Patent Application
20240256822
The present disclosure relates to computation using nanoparticles. Specifically, it relates to nanoparticle computation using bioinformation as an input.
Across many length scales, matters have been merged with computation, from micro-sized droplets and microparticles to molecular machines and biomolecules, such as enzymes and nucleic acids. However, implementing complex computation in nanoscale objects, especially in nanoparticles, remains unexplored, despite a wide range of potential applications that would benefit from controlling their potentially useful plasmonic, photonic, catalytic, and material properties.
A common approach to using nanoparticles as substrates for computation is functionalizing the “core” structures with stimuli-responsive surface ligands. A group of surface-modified nanoparticles can then carry out elementary logic operations, responding to various chemical and physical inputs, and ideally, individual nanoparticles should be used as modular “nano-parts”, and desired computation should be implemented in a plug-and-play manner. However, such an existing method has been limited to installing few logic computations that are applicable to controlling only simple outputs, such as aggregation/dispersion of particles and release of surface molecules. This limitation is because of the difficulties in modular wiring of multiple logic gates in the solution phase, where inputs, logic gates, and outputs all diffuse uncontrollably in the three-dimensional space.
In particular, the following constraints have imposed limitations on computing with nanoparticles.
First, logic-embedded particles are irreversibly altered after one operation and are indiscriminately mixed with unreacted inputs in one bulk solution. The lack of compartmentalization prevents the implementation of more than one computational task per test tube.
Second, it is difficult to control or characterize structure changes, dynamic interactions, and output signals of freely diffusing individual particles in the 3D space. In most cases, only an averaged signal is obtained as the final readout, in which particle-by-particle responses of the computation are averaged.
These constraints, thus, impose limitations on employing nanoparticles as modular and composable parts that can be reconfigured to implement a desired computation in a “plug-and-play” manner.
In order to construct a complex and reliable nanoparticle circuit, it is necessary to move beyond a method that once relies on a solution step to a scalable integrated platform with in-situ readout and control functions. In addition, the desired nanoparticle circuit should be systematically designed and constructed based on the digital design principle.
Nanoparticle systems equipped with computing capability can achieve complex functions that are not attainable in a simple collection of individual nanoparticles. Such a “nanosystem” autonomously performs complex tasks in response to stimuli. Then, the nanosystem can be a system that can direct the flow of matter and information at a nanoscale.
Processing molecular information with nanoparticles allows for incorporating the rich and powerful functions of nanoparticles into algorithmic and autonomous controls on molecular computing processes. In the present disclosure, a platform, termed “lipid nanotablet”, is provided to construct a widely applicable system that can create nanoparticle circuits. The lipid nanotablet is a technology that constructs nanoparticle logic gates and circuits at the level of a single particle in a two-dimensional lipid bilayer. Such a lipid bilayer-based nanoparticle computing platform (with computer system/software as a basis for use) is hereinafter referred to as a lipid nanotablet (LNT).
The lipid nanotablet uses lipid bilayers as a chemical/biological board, on which surface-modified nanoparticles are placed to enable the lipid bilayers to react with molecular information, to host nanoparticles, and employ them as units of computation. A single-nanoparticle logic gate senses molecular inputs and triggers particle assembly or disassembly. Through the present disclosure, a set of Boolean logic operations such as AND, OR, and NOT computations, fan-in/fan-out of logic gates, and a circuit such as a multiplexer can be provided. As described, implementation of nanoparticle circuit modules on the lipid nanotablet may open previously unknown opportunities in information-processing nanosystems.
A lipid nanotablet according one aspect of the present invention includes: a supported lipid bilayer to which a plurality of nanoparticles are integrated in nanoparticle units; an immobile nano-receptor that includes at least one first surface molecule tethered to a surface and is immobile among the plurality of nanoparticles; and a nano-floater that includes at least one second surface molecule tethered to a surface and is mobile among the plurality of nanoparticles, wherein an interaction between the nano-receptor and the nano-floater is controlled according to a reaction result of the at least one first surface molecule and the at least one second surface molecule with respect to inputs, and a logic result based on the interaction is provided.
