to the selectivity observed for hole transport through G. The orthogonality of the modulation of these hole-transport properties by complementary pyrimidine bases is promising for the design of a new molecular logic gate. The logic gate system was executed by hole transport through short DNA duplexes, which consisted of the "logic gate strand", containing hole-transporting nucleobases, and the "input strand", containing pyrimidines which modulate the hole-transport efficiency of logic bases. A logic gate strand containing multiple (MD)A bases in series provided the basis for a sharp AND logic action. On the other hand, for OR logic and combinational logic, conversion of Boolean expressions to standard sum-of-product (SOP) expressions was indispensable. Three logic gate strands were designed for OR logic according to each product term in the standard SOP expression of OR logic. The hole-transport efficiency observed for the mixed sample of logic gate strands exhibited an OR logic behavior. This approach is generally applicable to the design of other complicated combinational logic circuits such as the full-adder.     

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.