Advanced Smart Nanomaterials with Integrated Logic-Gating and

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Advanced Smart Nanomaterials with Integrated Logic-Gating and Biocomputing: Dawn of Theranostic Nanorobots Andrey A. Tregubov,† Petr I. Nikitin,‡ and Maxim P. Nikitin*,† †

Moscow Institute of Physics and Technology (State University), 1A Kerchenskaya St, Moscow 117303, Russia Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilov Street, Moscow 119991, Russia

Chem. Rev. Downloaded from pubs.acs.org by 185.101.69.29 on 09/20/18. For personal use only.

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ABSTRACT: Accurate and precise drug delivery is the key to successful therapy. Monoclonal antibodies, which can transport therapeutic payload to cells expressing specific markers, have paved the way for targeted drug delivery and currently show tremendous clinical success. However, in those abundant cases, when a disease cannot be characterized by a single specific marker, more sophisticated drug delivery systems are required. To enhance targeting accuracy, diverse smart materials have been proposed that can also react to stimuli like variations of pH, temperature, magnetic field, etc. Furthermore, over the past few years a new category of smart materials has emerged, which can not only respond to virtually any biochemical or physical stimulus but also simultaneously analyze several cues and, moreover, can be programmed to use Boolean logic for such analysis. These advanced biocomputing agents have the potential to become a basis for future nanorobotic devices that could overcome some of the grand challenges of modern biomedicine. Here, with a brief introduction to the multidisciplinary field of biomolecular computing, we will review the concepts of nanomaterials with built-in biocomputing capabilities, which can be potentially used for drug delivery and other theranostic applications.

CONTENTS 1. Introduction 1.1. Scope 2. Overview of Boolean Logic Gates 3. Logic-Gated Nanoagents for Specific Biomedical Applications 3.1. Biomolecular Logic-Gated Agents for Selective Cell Targeting 3.2. Logic-Gated Nanoparticles for Sensing and Imaging 3.2.1. Luminescence-Based Logic-Gated Systems 3.2.2. Logic Gates Fabricated Using Assembly/Disassembly of Nanoparticles Exhibiting Surface Plasmon Resonance 3.2.3. Logic-Gated Magnetic Nanoparticles 3.3. Logic-Gated Nanoparticles for Therapeutic Tasks 3.3.1. Logic-Gated Systems for Controlled Release of Payload 3.3.2. Logic-Gated Nanoparticles Able to Alter Chemical Composition of Microenvironment by Means of Catalyzed Transformation 4. Current Challenges and Future Perspectives 4.1. Design Aspects Composition of the Logic Gating Mechanisms: Suitability for in Vivo Operation All-in-One Agents vs Logic-Gate Cascading via Agent Cross-Talk © XXXX American Chemical Society

Importance of the Complete Set of Logic Functions Logic Functions for Same Inputs and with Same Output Readout Sensitivity to Inputs Range of Analyzable Input Types True Inertness of the Agent in the Inactive State General in Vivo Concerns 4.2. Issues beyond Materials Science Suitable Inputs Methods for in Vivo Tracking of the Assembled Agents Computer Simulations Personalized Medicine vs Reproducibility: OnDemand Composition, Threshold, and Sensitivity 5. Conclusion Author Information Corresponding Author ORCID Notes Biographies Acknowledgments List of Abbreviations References

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DOI: 10.1021/acs.chemrev.8b00198 Chem. Rev. XXXX, XXX, XXX−XXX

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“nanorobots”. The present review also has some limitations. Only the biocomputing agents that can operate outside cells (so-called extracellular biocomputing) are covered to address the primary challenge in drug delivery, namely, proper identification, attachment, and release of drug to the diseased cells. Besides, only colloidal nanoparticle-based logic-gated systems are considered due to their potential to be incorporated into autonomous multifunctional agents. We also excluded comprehensively reviewed elsewhere12,13 conventional stimuli-responsive materials such as pH- and temperature-responsive polymers, which could be regarded as materials that implement the primitive OR-gate. Solely those stimuli-responsive nanoparticles are surveyed, whose logic gating is strictly defined by the authors. In addition, these systems must be responsive to two or more inputs, and their behavior is studied in all possible inputs combinations [e.g., (0,0), (1,0), (0,1), (1,1) for two input system].

1. INTRODUCTION In 1959, Nobel Prize Laureate Richard Feynman in his lecture “There’s Plenty of Room at the Bottom”1 envisioned the use of molecules to perform calculations, which later gave birth to the subarea of unconventional data processing called biomolecular computing, or biocomputing. The landmark in physical realization of these systems was the 1994 work by Adleman who had successfully demonstrated DNA capabilities to solve the Hamiltonian path problem.2 Since then, these systems have evolved significantly and now can perform basic arithmetic operations, such as addition/subtraction,3−5 calculate square root,6 simulate keypad lock,7 and even play logic games.8,9 Today, however, the biocomputing systems are of primary interest not as an alternative to silicon-based computers but rather as a promising development of the nanotechnologybased theranostics that can completely change our approach to targeted drug delivery. Currently, the most advanced drug delivery systems with active targeting are based on the use of monoclonal antibodies as guiding ligands.10,11 Indeed, the high specificity of antibody−antigen interactions allows precise tagging of the diseased cells that overexpress a certain receptor. On the other hand, many medical disorders cannot be unambiguously characterized by a single specific marker, and, as a result, autonomous drug delivery vehicles are needed that are capable of comprehensive analysis of cell surface markers and/or microenvironment. Such analysis can be readily performed using simple yet powerful language of Boolean algebra. In Boolean notation, the presence or absence of a certain marker can be specified as the Boolean TRUE or FALSE value, respectively. Therefore, processing of such input data with some sort of on-board computer based on biomolecular logic circuits may facilitate differentiation of the diseased cells and tissues from their healthy counterparts, thereby significantly improving therapy effect and/or precision of diagnostics. Recent progress in biomolecular computation has brought logic-gated nanosystems not only for identifying challenging cellular targets but also for releasing drugs in a precisely regulated fashion and broadcasting the information about the local microenvironment. In this respect, there is a great interest in merging these task-specific nanoagents into an all-in-one theranostic platform to afford powerful nanorobotics devices that surpass all state of the art drugs.

2. OVERVIEW OF BOOLEAN LOGIC GATES Most biological processes, especially those relevant to medical disorders, are inherently sophisticated. Their precise mathematical description requires complex nonlinear dependences on many variables. However, in practice, due to significant patient-to-patient differences, we often have to simplify the system’s performance to primitive TRUE/FALSE behavior. For instance, although it is possible to precisely measure the concentration of a certain tumor marker, the variability of this parameter from person to person impels us to introduce a certain ≪threshold≫ to distinguish between the two possible ranges of the marker concentrations: the “safe” level (FALSE or “0” values) and the “dangerous” one (TRUE or “1” values). Simultaneous analysis of a multitude of such physiological parameters can be conveniently performed using Boolean logic. This branch of algebra has already demonstrated its power in processing of another type of information that takes only two values of 0 or 1, namely, in electronic circuits, where these values are defined as low (e.g., 0.5 V) and high (e.g., 5.0 V) voltages, respectively. Every circuit is composed of elementary units called “logic gates”, each of them implementing a certain Boolean logic function over a number of inputs in order to compute and yield an output, which is transferred to the next gate as input. Let us consider the variety of Boolean functions (gates) possible for two variables (see Table 1). Four different sets of values of variables (inputs) are available, namely, “0/0”, “0/1”, “1/0”, and “1/1”. These can produce totally 42 = 16 unique sets of outputs (each output can also be either 0 or 1). Hence, there are 16 unique Boolean functions, which can be divided into three groups: nullary, unary, and binary. The “nullary” (i.e., input-independent) group comprises two simplest logic gates PASS 0 and PASS 1, each of these yields the same output (0 and 1, respectively) regardless of the input value. The unary (dependent on one of the inputs and independent from the other) functions encompass YES and NOT gates: the former generates output that equals the input, while the latter produces an inverse value. Accordingly, the two-input system of Boolean functions has two YES/NOT pairs of logic gates for each input. Other binary gates simultaneously depend on both inputs. Among the two-input functions, we would mention the OR logic gate that yields 1 output when at least one of the inputs equals 1, and the AND logic gate that generates 1 output if and only if both inputs are 1. It is important that the basic YES, NOT, AND, and OR functions form a functionally

1.1. Scope

The purpose of this review is to summarize the concepts of nanomaterials with built-in biocomputing capabilities reported over the last 10 years, which can be potentially used for various theranostic applications. First, in order to make this review available for the broad circle of researchers, a general introduction to the Boolean logic is given. Next, we survey the strategies for construction of biocomputing agents that can control their binding with a cell target, which is the foremost problem of drug delivery. Importantly, this group uniquely features several concepts that allow implementing any Boolean logic gate for a given number of inputs. Then, we summarize the highly diverse examples of inorganic colloidal logic-gated nanomaterials that perform specific theranostic actions (e.g., sensing/imaging based on luminescent, plasmonic, magnetic principles, as well as controlled release and catalyzed transformations). Finally, we discuss the prospects of the in vivo applications of the biocomputing nanoagents and the main challenges on the way to creation of theranostic B


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the time being, a robust implementation of a system of truly digital biocomputing was reported in the work.6 The authors introduced a special thresholding mechanism able to suppress the output signal to zero or amplify it to one if the respective input signal is below or above the certain threshold. Thus, this system always provides strict 0 and 1 values of output regardless of small fluctuations in concentrations of input. Most of other biocomputing systems including those with the use of nanoparticles, though, are yet analog (i.e., the values of their inputs and outputs are continuously distributed between 0 or 1). Nevertheless, description of such systems in terms of Boolean algebra greatly simplifies the narrative and facilitates comprehension of each concept. In the future, perhaps, one will be able to take advantage of the complex behavior of the analog systems and use the intricate relationships between their outputs and inputs. In most state-of-the-art cases, though, the Boolean terms would be sufficient to demonstrate the system abilities for data processing. It is important to point out that Boolean logic has been successfully applied in design of diverse biocomputing structures based on different biochemical entities (e.g., organic fluorophores,15,16 molecular machines,17−19 dendron,20 and micelle-like21 assemblies, peptides,22,23 enzymes,3,24 other proteins,25,26 DNA,27−32 and living cells33,34). Below, we will review those of them, which are incorporated into nanoagents or targeted delivery systems and are promising for development of a new generation of intelligent theranostic materials.

complete set of logic gates (i.e., any arbitrary Boolean function can be expressed as a combination of these basic functions). For instance, the INHIBIT function can be expressed as the AND logic gate with one of its inputs inverted by the NOT function [i.e., YES(X) AND NOT(Y)]. The XOR (exclusive OR) function can be obtained by integration of outputs of such two INHIBIT gates into the resulting OR logic gate: [YES(X) AND NOT(Y)] OR [YES(Y) AND NOT(X)] (see Figure 1). The NOR, NAND, IMPLICATION, and XNOR

Figure 1. Realization of XOR function through two INHIBIT logic gates. Wires denote inputs/outputs; circles represent signal inversion.