The lipid nanotablet may include a YES gate that generates a logic result based on an assembly reaction in which the at least one first surface molecule and the at least one second surface molecule are combined by inputs and thus the nano-receptor and the nano-floater are tethered.
The input may include a DNA input, and the at least one first surface molecule and the at least second surface molecule respectively comprise surface DNA ligands, and the surface DNA ligand of the nano-receptor and the surface DNA ligand of the nano-floater may be hybridized in response to the DNA input.
The lipid nanotablet may include a YES gate that generates a logic result based on a disassembly reaction that removes the tethering between the nano-receptor and the nano-floater by inputs, while the nano-receptor and the nano-floater are tethered by combination of the at least one first surface molecule and the at least one second surface molecule.
The input may include a DNA input, and the DNA input may remove a DNA bond through a toehold-mediated strand displacement in pre-dimerization of the nano-receptor and the nano-floater tethered through the DNA bond.
The lipid nanotablet may include an AND gate that generates a logic result through tethering of the nano-receptor and the nano-floater by combinations of a first input to the at least one first surface molecule and a second input to the at least one second surface molecule.
The at least one first surface molecule and the at least one surface molecule may include conformation-switchable first and second DNA hairpins, and the first DNA hairpin may be opened by hybridization with the first input and thus a first binding domain is exposed, the second DNA hairpin may be opened by hybridization with the second input and thus a second binding domain is exposed, and the nano-receptor and the nano-floater may be tethered through hybridization of the first binding domain and the second binding domain.
The at least one first surface molecule may include third and fourth surface molecules, the at least one second surface molecule may include fifth and sixth surface molecules, and the lipid nanotable may include an OR gate that generates a logic result from tethering of the nano-receptor and the nano-floater by at least one of a combination of a first input and the third and fifth surface molecules and a combination of a second input and the fourth and sixth surface molecules.
The third to sixth surface molecules may be DNA ligands and may include first to fourth binding domains, and the first input may be hybridized with the first and third binding domains and the second input may be hybridized with the second and third binding domains.
The at least one first surface molecule may include third and fourth surface molecules, the at least one second surface molecule may include fifth and sixth surface molecules, the third surface molecule and the fifth surface molecule may be combined, the fourth surface molecule and the sixth surface molecule are may be, and the third surface molecule and the fifth surface molecule are may be, the fourth surface molecule and the sixth surface molecule may be combined, and the lipid nanotablet may include an AND gate that generates a logic result by removing the combination of the third and fifth surface molecules and removing the combination of the fourth and sixth surface molecules
First DNA binding between the third surface molecule and the fifth surface molecule and second DNA binding between the fourth surface molecule and the sixth surface molecule may expose a first toehold domain and a second toehold domain, the first toehold domain may be a recognition area of the first input, the first input may remove the first DNA binding through strand displacement, the second toehold domain may be a recognition area of the second input, and the second input may remove the second DNA binding through strand displacement.
The at least one first surface molecule and the at least one second surface molecule may be combined, and the lipid nanotablet may include an OR gate that generates a logic result by removing the at least one first surface molecule and the at least one second surface molecule by at least one of a first input and a second input.
The combination of the at least one first surface molecule and the at least one second surface molecule may be DNA binding, the DNA binding may include first and second toehold domains, and when the first toehold recruits the first input, the first input cleaves the DNA bonding through strand displacement with at least one first surface molecule or when the second toehold domain recruits the second input, the second input may cleave the DNA binding through strand displacement with the at least one second surface molecule.
Interaction between the nano-receptor and the nano-floater may be a first logic gate that is controlled by a first input and a second input, interaction between another one first nano-receptor and another one first nano-floater among the plurality of nanoparticles may be a second logic gate that is controlled by a third input and a fourth input, and the lipid nanotablet may include a third logic gate that generates a logic result based on a first logic result of the first logic gate and a second logic result of the second logic gate.