(exclusively NOR) functions can be derived by inverting the output of OR, AND, INHIBIT, and XOR logic gates, respectively. It should be taken into account that the YESNOT-AND-OR is not the only functionally complete set. For example, in electronics a NAND function can be used alone to construct any other gate. The same is true for the NOR gate or the (AND, NOT) set. In practice, Boolean logic can be applied to any type of information that can be expressed as 0 and 1. For example, before the introduction of semiconductors, electronic computers were based on vacuum tubes that processed high/low voltages as Boolean 1/0 values, respectively. An even earlier device, Konrad Zuse’s Z1 (the first freely programmable machine), employed mechanical logic gating where forward linear movement of a small rod or metallic plate was defined as 1, and no movement was interpreted as 0 (or vice versa depending on component).14 Generally, it is possible to construct the logic gate in virtually any form of an electronic, mechanical, optical, magnetic, and biochemical system. In this respect, Boolean algebra can be readily used for functional description of the biomedical devices that can be programmed to be susceptible to certain compounds (e.g., cancer markers) or external stimuli, such as light or magnetic field. By convention, we can think of the presence of these factors as a Boolean true, while their absence as the Boolean false. In turn, the biological response such as drug release could be regarded as the true output, while the absence of such response as the Boolean false. Similar to their electronic macro counterparts, the biocomputing systems can be both analog and digital. For

3. LOGIC-GATED NANOAGENTS FOR SPECIFIC BIOMEDICAL APPLICATIONS Today, nanotechnology offers exciting opportunities in diagnostics and treatment of many diseases including those of high social impact, such as cancer. Remarkably, many properties of colloidal inorganic nanomaterials, valuable for diagnostics and therapy, are not available in molecular, macromolecular, and micron-size systems. For example, plasmonic gold nanoparticles, capable of absorbing nearinfrared light, can be employed as heat-emitting probes inducing cancer cell death.35 Nanoparticles exhibiting sizedependent luminescence (e.g., quantum dots) can be used as highly efficient imaging agents.36 Mesoporous inorganic silica or metal organic framework nanoparticles, loaded with a chemotherapeutic agent, can release their cargo on-site using chemical and/or physical triggers, thereby minimizing the nonspecific uptake and side effects.37,38 Furthermore, nanosized materials are a perfect platform for development of multifunctional agents. Synergistic combination of various particles, targeting receptors, payloads, etc. in a single entity can significantly increase the chances of successful disease treatment.39,40 All these features make colloidal nanoparticles probably the most promising building blocks for theranostic systems of the future.

Table 1. Basic Boolean Logic Gates with Single and Two Inputs

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Figure 2. Scheme of logic-gated tagging cell surface with molecular automata employing strand displacement reactions as cross-talk language. Reprinted with permission from ref 44. Copyright 2013 Springer Nature.

while the others are still on their way to achieve functional completeness. Douglas and co-workers designed nanorobotic DNA origami-based devices for smart delivery of signaling molecules to cell surfaces.41 These devices represented hexagonal nanobarrels with dimensions of 35 × 35 × 45 nm assembled using DNA origami technology. The barrel was composed of two domains that were covalently linked in the rear side by single-stranded scaffold hinges and could be closed by means of hybridization between ssDNA staples at the front side. Specifically, the front side of one domain bore DNA aptamer strands, while that of the other domain had ssDNAs partially complementary to these aptamers. In the presence of their targets (inputs, exogenously expressed surface cell markers), the aptamers folded into rigid structures leading to dissociation of the lock duplexes, followed by opening of the nanobarrel. As a result, the internal surface, which was covalently modified with 5 nm AuNPs and Fab antibody fragments able to attach to protein markers on the surface of the cell of interest, became available for interaction. These agents could be programmed to activate in response to a single input by using the same aptamer sequence in the right and the left sides of one domain. On the other hand, the use of different aptamer sequences at these sides enabled their programming to be responsive to two inputs. Such devices remained inactive until both aptamer locks were opened. This opening mechanism was consistent with Boolean AND, with possible inputs of cell surface antigens not binding or binding (0 or 1, respectively) to aptamer locks and possible outputs of remaining closed or a conformational rearrangement to expose the payload (0 or 1, respectively). Later, the proposed design of such nanorobotic device with some alterations was successfully tested in vivo using animal with low systemic nuclease activity (Blaberus discoidalis).42 In this work, the authors employed soluble protein inputs (PDGF-BB and VEGF165) and modified DNA lock so it could be opened as a result of either recognition of a target with its aptamer strand or via toehold-mediated strand displacement reactions. Such modification enabled reprogramming of the AND-gated nanorobot into devices capable of implementing other Boolean operations (OR, NOR, XOR, NAND, NOT, CNOT, and half adder). For example, the OR-

Over the past decade, significant research efforts have been focused on the design of nanomaterials whose properties and, therefore, behavior is regulated in a programmable fashion. In relation to biomedicine, this development is represented by the concept of nanoagents capable of implementing logic-gated targeting, sensing, imaging, and therapy. Here, we present these highly diverse nanomaterials sorted into three groups according to their functional properties promising for biomedicine. We will first survey the systems that control their attachment to cells based on analysis of cell surface markers, as well as of soluble inputs. Then, we will consider logic-gated luminescent, plasmonic, and magnetic nanoparticles that can potentially be utilized as reporting modules. In the third group, we will cover the control release systems and nanomaterials, which can alter chemical composition of the target (micro)environment through the regulated catalysis. Some of the examples in the second and the third group, though, may not be directly related to biomedicine, but rather to, for example, environmental monitoring and were included in this section for completeness. Finally, each subsection devoted to a certain group of logic-gated nanomaterials is concluded with a table briefly summarizing the examples surveyed in the text to facilitate overview and comparison of these logic-gated systems by a number of parameters, such as employed input sensing interfaces, logic gating mechanism, demonstrated logic gates, nature of the output readout, etc. 3.1. Biomolecular Logic-Gated Agents for Selective Cell Targeting

One of the grand challenges of drug delivery is how to target a diseased cell if it can be distinguished only by multiple surface markers or soluble cues in its vicinity (e.g., elevated concentrations of the tumor angiogenesis factors). To address this problem, we need significantly more sophisticated delivery systems capable of autonomous analysis of their environment. Recently, a few logic-gated systems capable of implementing such analysis in order to attach to proper cells have been reported. Most importantly from the computing point of view, some of these systems can already implement a complete set of Boolean functions (and hence, in principle, any logic gate), D


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Figure 3. Principles of logic-gated cell targeting: (a) scheme of AND-gated tagging of diseased cells in the presence of nontargeted ones and (b) mechanism of strand displacement cascade of the AND gate and the truth table. Reprinted from ref 46. Copyright 2015 American Chemical Society.

respectively. The cascade of displacement reaction was triggered by ssDNA 0, which displaced ssDNA 2 from the 1•2 complex via interaction with toehold T1. Then, the liberated ssDNA 2 substituted ssDNA 4 from the 3•4 complex via another toehold-mediated DNA strand displacement reaction because a fragment of ssDNA 2 was complementary to the T3 region of 3•4. The yielded ssDNA 4 could trigger another strand displacement and thereby extend the cascade further. The displacement of ssDNA 4 with ssDNA 2 was possible only when αCD45-1•2 and αCD20-3•4 automata were colocalized on the surface of B cells via antigen−antibody interactions αCD45:CD45 and αCD20:CD20, respectively. As a result, the surface of B cells was tagged with complexes αCD45-2•3 and αCD20-3. In contrast, cells T lacked the marker CD20, and therefore, the surface of these cells was eventually tagged with only complex αCD45-2. Thus, these automata generated two different outputs depending on the presence or absence of cell surface markers. This scheme enabled fabrication of a functionally complete set of Boolean logic gates (YES, NOT, AND, and OR). Using automaton YESCD45YESCD3, the authors demonstrated tagging fluorescein-labeled cells with antifluorescein antibodies-modified magnetic microparticles for cell separation. Tan and co-workers employed both DNA aptamer−target interactions and toehold-mediated DNA strand displacement reactions to fabricate a series of “DNA nano-claws” for autonomous analysis of multiple surface cell markers simultaneously.45 For example, the Y-shaped nanoclaw was composed of oligonucleotide scaffold bearing two aptamercomplementary strand capture dsDNA toes and one effector dsDNA toe with built-in logic gating based on toeholdmediated DNA strand displacement reactions. The capture toes had dual purpose: first, locate the specific surface marker

gated device was obtained using the AND-gated nanorobot E activated upon the simultaneous presence of the soluble protein inputs plus two additional robots P1 and P2. Each of these devices was designed to get opened in the presence of respective protein input and comprised a DNA key inside its internal hollow, which upon barrel opening became able to cleave the dsDNA complex composed of aptamer to the other protein and its complementary strand. We should note that a recent work by Zhao and co-workers has demonstrated successful application of a related DNA origami-based nanorobot for tumor targeting in mammals, though, yet without logic-gating.43 This device, responsive to a single nucleolin input (effectively a YES-gate), represented DNA origami sheet loaded with thrombin and then folded and locked using nucleolin-binding aptamer. In the presence of nucleolin-positive tumor vascular endothelial cells, this device exposed its payload, thereby causing tumor vessels thrombosis followed by tumor tissue necrosis and at the same time sparing healthy blood vessels. Rudchenko and co-workers developed molecular automata based on toehold-mediated DNA strand displacement reactions able to autonomously analyze surface marker profile of T lymphocytes in the presence of the related cells and tag these cells with DNA label which can be used for delivery of functional nanoparticles.44 Figure 2 illustrates the basic design principles for these automata capable of tagging lymphocytes with targeted cluster differentiation (CD) markers characteristic of B cells (CD45+CD20+) cells, in the presence of nontargeted CD45+CD20− cells (CD45+CD3+, T cells). The antibodies against targeted markers CD45 and CD20 (αCD45 and αCD20, respectively) were conjugated with a set of partially complementary dsDNA complexes (1•2, 3•4, respectively) containing toehold regions T1, T3, and T5, E


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Figure 4. Conceptual designs of biocomputing agents for YES/NOT/AND/OR complete functional set of Boolean logic. (a and b) Assembly and performance of (a) YES gate and (b) NOT gate. (c) Designs of three different AND-type logic gates based on YES/NOT gates. (d) OR gates are realized by mixing the structures for YES/NOT/AND functions. Input-processing interfaces in (a−c) are denoted as locks to illustrate the indifference of the concept to the biochemical nature of interfaces and inputs. Rectangular and round locks represent interfaces that process different inputs. Reprinted with permission from ref 47. Copyright 2014 Springer Nature.

arrangement of the self-assembled nanoparticles and biomolecules rather than into biochemical peculiarities of the involved interfaces. Accordingly, the proposed concept is not limited to a specific input-processing interface and can operate via a wide range of interfaces based on antibodies, DNA, chemical bonds, etc. Biocomputing agents based on antibody− antigen, lectin-glycoprotein, streptavidin-(imino)biotin, and other interfaces for processing ions, small molecules, and protein inputs have been demonstrated.47,48 Generally, the biocomputing agent consists of two key elements: a core particle to be delivered to the target and a structural component on the core particle surface that realizes logic gating. This component comprises an output receptor that can bind to a target (e.g., a biomolecule specific to a certain cellular marker) and an input-sensitive interface that regulates (i.e., either allows or forbids) attachment of the core particle to the cellular target via the output receptor. There are two essentially different ways of allocation of this interface on the core particle surface depending on the type of single-input logic (YES or NOT). In the YES-gated nanoagent (Figure 4a), the interface represents an array of “shielding” particles assembled via input-susceptible bonds on the surface of the core particle. The shielding particles realize steric hindrance that prevents interaction of the output receptor on the surface of the core particle with the target. In the presence of input that cleaves the bond between the core and the shielding particles, the YES-gated agent disassembles so that the core’s output receptor becomes available for binding to the target. In the NOT-gated agent, the output receptor is immobilized on the core particles via the input-susceptible interface (Figure 4b). Once the input is introduced, it releases the output receptor from the core particle, thereby forbidding binding of the core particle to its target. Accordingly, the core particle can bind the output receptor’s ligand only if no input is present.

and then release effectors for activation of the effector toe. Specifically, binding of DNA aptamers to their respective targets (surface markers) resulted in dissociation of aptamercomplementary strand dsDNA complexes forming capture toes, and two released complementary strands launched DNA strand displacement cascade in the effector toe. As a result, the effector toe duplex bearing fluorophore and its quencher became cleaved as was visualized by restoration of fluorophore emission. The overall process followed Boolean AND logic. The same research group also reported more advanced logicgated cell-tagging interfaces.46 Figure 3 shows modular design of these agents and principles of their logic gating. First, DNA aptamers bind to their respective targets located on surfaces of different cells, thereby marking these cells with short ssDNA tags linked to these aptamers. Different aptamers could have the same or different ssDNA tags depending on logic operation executed by these interfaces. These tags are then analyzed by the effector, which is a dsDNA complex carrying drug or dye whose sequence was designed based on ssDNA tags profile on the cell of interest. Specifically, the cascade of toeholdmediated DNA strand displacement reactions results in labeling of the cell of interest with dye or drug, thereby signaling selective recognition. Along with basic AND, OR, and NOT, this scheme enabled execution of more complex logic operations, such as logic gates requiring three and even four inputs and a concatenated gate. The Nikitin group presented a concept of biocomputing “based on particle disassembly” for fabricating nanoagents that can target specific cells as a result of logic analysis of soluble markers in the microenvironment.47 The approach stands out due to its unique flexibility towards the biochemical nature of the components of the agents. This is currently the only approach that provides the functionally complete set of Boolean logic functions and is not inherently DNA-dependent. The biocomputing programs are hard-coded into the spatial F


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Table 2. Summary of Biocomputing Systems for Logic-Gated Cell Targeting

means that the agent’s performance is fully independent from presence and/or concentration of other agents, which may be crucial for the in vivo conditions (when concentration is limited due to safety/toxicity considerations). To conclude this subsection, the brief summary of the surveyed biocomputing systems that offer logic-gated cell targeting is given in Table 2.

These basic YES and NOT logic gates can then be combined according to AND-type and OR-type logic to derive multipleinput logic gates (e.g., AND, NOR, INHIBIT, IMPLICATION - see Figure 4c, d) and, in principle, allow implementation of any logic gate. Such flexible architecture based on biomolecule-driven selfassembly49−51 allowed fabrication of biocomputing devices using a great variety of the core nano- or microparticles (magnetic, plasmonic, luminescent, etc.47,48,52) regardless of their surface properties, which indicates the versatility of the approach with respect to the type of cargo that can be delivered to cells. A notable feature of this approach is complete self-sufficiency of each biocomputing agent. This

3.2. Logic-Gated Nanoparticles for Sensing and Imaging

Besides targeting, biocomputing agents also have another important task: transmission of results of microenvironment analysis to biomedical personnel. In some cases, the amount of biochemical information may be rather large and hard to transmit. Herein, Boolean algebra is an excellent tool for data G


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The immobilization of a molecular chemosensor onto nanoparticles surface aims to protect the former from nonspecific interaction (e.g., binding to cellular proteins), which can affect their fluorescent properties, when used in vitro and in vivo. In this respect, silica nanoparticles (SiO2NPs) are a good surface substrate for molecular chemosensors because it is an optically transparent material, and its surface can be readily functionalized with organic molecules via alkoxysilane chemistry. The presence of a spacer between the surface and the probe prevents the possible influence of the surface on fluorescent properties of the probe, so the SiO2NPs-tethered fluorescent probes retain the functionality of their parent homogeneous counterparts. The supported probes have significantly lower cellular toxicity than their homogeneous counterparts, which is important in bioimaging.55 Ma and co-workers prepared a series of SiO2NPs-supported molecular INHIBIT56−59 and IMPLICATION60 logic gates. The true output of INHIBIT logic gates was obtained as a result of disruption of PET between fluorophore and receptor by coordination of the first metal ion input to the latter. In all other situations (absence of inputs, presence of the second input alone, simultaneous presence of both inputs), these systems yielded the false output as was expected for INHIBIT logic operation. The second input alone either did not interact with the receptor or was a strong electron acceptor increasing PET. When both inputs were introduced simultaneously, the second input either scavenged the first one (e.g., formation of precipitation or an inactive complex) or displaced it from the receptor unit. For example, this research group reported a fluorescent sensor for Hg2+ based on rhodamine derivative modified SiO2NPs (R3-SBA-15).57 In the absence of inputs, R3-SBA-15 showed almost no fluorescence emission. When Hg2+ was introduced alone, a strong enhancement of fluorescence was observed. This was due to restoration of the conjugated xanthene structure because of the cleavage of the spiro N−C bond upon binding Hg2+ to N and O donor atoms. Upon addition of S2− without Hg2+, it had no effect on the fluorescence intensity of R3-SBA-15. However, when both inputs were introduced together, the scavenging of Hg2+ with sulfide prevented the coordination of Hg2+ to donor atoms of the fluorophore, and as a result, the false output (no fluorescence) was obtained. The functioning of the system mimicking IMPLICATION logic was based on reversed principle: in the absence of inputs, Fe3O4@SiO2 core−shell nanoparticles functionalized with fluorophore bearing 1,8naphthalimide and bis(2-pyridylmethyl)amine moieties showed strong emission. The first input (pyrophosphate ion) alone did not interact with the fluorophore, so its fluorescent properties remained intact and the true output was also observed. On the other hand, the second input (Cu2+, a good electron acceptor due to the empty d shell) alone became chelated with donor atoms of bis(2-pyridylmethyl)amine thereby making the probe nonemissive. Finally, when pyrophosphate and Cu2+ coexisted in the medium, their mutual interaction prevented Cu2+ binding to the receptor unit, so the probe remained fluorescent. Badiei group reported an XOR-gated sensor for Fe3+, Al3+, and CN− based on salicylimine-modified SiO2NPs.61 The presence of electron-rich and electron-deficient areas in salicylimine molecules facilited ICT, so the nanoparticles dyed with these molecules showed low emission in the absence of inputs. The addition of either Al3+ or CN− alone led to

compression: the information can be analyzed by an agent itself and then reported to a doctor in the encoded state. This type of reporting can be implemented by systems whose state can be interrogated in the noninvasive fashion: namely by means of reading out their variable optical (luminescence and surface plasmon resonance) or magnetic properties. 3.2.1. Luminescence-Based Logic-Gated Systems. Sensing and imaging based on luminescent phenomena (fluorescence, phosphorescence, and (electro)chemiluminescence) has superior characteristics over those using colorimetric and electrochemical techniques due to unique attributes such as ultrahigh sensitivity (up to a single molecule), very high speed of response (as fast as 10−8−10−10 s), high spatial resolution, versatility (usage in any environment), and noninvasiveness. In particular, these methods have received considerable attention as detection and visualization tools for obtaining information characterizing biological processes both in vitro and in vivo using a series of nanoparticles as reporters.36,53 Moreover, more advanced logic-gated sensing and imaging probes can be produced by introducing logic gating mechanism regulating luminescent properties of these materials. For example, in ratiometric sensing it is more reasonable to employ one analog logic-gated sensor rather than two separate probes for each analyte. The incorporation of logic gating into luminescent systems largely depends on exact mechanism of quenching of emission of nanomaterials or other luminescent systems by nanomaterials. The most common ones include photoinduced electron transfer (PET), intramolecular charge transfer (ICT), and fluorescence resonance energy transfer (FRET). These mechanisms have been thoroughly described elsewhere.54 3.2.1.1. Emitting Nanoparticles. 3.2.1.1.1. Dyed Nanoparticles. 3.2.1.1.1.1. Silica-Supported Molecular Chemosensors. Molecular fluorescent chemosensors are a class of molecular devices composed of fluorophore and receptor unit(s). The binding of an analyte to the receptor adjusts the fluorescent properties of the fluorophore unit, and, as a result, the quantitative information can be extracted from the changes of the fluorescence intensity. The quenching of the fluorescence of these systems is regulated by PET or/and ICT processes. PET and ICT systems are structurally different from each other. The design of PET probes implies the separation of electronic systems of receptor and fluorophore by spacer. In the absence of analyte, such PET probe is nonemissive due to electron transfer between the receptor and fluorophore. The binding of analyte to the receptor disrupts this process, leading to the fluorescence enhancement. In contrast, ICT probes integrate the receptor and fluorophore within the same electronic system. ICT systems show highly shifted emission band (bathochromic shift) in the absence of analyte. The binding of the latter to the receptor results in inhibition of the charge transfer and, as a result, the probe shows normal fluorescence emission. The advantage of molecular chemosensors is that their structure can be readily modified for the desired performance using synthetic chemistry, which provides the opportunity to precisely regulate PET and ICT processes. Metal ions are ideal targets for such probes due to their specific affinity to particular ligands (receptors). Therefore, employing metal−ligand and metal− other analytes interaction it is possible to regulate these quenching processes in a logical manner which enables fabrication of logic-gated sensors. H


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significant enhancement of fluorescence which was due to disruption of ICT upon interaction of these inputs with salicylimine (chelation of Al 3+ with salicylimine and nucleophile addition of CN− to the imine site, respectively). However, when these inputs were introduced simultaneously, their mutual interaction produced a new compound unable to interact with salicylimine, and, therefore, the nanoparticles remained weakly fluorescent. Bozdemir and co-workers reported a series of molecular logic gates based on bitopic BODIPY derivatives containing both ICT and PET donors.62 Specifically, BODIPY was functionalized with dipicolylamine (DPA − a selective Zn2+ chelator) at the 8-position and with the dithiaazacrown (a ligand with high affinity to Hg2+) group at the 5-position. In the absence of all inputs, the bitopic BODIPY showed weak fluorescence intensity. The presence of either Zn2+ or Hg2+ alone did not lead to significant fluorescence intensity enhancement because ICT from dithiazocrown to BODIPY and PET from DPA to BODIPY remained unforbidden, respectively. However, when Zn2+ and Hg2+ were introduced simultaneously, both PET and ICT became inhibited resulting in strong enhancement of fluorescence. This behavior was in accordance with an AND logic gate (emission at lower wavelength). Choi and co-workers modified this BODIPY derivative by replacing dithiaazacrown group with bis(chloromethyl)aniline and attached it onto SiO2NPs surface via coupling between chloromethyl groups and silica-anchored thiol groups.63 This SiO2-supported BODIPY probe was used to fabricate NAND and OR fluorescent logic gates with Zn2+/ Hg2+ and Ni2+/Cu2+ as inputs, respectively (emission at higher wavelength as an output). 3.2.1.1.1.2. Miscellaneous Dyed Nanoparticles. In addition to SiO2-supported molecular chemosensors, a series of other nanoparticles modified with luminescent compounds were also used in fabrication of logic gates. Wang and co-workers demonstrated fluorescent ratiometric ClO−/SCN− sensing using a coumarin-rhodamine CR1-Eu probe attached to magnetic Fe3O4 nanoparticles via coordination to dipicolinic acid ligand on their surface.64 This sensor exhibited three emissions: coumarine (455 nm), rhodamine (550 nm), and Eu3+ (616 nm) upon excitation of its acetonitrile solution at 415, 225, and 274 nm, respectively. In aqueous solutions, this probe was sensitive to ClO−. The intensity of emission peaks at 592 and 616 nm obtained under excitation at 274 nm became decreased significantly, while that at 440 nm was slightly enhanced. This was due to leaching of the CR1-Eu complex from the surface of Fe3O4 nanoparticles followed by formation of a new CR1-Eu-ClO complex. On the other hand, in the presence of SCN− ions, this process could be reversed due to oxidation of the latter with ClO− to afford NCSO− followed by reattachment of CR1-Eu complex onto magnetic particles surface. This pattern was consistent to INHIBIT logic with ClO− and SCN− as input and emission at 616 nm as the true output. Another example of the use of luminescent Ln3+ complexes tethered to nanoparticles for logic gates construction was reported in the recent work by Gunnlaugsson and coworkers.65 Complexes of Eu3+ and Tb3+ with cyclene ligand were attached to AuNPs via dodecanethiol spacer. This linkage ensured sufficient separation of Ln3+ emitters from the surface of AuNPs which are good FRET acceptors. The obtained probes showed different luminescent response to pH, solvated dioxygen, and aromatic “antennae” ligands. At strong acidic