The nano-receptor and the nano-floater may be tethered by at least one of the first input and the second input, the tethering of the first nano-receptor and the first nano-floater may be disassembled by the third input and the fourth input, and the first logic output may be a logic OFF output of the third logic gate and the second logic output may be a logic ON output of the third logic gate.
The at least one first surface molecule may include third and fourth surface molecules, the at least one second surface molecule may include fifth and sixth surface molecules, and the lipid nanotablet may include an INHIBIT gate that generates a logic result by removing the combination between the third surface molecule and the fifth surface molecule.
The second input may combine the fourth surface molecule and the sixth surface molecule.
The first input may remove DNA bonding between the third surface molecule and the fifth surface molecule.
Interaction of the nano-receptor and the nano-floater may be controlled as third and fourth surface molecules tethered to the at least one first surface molecule and the surface of the nano-receptor and fifth and sixth surface molecules tethered to the at least one second surface molecule and the surface of the nano-floater interact with each other.
At least one of a first input and a second input may remove a combination between the at least one first surface molecule and the at least one second surface molecule, at least one of a third input and a fourth input may remove a combination between the third surface molecule and the fifth surface molecule, and, at least one of a fifth input and a sixth input may remove a combination between the fourth surface molecule and the sixth surface molecule.
At least one of a first input and a second input may remove a combination between the at least one first surface molecule and the at least one second surface molecule, a third input may form a combination between the third surface molecule and the fifth surface molecule, and a fourth input may form a combination between the fourth surface molecule and the sixth surface molecule.
A first input may remove a combination between the at least one first surface molecule and the at least one second surface molecule, a second input may remove a combination between the third surface molecule and the fifth surface molecule, and a third input may form a combination between the fourth surface molecule and the sixth surface molecule.
A first interaction between the nano-receptor and the nano-floater may be controlled by a first input and a second input, among the plurality of nanoparticles, a second interaction between another one first nano-receptor and another one first nano-floater may be controlled by a third input and a fourth input, and the first input and the third input may be of the same type and the second input and the fourth input are of the same type.
A color of an image signal detected according to the first interaction and a color of an image signal detected according to the second interaction may be different from each other.
The tethering between the nano-receptor and the nano-floater may be disassembled by the first input and the second input, and the tethering between the first nano-receptor and the first nano-floater may be disassembled by the third input and the fourth input.
According to another aspect of the present invention, a lipid nanotablet in a supported lipid bilayer to which a plurality of nanoparticles are integrated in nanoparticle units, includes: a first nano-receptor; a second nano-receptor; a first nano-floater that interacts according to at least one input for the first nano-receptor; and a second nano-floater that interacts according to a first input for the second nano-receptor, wherein wiring between a first logic gate including the first nano-receptor and a second logic gate including the second nano-receptor may be determined based on the first nano-floater and the second nano-floater.
The first nano-floater and the second nano-floater may be of the same type, and the first logic gate and the second logic gate may be AND wired.
The at least one input may include two inputs, and the lipid nanotable may output a result of interaction of the first nano-receptor and the first nano-floater according to the two inputs and a result of interaction of the second nano-receptor according to the first input.
Tethering between the first nano-receptor and the first nano-floater may be disassembled by the two inputs and thus the first nano-floater may be released, and the first nano-floater and the second nano-receptor may be tethered by the first input.
Tethering between the first nano-receptor and the first nano-floater may be disassembled by at least one of the two inputs and thus the first nano-floater may be released, and the first nano-floater and the second nano-floater may be tethered by the first input.
Tethering between the first nano-receptor and the first nano-floater may be disassembled by one of the two inputs and thus the first nano-floater may be released, the first nano-floater and the second nano-floater may be tethered by the first input, and the first nano-receptor and the first nano-floater may be tethered by the other one of the two inputs.
The first nano-floater and the second nano-floater may be of different types, and the first logic gate and the second logic gate may be OR wired.
The at least one input may include two inputs, and the lipid nanotable may output a result of interaction of the first nano-receptor and the first nano-floater according to the two inputs and a result of interaction of the second nano-receptor and the second nano-floater according to the first input.
Tethering between the first nano-receptor and the first nano-floater may be disassembled by the two inputs and thus the first nano-floater may be released, and, the second nano-receptor and the second nano-floater may be disassembled by the first input and thus the second nano-floater may be released.
Tethering between the first nano-receptor and the first nano-floater may be disassembled by at least one of the two inputs and thus the first nano-floater may be released, and the second nano-receptor and the second nano-floater may be disassembled by the first input and thus the second nano-floater may be released.
According to another aspect of the present invention, a lipid nanotable in a supported lipid bilayer to which a plurality of nanoparticles are integrated in nanoparticle units, includes: a first logic gate where a first nano-receptor and a first nano-floater interact with each other according to a selected input and a first input; and a second logic gate where a second nano-receptor and a second nano-floater interact with each other according to the selected input and a second input, wherein one of the first logic gate and the second logic gate may release a nano-floater that corresponds according to a corresponding input among the first input and the second input according to the selected input.
The first nano-receptor and the first nano-floater may be disassembled by the first input, the first nano-receptor and the first nano-floater may be assembled by the selected input, and the second nano-receptor and the second nano-floater may be disassembled by the second input and the selected input.
The lipid nanotablet may further include: a first surface molecule and a second surface molecule that are tethered to the surface of the first nano-receptor; and a third surface molecule and a fourth surface molecule that are tethered to the surface of the first nano-floater, wherein a combination between the first surface molecule and the third surface molecule is removed by the first input, and the second surface molecule and the fourth surface molecule are combined by the selected input.
The lipid nanotablet may further include: a fifth surface molecule and a sixth surface molecule that are tethered to the surface of the second nano-receptor; and a seventh surface molecule and an eighth surface molecule that are tethered to the surface of the second nano-receptor, wherein a combination of the fifth surface molecule and the seventh surface molecule may be removed by the second input, and the sixth surface molecule and the eighth surface molecule may be removed by the selected input.
A nanobio computing method in a supported lipid bilayer to which a plurality of nanoparticles are integrated in nanoparticle units, according to another aspect of the present invention, includes: generating a plurality of interactions between a plurality of immobile nano-receptors in the lipid bilayer and a plurality of mobile nano-floaters in the lipid bilayer according to inputs; generating a plurality of signals based on the plurality of interactions; tracking signals generated only from the plurality of nano-receptors among the plurality of signals; and determining a logic result based on the tracking result.
The tracking may include: detecting a signal higher than a detection parameter in generated image data by dark-field microscopy; generating a segmented signal by distinguishing a boundary of the detected signal; providing positions of nanoparticles by localizing a center of the segmented signal; identifying a nano-receptor by comparing the positions of the nanoparticles through a plurality of frames; and sampling a signal corresponding to the identified nano-receptor.
An increase in intensity of a signal in the tracking result may indicate assembly of a nano-receptor corresponding to an input among the plurality of nano-receptors and a nano-floater corresponding to the input among the plurality of nano-floaters.
A decrease in intensity of the signal in the tracking result may indicate disassembly of a nano-receptor corresponding to an input among the plurality of nano-receptors and a nano-floater corresponding to the input among the plurality of nano-floaters.
A lipid nanotablet that can systematically design and build circuits based on digital design principles with a scalable integrated platform having real-time readout and control functions, while overcoming the limitations of a solution-based existing approach that can only drive one kind of computation at a time, can be provided.