conditions, the emission of Eu3+ (592 nm) was quenched and subsequently became enhanced with the increase of pH independently from the presence of O2 in solution. In contrast, the Tb3+ emission (545 nm) was quenched in aerated strong acidic solution, reached maximum with the increase of pH until 7, and became quenched again with further basification up to pH = 12. In the latter case, deaeration led to increase of fluorescence intensity; however, its profile against pH remained the same. The difference in pH response of AuNPLn3+(cyclene) device was exploited to construct XOR (true output, Tb3+ emission) and NAND (true output, Eu3+ emission) gates both with degenerate H+ input and could be operated parallelly (half-subtractor). The AuNP-tethered Eu3+ and Tb3+ complexes also showed luminescence enhancement in the presence of aromatic “antennae” ligands: 4,4,4-trifluoro1-(naphthalene-2-yl)-butane-1,3-dione (nta) and 4(dimethylamino)benzoic acid (DMAB), respectively, which were able to displace quenching water molecules from the coordination sphere. It was also found that nta could displace the DMAB ligand because its complexes with Eu3+ and Tb3+ have less strained geometry. On the basis of these luminescence enhancement phenomena, the authors fabricated logic gates INHIBIT (true output, Tb3+ emission) and TRANSFER (true output, Eu3+ emission) integrated in parallel fashion with nta and DMAB as inputs. Zink and co-workers developed AND-gated “controlled inlet” nanoparticles for sensing perchlorate ions in water.66 The authors loaded hollow SiO2NPs with [Pt(tpy)Cl]PF6 (tpy = 2,2′:6′,2″-terpyridine) complex which is sparingly soluble in water. The exposure of this compound to ClO4− leads to counterion exchange, and the resulting [Pt(tpy)Cl]ClO4 complex becomes intensively fluorescent. The external pores of these SiO2NPs were gated by supramolecular complexes formed by host−guest interactions between α-cyclodextrins (α-CDs) and N-phenylaminomethyltriethoxysilane. These caps inhibited diffusion of perchlorate ions inside internal cavity of SiO2NPs, and, as a result, these particles remained nonfluorescent. However, upon acidification of the medium, αCDs could get detached from protonated N-phenylaminomethyltriethoxysilane stalk, thereby making internal pores accessible for perchlorate inlet. The formation of emissive complex upon simultaneous presence of H+ and ClO4− was consistent to Boolean AND logic with these ions as inputs. This logic-gated sensor worked according to inverse principle of functioning of controlled release systems which will be covered in the next section of the present review. 3.2.1.1.2. Luminescent Nanoscale MOF. Metal organic frameworks (MOF) are a subclass of coordination polymers which are periodic structures formed by self-assembly of metal ions or their clusters and multitopic (i.e., capable of linking more than one metals) organic ligands. The miniaturization of these materials to the nanoscale level has paved the way toward their application in biomedicine.67 Indeed, the formidable MOF toolbox allows fabricating MOF nanoparticles with low cytotoxicity, which in conjunction with their high surface area and mesoporous texture makes these systems highly attractive as prospective drug carriers. Aside from controlled release applications (see the next section), nanosized MOF are also interesting as magnetic resonance and luminescence imaging probes. If the existence of magnetism of certain MOF is explained by the presence of paramagnetic metal ions such as Gd3+ and Mn2+, in the framework, the origin of luminescence of these materials can be multifaceted. In general, it can arise I


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from either organic linkers or f-element metal ions (lanthanide and actinide). In more specific cases, this property can appear as a result of confinement of dyes inside the framework. This effect was first demonstrated in works by Kimizuka and coworkers who reported water dispersible coordination polymer nanoparticles (CPNPs) obtained by adaptive self-assembly lanthanide ions and nucleotides [adenosine 5′-monophosphate (AMP) and guanosine 5′-monophosphate (GMP)].68,69 More specifically, these CPNPs provided hydrophobic internal compartments for guest molecules restricting their conformational mobility which facilitated enhancement of their fluorescence. This phenomenon along with assembly/disassembly of Ln-based CPNPs and their inherent fluorescence was exploited by a few other research groups in design of fluorescent logic gates. Qu and co-workers demonstrated logical regulation of fluorescence of N-methyl mesoporphyrin IX (NMM) entrapped inside CPNPs which were obtained by self-assembly of lanthanide ions (Eu3+ and Tb3+) and GMP.70 This dye had low fluorescence in water solution due to solvent-induced quenching; however, its confinement inside a hydrophobic internal compartment of CPNPs led to dramatic fluorescence enhancement. This event was defined as the true output of an AND logic gate with Eu3+ and NMM as inputs and GMP as gate machinery. Similar results were obtained when using Tb3+ instead of Eu3+, so using these ions as input and GMP/NMM as a processing unit, an OR logic gate was obtained. The lanthanide/GMP framework, however, was prone to collapse in the presence of strong chelators such as EDTA or at either acidic/basic conditions. As a result, the dye was released, and its fluorescence became quenched with water molecules. These observations were used to fabricate INHIBIT and XNOR logic gates. The INHIBIT one was fabricated using NMM/GMP and Eu3+/EDTA as a gate machinery and inputs, respectively. The XNOR one was obtained employing CPNPs confining NMM dye and HCl/NaOH as inputs. The true output was achieved either in the absence or in the presence of both inputs together because at the neutral pH values the dye remained entrapped inside CPNPs and, therefore, was highly fluorescent. In contrast, when either input was present, the decrease or increase of pH resulted in collapse of the framework accompanied by release of the dye. Similarly, a NOR gate was obtained using EDTA and HCl as inputs. These CPNPs possessed good water dispersibility, which ensured successful running the gates AND and OR in vitro for imaging A549 cells. In addition, these logic gates could be concatenated to afford three-input INHIBIT → INHIBIT, OR → INHIBIT, and XNOR → AND circuits. The subsequent work of this research group demonstrated construction of PASS0, PASS1, YES, NOT, AND, OR, and IMPLICATION logic gates with Tb3+ and OH− as inputs by excluding one or more components from the system composed of NMM, GMP, H+, Eu3+, and EDTA (Figure 5).71 On the contrary, Yao and co-workers reported construction of logic gates using regulation of fluorescence of namely lanthanide/GMP-based CPNPs rather than entrapped guest molecules within the framework.72 These particles exhibited different emissive properties depending on lanthanide ion used in their preparation. CPNPs formed as a result of self-assembly between Tb3+ and GMP showed strong green emission at neutral pH due to hydrophobic compartments of the framework which prevented interaction between Tb3+ and water leading to quenching. On the other hand, those CPNPs

Figure 5. Design of logic gates based on fluorescence switching regulated by self-assembly of 5′-GMP and lanthanide ions. Reprinted from ref 71. Copyright 2014 American Chemical Society.

formed by Eu3+/GMP and Ce3+/GMP were weakly emissive at neutral pH due to the quenching with water. In the presence of dipicolinic acid, these CPNPs showed completely different fluorescent properties. The Tb3+/GMP particles lost their green emission and obtained a blue one at pH lower than 4, while Eu3+/GMP and Ce3+/GMP frameworks showed strong emissions in red and blue regions. The fluorescence enhancement of the latter ones was due to ICT from carboxyl group of dipicolinic acid to lanthanide ions. Employing competitive interaction between lanthanide ions and GMP and other molecules, the authors developed a series of fluorescent logic gates. A concatenated INHIBIT → INHIBIT gate was derived using GMP as gate machinery and Tb3+, EDTA, and dipicolinic acid as inputs. In accordance with this logic, the true output (green emission of Tb3+/GMP CPNPs) could be achieved only upon addition of Tb3+ without other inputs, which either quench the green emission or induce the collapse of CPNPs as a result of chelation of Tb3+. The green emission of Tb3+/GMP CPNPs could also be quenched by Cu2+ and Hg2+, which are strong electron acceptors. This effect could be reversed by addition of cysteine capable of scavenging these metal ions. Such competitive interactions could be described by NOR → OR logic gate with Cu2+, Hg2+, and cysteine as inputs and Tb3+/CPNPs as gate machinery. The addition of EDTA to Eu3+/GMP particles treated with dipicolinic acid led to further increase of red emission, which was defined as the true output in AND → NOR logic gate with dipicolinic acid, EDTA, and Cu2+ as inputs. Finally, using green emission of Eu3+/GMP in the presence of dipicolinic acid as one output, and blue emission of the same particles at pH < 3.5 as another one, the authors obtained AND and YES logic gates with dipicolinic acid and H+ as inputs run in parallel fashion. J


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3.2.1.1.3. Fluorescent Noble Metal Nanoclusters. Fluorescent noble metal nanoclusters (NCs) represent a subclass of the respective metal nanoparticles with a core size below 2 nm and are typically composed of several to tens of metal atoms. Due to their small size, the nanoclusters have fluorescent behavior similar to organic fluorophores. In comparison to molecular probes and quantum dots, noble metal nanoclusters are characterized by low cytotoxicity, biocompatibility, and high resistance toward photobleaching. These characteristics make these nanomaterials highly attractive as reporting probes for fluorescent sensing and imaging in vitro and in vivo.73 The synthesis of these systems is based on the use of different templates such as polymers, proteins, and DNA which protect nanoclusters from aggregation. The choice of a template determines the fluorescent properties of these nanomaterials.74 In addition, the change in conformation/structure of the protecting template may have dramatic effect on fluorescence, and these event processes can be exploited in incorporation of logic gating mechanism. The Qu research group constructed a complete set of binary logic gates (AND, OR, INHIBIT, IMPLICATION, NOR, XNOR, XOR, and NAND) by tuning fluorescent properties of AgNCs with various DNA used as stabilizers for these AgNCs.75 The authors prepared AgNCs by reduction of AgNO3 with NaBH4 in the presence of poly(acrylic acid) (PAA) as a template. The fluorescence of AgNCs was controlled by three DNA sequences: ssDNA-1, ssDNA-2, and ssDNA-3 (Figure 6). ssDNA-1 contained the C-rich region a and region b that was complementary to region c of ssDNA-2. The latter, in addition to the region c, also comprised the G-rich region d. ssDNA-3 was composed of the region c, complementary to region b of ssDNA-1, and the

T-rich region e. The C-rich fragment of ssDNA-1 had high affinity to AgNCs and was able to remove AgNCs from the PAA scaffold. The so-obtained ssDNA-1/AgNCs complex had dim red fluorescence. When ssDNA-2 was added to the ssDNA-1/AgNCs device, the hybridization between regions b and c of ssDNA 1 and ssDNA-2, respectively, produced a dsDNA complex where the G-rich region of DNA-2 became immediately adjacent to AgNCs stabilized by the C-rich region a of ssDNA-1. The newly formed DNA scaffold resulted in an increase of red light emission of AgNCs. When ssDNA-3 was added to ssDNA-1/AgNCs agent instead of ssDNA-2, the hybridization between their respective c and b regions produced a different dsDNA complex, and, as a result, the T-rich region of ssDNA-3 and AgNCs stabilized by the C-rich region of ssDNA-1 were put in close proximity to each other. This structure resulted in transformation of dim-red AgNCs into a bright green light emitter. An AND logic gate was fabricated using ssDNA-1 and ssDNA-2 as inputs and PAAtemplated AgNCs as gate machinery. When ssDNA-1 was added without ssDNA-2, it captured AgNCs from PAA scaffold; however, the fluorescence intensity was below the threshold defined as the output. If ssDNA-2 was added in the absence of ssDNA-1, it could not capture AgNCs from PAA scaffold because of the lack of C-rich fragment in the sequence, and, as a result, AgNCs remained nonemissive. Only when both sequences were added, the hybridization between ssDNA-2 and ssDNA-1 bearing AgNCs produced the dsDNA with the structure facilitating bright red emission defined as the output. Using PAA-templated AgNCs as a gate machinery and various input ssDNA sequences, INHIBIT, OR, and XOR logic gates were fabricated. NOR, XNOR, and NAND logic gates were obtained employing various emissive dsDNA-AgNCs devices and the respective ssDNA inputs. The introduction of these inputs led to the formation of nonemissive ssDNA/AgNCs complexes depending of logic function and inputs combinations. In addition, ssDNA-1/ AgNCs complex and input ssDNA-2 and ssDNA-3 were used to fabricate sequential logic gates and keypad lock. Zhang and co-workers reported fluorescent detection of PDGF-BB exploiting PET between DNA-AgNCs system and G-quadruplex/hemin complexes.76 The fluorescent probe comprised three parts: the aptamer sequence against PDGFBB, the fluorophore DNA-templated AgNCs as the donor, and the G-quadruplex sequence able to form horseradish peroxidase (HRP)-mimicking DNAzyme acting as the electron acceptor, and it was self-assembled into a hairpin structure. The hairpin loop contained 12 additional cytosine bases to stabilize AgNCs. In the presence of PDGF-BB alone, the recognition of this target with the respective aptamer sequence induced the dissociation of dsDNA yielding the release of the G-quadruplex sequence part. In the presence of hemin and K+, this fragment folded into the HRP-mimicking DNAzyme thereby being immediately adjacent to AgNCs. This transformation enabled PET from AgNCs to Fe3+ core of hemin, which led to a decrease of the fluorescence emission. Thus, using PDGF-BB and hemin as inputs, a fluorescent AND logic gate was produced. Another method of constructing fluorescent logic gates using AgNCs was reported by Wang and co-workers, who employed folding of DNA strands into secondary structures in the presence of cations (Figure 7).77 The protecting DNA containing G-rich, poly-C, and C-rich regions was selfassembled into a hairpin structure. The poly-C hairpin loop