The invention disclosed in the present disclosure can be applied to overall nanoparticle computing based on a lipid membrane to which nanoparticles are attached rather than being limited to DNA molecules. Molecules that can be attached to the nanoparticle may include various chemical ligands such as DNA, RNA, proteins, peptides, metal chelators, and the like. That is, the present invention can be applied not only to biocomputing but also to nanoparticle-lipid platform-based molecular computing.
A cell membrane biologically serves the same function as a circuit board of an electronic circuit. The cell membrane compartmentalizes a receptor from an information-rich external fluid of the cell while hosing various receptor proteins in a computation unit, and performs complex functions by guiding receptors to interact laterally on the two-dimensional fluid surface. Each receptor, which is an active constituent of a biological circuit, takes chemical and physical cues as “inputs” such as binding events with ligands and changes in membrane voltage, and generate “outputs” such as conformational changes and dimerization/dissociation reactions. The membrane may allow many different computing processes to occur in parallel.
Taking inspiration from cellular membranes, a lipid nanotablet (LNT) includes a supported lipid bilayer (SLB) to which light-scattering plasmonic nanoparticles are tethered, and performs logic computation using the SLB. To perform the computation, it is programmed that the SLB-tethered nanoparticles interact with one another using surface ligands.
SLBs, which have been widely used as synthetic mimics for cell surfaces, are used as “chemical circuit boards” here, and nanoparticles that perform computation are placed on the surface. Tethering nanoparticles a lipid bilayer enables the following.
First, particle-to-particle interactions are confined to occur only through lateral diffusion at a 2D reaction space. Second, parallel in situ tracking and analysis of the nanoparticle interactions can be achieved with single-particle resolution because a large number of light-scattering nanoparticles are confined in the focal plane of dark-field microscopy (DFM). Third, nanoparticles are compartmentalized from solution containing molecular inputs. “Nano-bio” computing, which is an unconventional way to carry out computation with single nanoparticles, can be provided by using such features. The nano-bio computing occurs at the interface of the nanostructures and biomolecules. In order to prove this, in the present disclosures, nanoparticles are tethered to the lipid bilayer by using strong biotin-streptavidin interaction, and uses DNA as molecular inputs DNA and surface ligands. However, the surface ligand of the present invention is not limited to DNA, and molecules that can provide bondings capable of controlling interactions between nanoparticles by reacting specifically to molecular inputs, for example, antigen-antibody bonding, ligand-receptor bonding, chelate bonding, covalent bonding, hydrogen bonding, van der Waals bonding, bonding by hydrophobic interactions, electrostatic bonding, or bonding by chemical reaction, and bonding molecules capable of providing functional bonding, electrostatic bonding, or chemical bonding may be applied.
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DNA sequence and experiment conditions related to single-nanoparticle logic computation are summarized in Tables 1, 2, and 13.
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It can be determined that Cr-patterned glass substrates help to find the same position in both images. The co-relationship of the two image shows that characterized scattering signals shown in
When two plasmonic nanoparticles are in close proximity, the nanoparticles exhibit a plasmon binding effect, and thus it is possible to determine whether a bright particle is a single nanoparticle or an aggregation of nanoparticles by analyzing the scattering intensity of the DFM image. Compared with the same position in the SEM image, it can be confirmed that bright plaques in the initial state in the DFM image are from single nanoparticles.
As basic constituents of LNT, two classes of nanoparticles, nano-receptors (NRs) and nano-floaters (NFs), are used. A surface DNA ligand of each of the nano-receptor and the nano-floater is designed with a method in which the receptor-floater interaction (e.g., through assembly or disassembly) is controlled according to a result of logic computation that takes DNA molecules from the solution.
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Floaters, of which biotin linker valency ranges from 0.4% and 0.5%, are highly mobile on an SLB with a diffusion coefficient of about 1.0 μm2/s.
Due to the high mobility, NFs can actively interact with NRs across space and time, while functioning as active units of computation. The surface DNA ligands mediate a receptor-floater interaction, taking DNA molecules as inputs and inducing assembly or disassembly of the receptor-floater complex as an output.