Figure 6. DNA-programmable regulation of fluorescence properties of AgNCs. Reprinted with permission from ref 75. Copyright 2012 Wiley-VCH. K


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histidine or cysteine. This observation could be explained by the fact that the interaction of these amino acids with Cu2+ produced more robust complexes than that with 11-MUA bearing the sole carboxyl group. On the other hand, histidine and cysteine could be scavenged by Ni2+ ions or Nethylmaleimide, respectively, so the restoration of the fluorescence induced by these amino acids could be reversed. Thus, two INHIBIT logic gates with the respective Ni2+/ histidine and N-ethylmaleimide/cysteine pair of inputs were fabricated. The true outputs of these INHIBIT logic gates could be integrated to afford an integrated OR logic gate for detection and differentiation of histidine and cysteine in the presence of Ni2+ and N-ethylmaleimide. Chattopadhyay and co-workers reported a logic device based on mercaptopropionic acid (MPA)-stabilized AuNCs whose fluorescence could be tuned by various stimuli such as temperature, pH, and metal ions.80 The MPA-stabilized AuNCs exhibited temperature-dependent fluorescence. At low temperatures (5 °C), the MPA-stabilized AuNCs showed maximum emission, while upon heating (70 °C), these NCs were almost nonemissive. This was due to a nonradiative deexcitation (internal conversion) pathway which became more favorable at higher temperatures. The fluorescence of these NCs could be also quenched in the presence of Cu2+, which upon binding to carboxy groups of MPA enabled PET. On the other hand, the decrease of pH prevented binding of Cu2+ to the carboxy group of MPA, so the fluorescence of MPA-stabilized AuNCs became restored. Therefore, employing Cu2+/H+ and H+/low temperature as inputs, IMPLICATION and OR logic gates could be obtained, respectively. Another design of logic gates employing noble metal nanoclusters as signal transducer is based on oxidation of surface atoms with reactive oxygen or nitrogen species leading to static quenching of fluorescence. Khashab and co-workers reported fluorescent sensing of antioxidants using Au(0)@ Au(I)-thiolate NCs.81 The fluorescence of these highly emissive NCs could be quenched by OH• radical generated from a Fenton reaction (Fe2+ + H2O2). Specifically, the reactive OH• radical induced the oxidation of Au(0) to Au(I) to afford a nonemissive state. Therefore, employing Fe2+ and hydrogen peroxide as inputs, and fluorescence quenching as the true output, a NAND logic gate was obtained. The quenching induced by OH• radicals, however, could be prevented by antioxidants (e.g., ascorbic acid, GSH, etc.) able to scavenge this radical. Thus, using the Fenton reagent (Fe2+ + H2O2) and ascorbic acid as inputs and fluorescence quenching as the false output, an IMPLICATION logic gate was produced. Yang and co-workers also employed oxidation of the surface of BSA-stabilized AuNCs to construct a fluorescent NAND logic gate.82 Specifically, the authors employed peroxynitrous (HOONO) acid, which could obtained in situ from nitrite and hydrogen peroxide, as an oxidizing agent for AuNCs to afford nonemissive clusters. On the other hand, in the absence of both or in the presence of either hydrogen peroxide or nitrite these NCs were highly fluorescent, which was fully consistent with Boolean NAND logic. 3.2.1.1.4. Quantum Dots. Quantum dots (QDs) are nanosized semiconductor devices typically composed of elements from groups II−VI or III−V of periodic system. Similar to fluorescent noble metal nanoclusters, these nanoparticles do not require the modification with fluorophores because their fluorescence is a result of a photonic quantum

Figure 7. Design of logic gates based on HP26-tuned fluorescent AgNCs. Reprinted from ref 77. Copyright 2011 American Chemical Society.

served as a stabilizing scaffold for synthesis of yellow- and redemissive AgNCs. The G-rich and C-rich fragments of this DNA scaffold were able to fold into a G-quadruplex and imotif structures in response to the presence of K+ and H+, respectively. The formation of these structures either separately or simultaneously resulted in remarkable change of the fluorescent properties of AgNCs. Employing these ions as inputs and different excitation wavelengths to achieve the desired output emission, logic gates NOT, NOR, and AND were constructed. Other gates, such as NOR and IMPLICATION, were obtained by alteration of the sequence of the protecting DNA scaffold. Finally, the introduction of logic gating into metal nanoclusters can be performed by exploiting degradation of NCs-protecting DNA scaffold leading to decrease of fluorescence of NCs.78 The fluorescence of DNA-templated AgNCs could be slightly enhanced in the presence of Cu2+, which interacted with the phosphate backbone and nucleobases inducing conformational change of DNA scaffold. This new adopted conformation of DNA scaffold facilitated the resistance of AgNCs to environmental quenching. The subsequent addition of ascorbic acid resulted in reduction of Cu2+ to metallic copper, so new DNA-templated AgCuNCs were formed. These NCs exhibited significantly stronger fluorescence emission in comparison with parent counterparts and those treated with Cu2+. Thus, using the DNA-templated AgNCs as a gate machinery and Cu2+ and ascorbic acid as inputs, an AND logic gate was obtained. On the other hand, the use of hydrogen peroxide could reverse this fluorescence enhancement. Specifically, when it coexisted with Cu2+, a highly reactive OH• radical was formed which induced the cleavage of DNA scaffold, thereby leading to loss of emitting ability of the clusters. Therefore, the system containing DNAstabilized AgNCs, Cu2+, H2O2, and ascorbic acid acted accordingly as a concatenated AND → INHIBIT logic gate. In addition to DNA, other scaffolds able to stabilize noble metal nanoclusters are also responsive to external stimuli, so the fluorescent properties of these emitters can be altered. Yang and co-workers prepared highly luminescent AuAg bimetallic nanoclusters stabilized by 11-mercaptoundecanoic acid (AgAuNCs@11-MUA).79 The carboxyl groups of 11MUA on the surface of AgAuNCs@11-MUA were able to bind to Cu2+, which enabled PET quenching the fluorescence. The Cu2+-induced quenching could be reversed by addition of L


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Figure 8. Two input, single output photonic logic gate schematic of the Tb-QD-A647 three-fluorophore system with four input states where the (a) initial input state (0,0) contains QD with no input A (A647) nor input B (Tb). The (b) subsequent states ((0,1), (1,0), (1,1)) contain distinct valences of input A and input B. (Inset) photoluminescence (PL) plots display the output PL intensity immediately (blue plot) and 55 ms (orange plot) after UV excitation. QD peak PL ∼ 625 nm; Tb peaks at ∼490, 550, 585, and 620; and A647 peak PL ∼ 670 nm. Note, the contribution of the Tb peak at 620 nm to the QD peak at 625 nm is deconvolved and accounted for in all logic gates. Immediate PL monitoring (blue) results in (a) either direct QD PL or in (b) QD-A647 FRET and emission from both. (b) Time-delayed PL monitoring (orange, indicated by the clock) results in a convolution of Tb, QD, and A647 PL. Changing the valence of TbN- or A647 M per QD provides for control over the magnitude, and to some extent, influence of these inputs on device emission. For time-gated monitoring, the Tb is preferentially excited at 339 nm while for immediate PL monitoring the QD is excited at 400 nm which excludes the Tb. Normalized (c) absorption and (d) emission spectra for the Tb, QD, and A647. Reproduced by permission of The Royal Society of Chemistry.88

effect.83 In comparison to molecular probes, QDs possess higher fluorescence quantum yield and greater resistance toward photobleaching. Also, QDs have a broader excitation spectra and sharper emission peaks. Fluorescence emission bands of QDs can be tuned by controlling the size of a QD nanoparticle during synthesis. These unique properties make QDs good candidates as fluorescent probes for bioimaging. Unfortunately, high toxicity of elements constituting many QDs in conjunction with their degradation in biological milieu hampers their application in biomedicine. The stability of QDs can be greatly enhanced by passivating the surface of QDs within organic capping ligands. The interaction between QDs-tethered molecules and other compounds might lead to a decrease of fluorescence via PET or static quenching mechanisms, and this is one common design of logic gates utilizing QDs as signal transducers. The Willner group reported a construction of fluorescent logic gates using CdS/ZnS QDs functionalized with C- and T-rich ssDNAs.84 In the presence of Ag+ and Hg2+, which have high affinity to C and T nucleobases, respectively, these ssDNAs folded into hairpin structures, resulting in short distance between metal ions and QD. These transformations facilitated PET from QDs to metal ions. An AND logic gate was constructed using a mixture of C-rich ssDNA@QD and Trich ssDNA@QD with Ag+ and Hg2+ as inputs. The quenching of fluorescence of either QD was defined as the false output, while the simultaneous quenching was established as the true

output. The latter condition was fulfilled only in the presence of both inputs. When the surface of QDs was functionalized with both C- and T-rich ssDNAs, the quenching occurred in the presence of either input which was in accordance to an OR logic gate. He and co-workers fabricated an IMPLICATION logic gate using GSH-capped CdTe QDs as signal transducer and Zn2+/diethylenetriaminepentaacetic acid (DTPA) as inputs.85 In the absence of inputs or in the presence of Zn2+ alone, these QDs showed strong emission. When DTPA was introduced without Zn2+, the fluorescence of these QDs became statically quenched due to binding DTPA to GSH capping ligand. In case of coexistence of DTPA with Zn2+ their interaction prevented binding the former to GSH and the subsequent quenching. Credi and co-workers reported different response of n-hexadecylamine-capped CdSe QDs to small and big amounts of tetracyanoethylene (TCNE) to fabricate logic gates with degenerate inputs.86 These amine-capped QDs exhibited a moderate fluorescence emission. The addition of a few equivalents of TCNE resulted in immediate fluorescence quenching followed by substantial fluorescence enhancement (up to by factor 2.5). The authors explained this phenomenon by interaction between capping amine and TCNE to afford (tricyanovinyl)-hexadecylamine (TCV-HDA). This reaction caused initial desorption of capping ligand from the QDs surface thereby worsening the surface passivation and decreasing the fluorescence efficiency. However, the product TCV-HDA could eventually be adsorbed by the surface of M


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photoluminescence intensities had a parabolic profile against A647 valence. In this case, the output 0 could be implemented at high and low A647 valence values, while the output 1 could be realized at average values of A647. Thus, a series of logic gates such as AND, OR, INHIBIT, NOR, XOR, NAND, YES, and NOT were fabricated by altering the valence of both A647 and Tb3+ complex around QDs. In addition, the system RESET could be implemented using proteases performing enzyme cleavage from QDs. Ma and co-workers designed a FRET-based biocomputing system employing monovalent DNA-functionalized multicolor QDs assembled to binary and ternary complexes by DNA hybridization.90 Three different QDs (ZnHgSe red, CdTe green, and ZnCdSe blue) with comparable PL intensities and well-separated emission spectra were functionalized with three chimeric DNA molecules as ligands. The hybridization between three monovalent DNA-QDs with red, green, and blue emissions and DNA template T produced a ternary QD complex (RGB) (Figure 9a). Each chimeric DNA contained a toehold for the strand displacement reaction. The disassembly of QD A from RGB complex could be triggered by a singlestranded fuel DNA a containing two different toeholds (1 and 2) at both ends. Specifically, due to the presence of the toehold region in the sequence of the monovalent chimeric DNA of

QDs resulting in improved surface passivation, which was accompanied by fluorescence enhancement. The addition of more equivalents of TCNE led to quenching due to PET from QDs to free TCNE molecules having good electron accepting properties. Using a different amount of TCNE as one input, the authors fabricated XOR and NAND logic gates. Besides photoluminescence, upconversion luminescence (UCL) of QDs was also exploited in construction of logic gates. For example, Gui et al. prepared MPA-modified CdTe QDs which were then conjugated with dopamine.87 In basic conditions, the catechol moiety of dopamine could easily be oxidized by ambient oxygen to afford quinone derivative. The latter is a good electron acceptor for QDs via provision of nonradiative channel (PET), thereby resulting in luminescence quenching. One INHIBIT logic gate was constructed using HCl and 1,1′-dimethyl-4,4′-bipyridinium dichloride (MV2+) as inputs and a UCL signal as an output. In the presence of H+, the UCL of QDs was restored because the quinonehydroquinone equilibrium was shifted to the hydroquinone side, and the latter is a poor electron acceptor. The addition of MV2+ to QDs led to additional UCL quenching because of contribution from PET mediated by electrostatic interactions between MPA and MV2+ (a good electron acceptor for QDs). When two inputs were introduced together, the UCL was also quenched. Another INHIBIT logic gate was fabricated using GSH and MV2+ as inputs. In the presence of GSH alone, the UCL was restored because of displacement of MPA-oxidized dopamine quencher with GSH, which has a much higher binding affinity to QD than MPA. When MV2+ was introduced either alone or together with GSH, the UCL was quenched because of PET mediated by electrostatic interaction between MPA and MV2+ or GSH and MV2+, respectively. Finally, an OR logic gate was produced using HCl and GSH as inputs. In this case, the presence of either or both inputs led to dramatic increase of UCL signal. Another method to regulate the fluorescence of QDs is FRET to other fluorescent materials (e.g., other QDs, fluorophores, etc.). Medintz and co-workers fabricated biophotonic logic devices based on assembly of peptides labeled with a luminescent Tb(III) complex and Alexa Fluor 647 (A647) fluorophore around QDs.88,89 The QDs central assembly point was employed as an optically active element capable to serve as both FRET donor (pairing with A647) and FRET acceptor (paring with Tb3+ complex) (Figure 8). When the system was excited with UV light of 400 nm, immediate photoluminescence emissions at 625 nm (QDs) and 670 nm (A647) corresponding to the QD → A647 FRET process were observed in luminescent spectra. The excitation of the system with UV light of 339 nm resulted in time-gated luminescence with emissions at 490 nm (Tb3+), 550 nm (Tb3+), 585 nm (Tb3+), 625 nm (QD), and 670 nm (A647), which was in accordance with the Tb3+ → QD → A647 FRET process. The presence and ratio of the Tb3+ complex to A647 influenced both direct and time-delayed photoluminescent spectra, thus producing 4 different input pairs defined as (0,0) (QDs with neither Tb3+ complex nor A647) and (1,0), (0,1), (1,1) (QDs plus Tb3+ complex and A647). The ratio of peak direct photoluminescence intensities observed at 670 and 625 nm had an almost linear profile against valence of A647, and the ratio values below and above 1 were defined as 0 and 1 outputs, respectively. The measurement of peak intensities of time-delayed photoluminescence against using different valences of A647 showed that the ratio of peak time-delayed

Figure 9. Schematic illustration of (a) fabrication of ternary QD complex and (b) its disassembly/reassembly triggered by strand displacement reactions. Reprinted with permission from ref 90. Copyright 2014 Wiley-VCH. N


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this ion has less effective electron-accepting properties in comparison to its oxidated counterpart. Therefore, the butylamine-modified GO could be used to discriminate Fe3+ from Fe2+. A fluorescent AND logic gate was obtained using Fenton’s reagent components (Fe2+ and H2O2) as inputs. The true output of this gate (quenching of the fluorescence of GO) could be achieved only upon the simultaneous presence of both components which resulted in Fe3+ formation. The authors also constructed a concatenated AND → OR logic gate using this Fenton’s reagent and Fe3+ as inputs. Similar results were obtained in the work where GO was functionalized with allylamine.100 Cui and He reported chemiluminescent logic gates using AuNPs@GO composite decorated with luminol and lucigenin.101 This hybrid nanomaterial exhibited strong chemiluminescence in the presence of Fe2+ at basic conditions due to oxidation of luminol and lucigenin with superoxide anion, which was produced as a result of interaction of Fe2+ with ambient oxygen. A chemiluminescent AND logic gate was fabricated using Fe2+ and NaOH as inputs. At higher concentrations, Ag+ could also induce chemiluminescence of this composite treated with NaOH, so an OR logic gate was obtained using the chemiluminescent nanocomposite plus NaOH as a signal transducer and Fe2+/Ag+ as inputs. On the other hand, Fe3+ was unable to induce the chemiluminescence of this system and could be obtained by oxidation Fe2+ with NaClO. This sequestering of Fe2+ was exploited to derive an INHIBIT logic gate using the nanocomposite plus NaOH as a signal transducer and Fe2+/NaClO as inputs. Similarly, another INHIBIT logic gate was obtained based on scavenging of Ag+ with cysteine. Carbon dots (CDs) are a relatively new class of fluorescent nanomaterials discovered in 2004 and represent ultrasmall graphene nanosheets.102 Like conventional QDs, CDs also exhibit photoluminescence as a result of a quantum confinement effect and their emission color can be tuned by varying experimental conditions upon their preparation. This nanomaterial is also resistant toward photobleaching and, more importantly, essentially nontoxic, which is particularly attractive for in vivo imaging applications. The fluorescence of CDs can be readily controlled, owing to the presence of abundant surface oxygen functionalities which can be converted to electron-donating or electron-withdrawing groups able to participate in PET. Coordination of metal ions to surface functional groups of CDs can either induce or disrupt PET responsible for CDs fluorescence quenching, and several examples of fabrication of fluorescent logic gates based on these phenomena are reported. For instance, Lin and Dhenadhayalan constructed a series of fluorescent logic gates using two different C dots: carboxyl- and amine-functionalized C dots (HOOC−C dots and H2N−C dots, respectively).103 In the presence of Fe3+, the fluorescence of HOOC−C dots was quenched due to PET enabled by coordination of Fe3+ to surface carboxyl groups, and this quenching was defined as a NOT gate. When S2O32− was added together with Fe3+, the quenching of HOOC−C dots fluorescence was prevented due to reduction of Fe3+ to S2O32− to afford Fe2+, a significantly less effective electron acceptor than Fe3+. The presence of S2O32− alone, on the other hand, had no effect on the fluorescence of HOOC−C dots. Therefore, using Fe3+ and S2O32− as inputs and HOOC−C dots as a signal transducer, an IMPLICATION logic gate was constructed. In contrast to HOOC−C dots, H2N−C dots were almost not emissive due to reductive PET

QD A, the toehold 1 initiated toehold-mediated DNA strand displacement reaction leading to detachment of QD A from RBG complex (Figure 9b). The obtained monovalent dsDNA/ QD A complex contained toehold region 2, so in the presence of antifuel DNA a′ another toehold-mediated strand displacement of QD A-tethered chimeric DNA occurred. The displaced QD A-tethered chimeric DNA were then hybridized back to the template T. The disassembly of QD A from the RGB complex led to a pronounced increase of PL emission of green QD B and a decrease of photoluminescence emission of red QD. This behavior corresponded to switching off FRET between QD A and B when a QD A was disassembled from QD B. Following this principle, a series of logic gates (AND, OR, NOR, NAND, INHIBIT, XOR, and XNOR) and a halfadder were fabricated using binary and ternary QD complexes as gate machineries and fuel/antifuel DNAs as inputs and PL signals of specific QDs as outputs. For example, an OR logic gate was constructed using a binary GB QD complex as a signal transducer. The true output (emission of blue QD) was achieved when either or both inputs inducing disassembly of the binary complex were in the system. Miao and co-workers also employed toehold-mediated DNA strand displacement reactions to fabricate fluorescent logic gates based on disassembly of Ag2S QDs from the surface substrate quenching their emission.91 The basic YES logic gate was obtained by hybridization between the Au electrodeanchored DNA molecule containing toehold region and its partially complementary strand attached to NIR Ag2S QDs. In the presence of miRNA which was fully complementary to Au electrode-bound strand, the Ag2S QDs-tethered DNA was displaced and, as result, the Ag2S QDs diffused away from the electrode which was accompanied by restoration of their fluorescence. Two and more input AND logic gates were fabricated by hybridizing DNA bound to Ag2S QDs with the end region of the DNA anchored to Au electrode which was opposite to the Au surface. Then, the remaining sequence of Au-electrode-bound DNA was blocked with the required number of complementary DNA strands only leaving the toehold region near the Au electrode surface necessary for triggering strand displacement cascade. A series of OR logic gates were obtained by hybridization between Au-electrodebound DNA with the one or more Ag2S QDs-anchored DNA, ensuring that all these duplexes obtained would have toehold regions, so the presence of either miRNA input would result in the displacement of the respective Ag2S QDs-anchored DNA. 3.2.1.1.5. Graphene and Carbon Dots. Graphene oxide (GO) is a water-soluble two-dimensional (2D) single atom thick material produced by powerful oxidation of graphite. Due to its unique surface properties, this material has received significant attention in many areas such as catalysis,92 separation,93 sensing,94 etc. GO has also fluorescent properties;95 however, the common GO is nonemissive due to the presence of oxygen functionalities which induce nonradiative recombination of localized electron−hole pairs.96,97 The functionalization of GO with alkylamines removes these nonradiative recombination sites, thereby enabling emissive states.98 For example, Zhang and co-workers fabricated fluorescent logic gates for intracellular imaging Fe3+, employing blue emissive butylamine-modified GO as a signal transducer.99 The fluorescence of this material could be quenched by Fe3+ (interacting with GO surface electrostatically) via PET mechanism. On the other hand, when Fe2+ was used instead of Fe3+, the butylamine-modified GO remained emissive because O


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Figure 10. Schematic illustration of construction of (a) AND and (b) OR logic gates based on disassembly of dyed-dsDNAs triggered by ssDNA inputs and following adsorption of yielded strands onto the GO surface. Reprinted from ref 107. Copyright 2012 American Chemical Society.