An action of the floater tethered to the SLB is binary at the level of a single particle. That is, for a given observation period, the floater either discretely switches its state (ON) through assembly or disassembly or it does not. When digital actions of each floater are controlled with Boolean logic, a receptor-floater pair can be implemented as a single logic gate.
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When an input DNA (Xa) induces an assembly reaction between the receptor and the floater, the floater-receptor pair is defined as an assembly YES gate. When the input DNA (Xd) is output through a strand displacement disclosed by a toehold domain T2 and thus the receptor-floater (R-F) pair induces disassembly, the floater-receptor pair is defined as a disassembly YES gate. In the drawing, “T” denotes a toehold domain, “A” and “B” are building domains, and function domains may be connected in the order of A-T-B or B-T-A. The arrow head indicates a 3′ end, and the asterisk “*” indicates complementarity. Each logic gate may be displayed in the reaction graph shown in
In the Assembly YES gate, a G-NF switches its conformation state from a diffusible monomer (“0”) to an immobile dimer (“1”) through association with a G-NR in response to a single-stranded DNA input (Xa) that can hybridize with the surface DNA ligands of both of the receptor and the floater.
In the Disassembly YES gate, a G-NF is initially bound to a G-NR via hybridization with an oligonucleotide (Xd*). This step is termed pre-dimerization. The G-NF is then released from the G-NR and switches its state from an immobile dimer (“0”) to a diffusible monomer (“1”) when a DNA input (Xd) removes the pre-existing DNA bond Xd* via toehold-mediated strand displacement.
DNA sequence and experimental conditions of the Assembly YES gate and the Disassembly YES gate shown in
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For inputs, the logic values “0” and “1” represent the absence and presence of an input DNA in the solution. For outputs, “1” represents a G-NF bound to a G-NR (i.e., an R-F dimmer) for an Assembly gate, and a diffusible, monomeric G-NF for a Disassembly gate. “0” indicates the floaters staying in their initial states.
The information on state-switching behavior of floaters can be obtained by tracking signal changes of receptors. For example, the Assembly YES gate produces output “1” when a G-NF assembles onto a G-NR through the input DNA resulting in a step-wise increase in G intensity of the G-NR.
“Nanoparticle reaction network” abstraction may be used to represent behaviors of logic-gated nanoparticles. The abstraction is based on a directed graph where a node is represented by a nanoparticle and an edge is represented by logic, inputs, and reaction types. As shown in
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For example, when an Assembly gate is composed of a G-NR and a G-NF and a Disassembly gate is composed of a B-NR and a G-NF, the two gates can be simultaneously executed. In the two signals, an increase in G intensity of a G-NR and a decrease in G intensity of a B-NR are readily discernible.
A sufficiently high density of nanoparticles needs to be maintained to ensure that a large number of logic-gated nanoparticle reactions can occur within a short period of time. Approximately over 4000 nanoparticles (>3700 receptors and 300 floaters) tethered to a unit area (180×180 μm2) are monitored for computing processes that typically last 15 minutes to 30 minutes.
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The numbers of receptors is set to be higher than that of floaters to minimize trimer and tetramer formation. Such a condition allows the floaters to switch exclusively between the monomer and dimer stages.
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Since it is difficult to track mobile signals of floaters in the high density setting, an image analysis pipeline method exclusively trace receptors is used. Ambiguous signals that do not fall in the scatter plot categories for red, green, or blue signal clusters are eliminated for analysis. In the image analysis pipeline method, a tracking algorithm is used to reconstruct videos that visualize receptor signals only. Such videos exhibit an enhanced signal-to-noise ratio, providing a clear view of how nanoparticle circuits operate at a single-particle level in real time. First, profiling the scattering signals of R, G, and B nanoparticles is performed through the corresponding analysis method, and the profile is used for signal classification.