from NH2 group to the CDs. This type of PET could be inhibited by coordination of Zn2+ to NH2 group, and the following enhancement of fluorescence emission was defined as a YES gate. Similar enhancement was observed in the presence of small amounts of PO43−. When Zn2+ and small amount of PO43− were added together, NH2−C dots showed slightly decreased emission but higher than that in the case of the absence of these molecules. This behavior was consistent with an OR logic gate with Zn2+ and a small amount of PO43− as inputs. This OR gate could be reprogrammed to an XOR logic gate by simply increasing the amount of PO43−. In the case of coexistence of these inputs, their interaction prevented disruption of PET, and therefore, the false output was obtained. Chattopadhyay and co-workers reported regulation of fluorescence of CDs using both Fe3+-induced PET and static quenching with picric acid to fabricate a series of binary logic gates, which could be concatenated into three and four input logic systems.104 Two NOT logic gates were obtained using quenching induced by either Fe3+ or picric acid, respectively. These gates could be integrated into a new logic gate, thereby yielding a NOR logic gate. The fluorescence of CDs quenched with Fe3+ could be recovered by scavenging Fe3+ with either cysteine (formation of Fe3+-cysteine complex) or ascorbic acid (reduction of Fe3+ to Fe2+), so this behavior was consisted with Boolean OR logic. When the fluorescence of CDs was quenched with both Fe3+ and picric acid, the restoration was possible only upon simultaneous removal of both quenchers, which corresponded to an AND logic gate. Specifically, Fe3+ could be reduced to Fe2+ with ascorbic acid (input 1), while picric acid could be removed by phase transfer operation (input 2). An IMPLICATION logic gate could be produced using competitive interactions between CDs, Fe3+, and ascorbic acid. Finally, a NAND logic gate was fabricated based on fluorescent quenching with Fe3+, which was obtained from a Fenton reagent [Fe2+ (input 1) plus H2O2 (input 2)]. These logic gates could be concatenated to afford three and four input logic circuits and could be operated not only in a liquid medium but also on a solid phase (e.g., nonfluorescent paper). Finally, Li and co-workers reported similar IMPLICATION fabricated using CDs, which was based on scavenging fluorescence-quenching Hg2+ with cysteine.105

Along with coordination of metal ions to surface functional groups of CDs, more complex interactions between CDs and other objects have been exploited in fabrication of logic gates. For example, the Qu research group constructed a few logic gates based on FRET between spermine-functionalized CDs (SC dots) and dsDNA intercalated with organic fluorophore.106 Specifically, the electrostatic interactions between positively charged SC dots and negatively charged phosphate backbone of dsDNA resulted in B-Z conformational transition of dsDNA. Upon this transition, SC dots were bound to the major groove of dsDNA, while the minor one could be intercalated with ethidium bromide. As a result, these two fluorescent systems became located in close proximity to each other and could communicate via FRET mechanism, which was a basis for construction of AND and NAND logic gates with dsDNA and ethidium bromide as inputs. The true output of the AND logic gate was defined as emission of ethidium bromide, while that of the NAND logic gate was defined as emission of SC dots. Furthermore, using iodide ions which were able to quench the fluorescence of SC dots and therefore disrupt FRET between SC dots and ethidium bromide, concatenated AND → INHIBIT and NAND → INHIBIT logic gates were fabricated. Besides being an emitter, GO is also a good FRET quencher, and this property in conjunction with its great adsorption capacity make this material an excellent platform for design of logic gates based on quenching of emissions of fluorophores or other nanoparticles. These examples will be surveyed in the next subsection. 3.2.1.2. Fluorescence-Quenching Nanoparticles. The other approach used in fabrication of fluorescent logic gates employing nanoparticles is based on exploitation of their ability to quench fluorescence of organic fluorophores or other emitters. One of the most common nanomaterial used for such logic gates design is graphene oxide (GO). Indeed, GO is an excellent FRET acceptor, and its high adsorption capacity can be used for capture of fluorescent probes. ssDNA bearing a fluorescent tag at the 3′ or 5′ end is the most typical example of such emitters. Specifically, GO is able to adsorb singlestranded oligonucleotides in noncovalent fashion via π−π stacking interactions between DNA nucleobases and hexagonal cells of graphene. As a result, the tag gets immediately adjacent P


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Figure 11. Operation mechanism of the developed half-adder and half-subtractor with the corresponding circuits. Reproduced by permission of The Royal Society of Chemistry.113

structure is another powerful method to control desorption of dyed ssDNA from GO surface. For instance, Wang and coworkers decorated the surface of GO with FAM-labeled adenosine- and thrombin-binding aptamers.108 A simple OR logic gate was constructed using adenosine and thrombin as inputs which triggered desorption of the respective oligonucleotide and restoration of fluorescence of the FAM label. This logic gate could be reprogrammed into an INHIBIT logic gate by substitution of one input (e.g., adenosine) with FAM-free aptamer sensitive to the second input (e.g., thrombin). Finally, metal ions-mediated base pairing can also effectively control desorption of ssDNA from GO surface. Luo and co-workers designed a logic-gated fluorescent sensor for Ag+ using GO bearing two ssDNA, which were almost complementary to each other with the exception of 11 cytosine pairs mismatches.109 One of these ssDNA comprised FAM label, which was nonemissive due to interaction of ssDNA scaffold with GO. The addition of Ag+ resulted in detachment of these ssDNAs from the GO surface due to formation of rigid duplex structures assembled by means of formation of C-Ag+-C pairs and was accompanied by restoration of the fluorescence of the FAM label. When Ag+ was added together with cysteine, their interaction prevented Ag+-mediated hybridization, so ssDNA remained on the surface of GO. Thus, using Ag+ and cysteine as inputs, an INHIBIT logic gate was obtained. Similar INHIBIT logic gates were reported in the works110,111 and were based on folding of dye-labeled ssDNA into hairpin structures induced by C-Ag+-C and T-Hg2+-T base pairings. Gui et al. reported room temperature phosphorescence logic gates constructed using both Watson−Crick and metal ions mediated base pairing to regulate desorption of T-rich DNAfunctionalized CDs from GO surface.112 An OR logic gate was obtained using Hg2+ and complementary strand as inputs, which induced folding of the T-rich ssDNA into a secondary structure. The phosphorescence of liberated CDs could be quenched by electron-accepting molecules, such as doxor-

to the surface and becomes involved in FRET. On the other hand, the stacking interactions can be disrupted by transformation of randomly coiled ssDNA to ordered rigid structures (e.g., duplexes through Watson−Crick or metal ions-mediated base pairing, i-motif, G-qudruplexes, DNA aptamer-target complexes, and others). These secondary structures cannot be held by GO and therefore diffuse away from the surface which is accompanied by restoration of fluorescence of the label. The Willner group utilized displacement of dyed strands from dsDNA complexes and its following capture with GO to construct turn-off AND and OR logic gates. The authors prepared two FAM- and ROX-labeled dsDNA complexes comprising single-stranded loop fragments.107 For example, the FAM-labeled dsDNA was obtained by hybridization between FAM-labeled ssDNA (1) with protective ssDNA (8) composed of fragments I, II, and IV (Figure 10a). The fragments I and IV were paired with the respective complementary regions of ssDNA (1), while the nonhybridized fragment II formed a loop. Due to their rigidity these secondary structures could not be adsorbed by GO surface, and, as a result, the quenching of fluorescence of labels was prevented. In the presence of specific DNA, these complexes were able to release the dye-labeled ssDNAs, which eventually got adsorbed by GO surface thereby becoming nonemissive. The sequences of protective ssDNAs were precisely tailored, so the obtained pairs of dye-labeled dsDNAs were able to get cleaved either simultaneously or separately in response to the presence of input DNAs. The simultaneous disassembly of dsDNA complexes achieved only when both DNA inputs were in the system was employed to derive the AND gate (Figure 10a), while that occurring in the presence of either or both inputs was used for construction of the OR gate (Figure 10b). The interaction between ssDNA and non-nucleic acid targets leading to the folding of the former into a rigid Q


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Figure 12. OR logic gate system with Hg2+ and Ag+ as inputs and ECL signal as output: (A) schematic representation of the logic gate. (B) ECL intensity vs time curves of the logic gate: (a) MCNTs/Ru-silica-labeled S1/Ru-silica labeled S2 complex. (b) MCNTs/Ru-silica-labeled S1/Rusilica-labeled S2 complex in the presence of 50 nM Hg2+. (c) MCNTs/Ru-silica-labeled S1/Ru-silica-labeled S2 complex with the addition of 10 nM Ag+. (d) MCNTs/Ru-silica-labeled S1/Ru-silica-labeled S2 complex in the presence of 50 nM Hg2+ and 10 nM Ag+. (C) The truth table of the OR logic gate. (D) The symbol of the OR logic gate. Reproduced by permission of the Royal Society of Chemistry.119

ubicin, able to bind to CDs surface. This behavior was consistent to a concatenated OR → INHIBIT logic gate with Hg2+, complementary strand, and doxorubicin as inputs. The platform dye-labeled ssDNA@GO was also employed for construction of more complex logic operations such as half adder and half subtractor. These arithmetic logic operations are obtained by integration of XOR with AND or INHIBIT logic gates in parallel fashion, respectively, and two-channel detection of outputs is required. For example, Wang and coworkers fabricated these parallel logic gates using enhancement of fluorescence of NMM entrapped within G-quadruplex as one channel, while the restoration of the fluorescence of ssDNA-tethered tag as the second one (Figure 11).113 An XOR logic gate for half-adder operation was implemented, employing the fluorescence of FAM label of t-DNA as an output and ssDNAs HA-1 and HA-2 as inputs. The introduction of either ssDNA to the system led to interaction with the dyed strand to yield a double-stranded structure which could not be held by GO, so the fluorescence of the FAM label became restored. On the other hand, when input molecules were added simultaneously, their mutual interaction produced more stable dsDNA than those formed between tDNA and HA-1 or HA-2. As a result, the FAM-labeled strand remained on the GO surface. At the same time, the Gquadruplex region of HA-1/HA-2 duplex became occupied by NMM which led to its fluorescence enhancement. The latter event was only possible when HA-1 and HA-2 were added together, which is consistent with Boolean AND for this half adder operation. Using different sequences of input DNA, it was possible to reprogram the AND logic gate into an INHIBIT gate, thereby implementing the half subtractor logic operation. More complex logic operations such as 2 to 1 and 4 to 2 encoders, 1 to 2 decoder, and comparator were reported

by the same group using similar system composed of NMM and ssDNA-templated [email protected] More examples of logic gates based on GO-induced quenching were reported in works by the Qu research group where competitive interaction between DNA and GO plus other nanomaterials were exploited. For example, the addition of AgNPs and cysteine to the system GO plus ssDNA could effectively control the adsorption/desorption of the latter on the surface.115 The authors prepared AgNPs using one ssDNA as a template, so this metallized DNA was unable to form a duplex pair with its complementary counterpart labeled with FAM. As a result, the latter became adsorbed by GO and its FAM tag lost the ability to emit due to FRET. When cysteine was added to this system, it displaced the metallized DNA from AgNPs, and the subsequent hybridization between ssDNAs followed by desorption of the dsDNA formed led to restoration of the fluorescence of the fluorophore. This result could be also achieved in the presence of DNA 1 which was complementary to the ssDNA with FAM. Therefore, using cysteine and DNA 1 as inputs, an OR gate could be obtained. This gate could be reprogrammed into an INHBIT logic gate by replacing input DNA 1 with DNA 2, which was able to form a more stable complex with the metallized DNA than the FAM-labeled one. Another INHIBIT logic gate was fabricated using N-ethylmaleimide (NEM, a cysteine scavenger) instead of DNA 1 or DNA 2 as an input. In another work by the same group, three value logic gates were constructed based on regulation of multiple emissions of upconversion nanoparticles with GO and fluorescent silica nanoparticles as FRET acceptors.116 More specifically, these particles were modified with ssDNA A and attached to the GO surface via noncovalent π−π stacking interactions. In the presence of complementary strand B, these nanoparticles were no longer bound to the GO R


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attached onto Au layer of magnetic Fe3O4@SiO2@Au nanoparticles via Au−S interactions. The hairpin conformation of the molecular beacon ssDNA brought the FAM tag very close to Au surface, so it became nonfluorescent. In the presence of either input ssDNA HA-1 or ssDNA HA-2, the formation of duplex structures resulted in sufficient separation of the FAM moiety from the surface to restore the fluorescence. However, when both ssDNAs were introduced simultaneously, they formed a more robust dsDNA complex, so the hairpin structure of the molecular beacon ssDNA was unaffected and the FAM label remained nonemissive. This behavior was equivalent to the XOR logic gate with HA-1 and HA-2 as inputs. In the presence of protoporphyrin IX, the dsDNA composed of HA-1 and HA-2 formed G-quadruplex able to enhance the fluorescence of protoporphyrin IX. This enhancement could be considered as an AND logic gate with the same inputs as the XOR gate. These gates function in parallel fashion which is a necessary condition for design of the half-adder. The half-subtractor was constructed using different input ssDNAs: HS-1 and HS-2. The XOR gate for the halfsubtractor was based on the same principle as for the halfadder: both HS-1 and HS-2 alone were able to straighten hairpin molecular beacon ssDNA; however, when they were added together, their mutual hybridization to afford another dsDNA prevented this transformation. In the meantime, the Grich HS-2 ssDNA alone could form the G-quadruplex with protoporphyrin IX exhibiting enhanced fluorescence. On the other hand, when HS-1 and HS-2 were in the system at the same time, the formation of this G-quadruplex was prohibited, which was in accordance to Boolean INHIBIT logic. Thus, both XOR and INHIBIT logic gates could also operate in parallel as required for half subtractor logic. The presence of magnetic core facilitated separation of the molecular beacon modified Fe3O4@SiO2@Au NPs. Therefore, applying thermal denaturation and magnetic separation followed by addition of a fresh portion of protoporphyrin IX enabled RESET for these arithmetic logic gates. A slightly different design of an XOR logic gate in comparison to that described above was reported by Wang and co-workers.122 The authors employed 13 nm AuNPs as a quencher for Cy5-labeled ssDNA probes (molecular beacon and adenosine apatamer). Both dye-labeled ssDNAs were attached to the AuNP surfaces via Au−S interaction. The tag of the molecular beacon was nonemissive due to the distance between this molecule and the Au surface was short enough to make FRET quenching favorable, while its counterpart linked to the adenosine aptamer was normally emissive because this strand possessed a randomly coiled conformation. Upon addition of adenosine alone, this aptamer folded into a compact structure bringing the Cy5 label connected to this aptamer close to the surface, which resulted in a decrease of the overall fluorescence intensity. On the other hand, when ssDNA complementary to molecular beacon was introduced without adenosine, the straightening of the molecular beacon brought the Cy5 label sufficiently away from the surface, so the overall fluorescence intensity increased. When both inputs were present simultaneously, the fluorescence intensity enhancement due to straightening of the molecular beacon and fluorescence intensity decrease due to aptamer folding nullified each other, and as a result, the intensity remained the same as in the absence of inputs. The output of this XOR logic gate was defined as an absolute intensity change. The important difference of this XOR logic gate from that fabricated in the previous work is the absence of

surface due to formation of dsDNA AB and, as a result, restored their ability to emit. The addition of fluorescent silica nanoparticles bearing ssDNA C resulted in formation of selfassemblies between these particles with upconverting ones due to displacement of strand B from AB duplex with strand C. Consequently, the green emission of upconversion nanoparticles was quenched via the FRET mechanism, whereas the red one was enhanced due to overlapping with that of fluorescent silica nanoparticles. This behavior was consistent to a three-value OR logic gate with red emission as an output. Employing different DNA sequences, this gate could be reprogrammed into an INHIBIT logic gate. Quenching fluorescence of organic probes by GO is also possible without ssDNA mediation because these fluorophores can interact with this surface via noncovalent π−π stacking. This phenomenon was used by Li and co-workers who reported reduced graphene oxide (rGO)-acridine orange (AO) system for selective fluorescent sensing Hg2+.117 In the absence of analytes, this system was almost nonemissive. The addition of Hg2+ alone led to displacement of AO from the rGO surface and restoration of its fluorescence. However, when Hg2+ was introduced along with cysteine, the displacement of AO from the surface of rGO was inhibited by formation of a robust Hg(cysteine)2, so AO emission restoration was prevented. This behavior was consistent with an INHIBIT logic gate. A similar gate based on disruption of π−π stacking interactions between GO and rhodamine 6G with Hg2+ and its inhibition with EDTA as a scavenger for Hg2+ was reported in the work.118 The principle of disruption of noncovalent π−π stacking between ssDNA and graphite basal plane via formation of secondary DNA structures to fabricate fluorescent logic gates can also be applied to carbon nanotubes (CNTs), which are in fact stapled graphene nanoribbons. For example, Ge and coworkers fabricated a series of electrochemiluminescent logic gates exploiting interactions between acid-eroded multiwalled CNTs (MWCNTs) and C-rich and T-rich ssDNAs attached to SiO2NPs dyed with electrochemiluminescent probe (Ru(bipy)32+).119 The randomly coiled C-rich and T-rich ssDNAs were able to wrap around MWCNTs as a result of π−π stacking. This resulted in a short distance between Ru(bipy)32+-doped SiO2NPs and the surface of MWCNTs, which was sufficient to enable quenching of the former via FRET and PET mechanisms. In the presence of Hg2+ and Ag+, the respective ssDNA was able to fold into a hairpin structure, and, thereby desorb from the surface of MWCNTs. Consequently, the electrochemiluminescence of Ru(bipy)32+-doped SiO2NPs became restored. Using MWCNTs decorated with both C-rich and T-rich ssDNAs attached to Ru(bipy)32+-doped SiO2NPs as a signal transducer and Hg2+/Ag+ as inputs, an OR logic gate was constructed (Figure 12). Other logic gates, such as INHIBIT and NOR, were fabricated, employing competitive interactions between ssDNAs comprising the electrochemiluminescent probe and label-free ssDNAs having similar sequences and iodide ions. Besides carbonaceous nanomaterials, gold nanoparticles with the size of 13−15 nm are also efficient fluorescence quenchers.120 Sun and co-workers reported resettable half adder and half subtractor fabricated using magnetic Fe3O4@ SiO2@Au nanoparticles.121 Two emitters, FAM and protoporphyrin IX, were used as reporters for these two output logic gates. The FAM emitter was linked to the 3′ end of the 5′ end of thiolated molecular beacon ssDNA containing a 14-base loop and a 6-base stem. This molecular beacon ssDNA was S


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citrate adsorbed on the surface. Upon increase of ionic strength of solution, the electrostatic repulsion becomes outweighed by van der Waals forces leading to aggregation of nanoparticles. However, the resistance toward aggregation at these ionic strength values can be significantly increased by coating AuNPs with molecules having protective capacity higher than that of citrate. Of these, ssDNA is one of the common AuNPs protective agent due to its unique conformational and electrostatic properties. Indeed, ssDNA is able to uncoil its bases, exposing them to Au surface, and van der Waals interactions between the exposed bases and gold surface atoms lead to physisorption of ssDNA onto AuNPs. At the same time, negatively charged phosphate residues enhance colloidal stability due to electrostatic repulsion.127 Similar to its counterpart captured by GO surface, this physisorbed strand is able to form rigid secondary structures (duplexes, aptamertarget complexes, i-motif, G-quadruplex, etc.) which then desorb from the Au surface due to their inability to uncoil bases. This principle has become very common in fabrication of colorimetric logic gates using unmodified AuNPs as the signal transducer. Ding and co-workers constructed XOR and AND logic gates employing transformation of AuNP-protecting C-rich ssDNA into rigid structures (i-motif and dsDNA) as a driving force of AuNP aggregation.128 An XOR logic gate was obtained using low pH and ssDNA-1 (complementary to the C-rich ssDNA) as inputs. In the absence of both inputs, the uncoiled C-rich ssDNA effectively stabilized AuNPs dispersion against aggregation at high ionic strength conditions. The decrease of pH without addition of ssDNA-1 resulted in aggregation of AuNPs at high ionic strength due to transformation of the protective C-rich ssDNA into the close-packed i-motif. The addition of ssDNA-1 was introduced at neutral pH also disrupted the colloidal stability of AuNPs due to hybridization between C-rich ssDNA and ssDNA-1. However, when both inputs were added together, the dispersion of AuNPs remained stable. Specifically, at these conditions the C-rich ssDNA folded into the i-motif, which was more stable than its duplex with ssDNA-1. In the meantime, the unhybridized ssDNA-1 became physisorbed on the AuNP surfaces, thereby preventing their aggregation. To construct an AND logic gate, AuNPs stabilized with both C-rich ssDNA and ssDNA-2 were used as a gate machinery and low pH and ssDNA-3 (complementary to ssDNA-2) as inputs. The acidification of the medium without addition of ssDNA-3 resulted in transformation of the C-rich ssDNA into an i-motif structure, while ssDNA-2 remained on the surface of AuNPs, thereby still protecting them against salt-induced aggregation. Similarly, addition of ssDNA-3 at neutral pH did not lead to AuNP aggregation because the C-rich DNA remained randomly coiled. Only when both acidification and addition of ssDNA-3 were applied together, the simultaneous desorption of both protecting ssDNAs resulted in AuNP aggregation. The aggregation of AuNPs due to folding of protective Crich DNA into a rigid i-motif at low pH was also employed as the signal transduction for enzyme logic gates, reported by the Qu group.129 An AND logic gate was constructed using dispersion of C-rich ssDNA protected AuNPs containing lactose and hydrogen peroxide as signal transducer, and βgalactosidase and glucose oxidase (GOx) as inputs. βgalactosidase catalyzed the hydrolysis of lactose to galactose and glucose, while GOx accelerated the oxidation of the latter product with hydrogen peroxide to afford gluconic acid. This

interaction between inputs. Zhang and co-workers employed 15 nm colloidal AuNPs as a quencher for the fluorescent probe to fabricate an AND logic gate based on toehold-mediated DNA strand displacement reactions.123 At first, thiolated ssDNAs A and B and ssDNA Q modified with Cy3 fluorophore in the middle of phosphate backbone were hybridized to afford a DNA self-assembly complex. This structure was attached onto 15 nm AuNPs via thiolate chemistry. This anchoring resulted in the inability of the Cy3 tag to emit. Both strands A and B hybridized with Q had toehold regions, so the respective part of the strand Q sequence could be displaced. When both input ssDNAs Ain and Bin, which were fully complementary to ssDNA A and B, respectively, were added to this DNA-AuNP agent, the strand Q was fully released which was accompanied by restoration of the Cy3 fluorescence. 3.2.2. Logic Gates Fabricated Using Assembly/ Disassembly of Nanoparticles Exhibiting Surface Plasmon Resonance. The processes of assembly/disassembly of noble metal nanoparticles exhibiting surface plasmon resonance phenomena have also attracted considerable attention in fabrication of signaling probes. Indeed, this type of nanomaterial can be employed in various biomedicinerelated applications: from very advanced SERS imaging124,125 to simple and inexpensive optical biosensing,126 which does not require sophisticated instrumentation for signal readout. In addition, biocomputing structures that comprise plasmonic nanoparticles monitored by a spectral shift of the localized surface plasmon resonance (LSPR) are convenient for biosensing and investigation of kinetic parameters of the reversible self-assembly and disassembly of the structures directly in liquids hardly realizable with other approaches.48 3.2.2.1. Homogeneous Assembly/Disassembly of Plasmonic Nanoparticles. Colloidal gold nanoparticles are a plasmonic substrate of choice due to combination of unique properties, such as ease of preparation, chemical stability, tunability of size and shape, straightforward surface modification, and distinctive optical properties. In particular, AuNPs show the sharp and strong adsorption band in the visible region due to localized surface plasmon resonance (LSPR). The LSPR of AuNPs can be altered by changing size or distance between AuNPs. For example, the small nanoparticles (

Advanced Smart Nanomaterials with Integrated Logic-Gating and

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