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DNA sequences and experiment conditions for the two input nanoparticle logic gates are summarized in Table 3 and Table 14.
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The design principles for the interface programming are schematically generalized and are illustrated in
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The assembly reaction is controlled by AND logic when bond-forming interactions require serial activation by two inputs, and by OR logic when bond-forming interactions are controlled in parallel.
The disassembly reaction is controlled by AND logic through a parallel disconnection paradigm and OR logic through a serial disconnection design.
The performances of the nanoparticle gates can be analyzed by counting output responses of the nanoparticle gates captured in dark-field videos. Whether or not a type of nanoparticle gate generates accurate digital output can be determined through a quantification process.
All four logic gates generated low output counts under the logical FALSE conditions and high output counts under the TRUE conditions. Specifically, ON/OFF levels over 5 folds, 88 folds, 93 folds, and 42 folds with fast response kinetics (t½<19 min, t½<5 min, t½<9 min, and t½<5 min) for the Assembly AND gate, the Assembly OR gate, the Disassembly AND gate, and Disassembly OR gate are provided, respectively.
The ON/OFF levels are evaluated by dividing the lowest output count obtained in the TRUE conditions by the highest output count obtained in the FALSE conditions. In Table 1, the response rate (%) (the number of floaters that react to inputs divided by the total number of floaters) is typically about 80% in the TRUE conditions.
The response rate, defined as the number of the floaters that reacted to the inputs divided by the total number of the floaters counted in the initial state, is typically over 80%. The Assembly AND gate exhibits minor output leaks at 1 AND 0 and 0 AND 1, presumably because the surface hairpins are in dynamic equilibrium between closed and opened states.
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Additionally, the LNT system is compatible with a “dual-rail” convention, where the Boolean values of a logic gate are represented by the presence of either one signal (“0”) or another (“1”). This formalism is used for systems where it is difficult to define the NOT function. With this representation, AND and OR gates are sufficient to compute any Boolean function.
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Interface programming may be expanded to enable nanoparticle logic gates to process INHIBIT logic (X1 AND NOT X2), and generate multiple outputs (fan-out) with multiple inputs (fan-in).
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Demonstration of an INHIBIT gate is significant because two-input AND, OR, and INHIBIT operations constitute a functionally complete set of Boolean functions.
Second, increasing the number of distinct DNA bonds, which can be disassembled, in a receptor-floater dimer enables the fan-in of a Disassembly gate.
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Third, a two-input Disassembly AND gate having three outputs is illustrated in
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As the complexity of reactions within the reactor-floater increases, incomplete reactions or spurious interactions also arise. Thus, relying on engineering the floater-receptor surface interface is not an efficient and scalable strategy for constructing complex circuits. Thus, in the present disclosure, nanoparticle “network programming”, which can connect two single particle logic gates with AND logic or OR logic, is used.
DNA sequence and experimental conditions are summarized in Table 5 and Table 16. Experiments are carried out at 25° C. in a 1× PBS buffer solution.
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First, network-level AND wiring can be demonstrated by enabling the use of the floater in the Disassembly and Assembly gates as shown in
In this network-level wiring scheme, the formation of a B-NR (R2)-G-NF (F1) dimer becomes an output of the AND-AND cascade circuit (X1 AND X2) AND X3.
Release of the G-NF F1 from the G-NR R1 is controlled by AND logic (X1 and X2). The released F1 may be tethered to R2 only when an assembly input X3 is present. The final circuit output is controlled by a three-input logic equation (X1 AND X2) AND X3. Green intensity in the first condition (1 AND 1) AND 1 is decreased in the receptor R1 and increased in the receptor R2 and thus is successfully cascaded by the G-NF F1. Only a decrease in green intensity is observed in the second condition (1 AND 1) AND 0. Lack of signal increase indicates that the released floater F1 is not tethered to another receptor. No reaction was seen in the two drawings below.
The AND-AND cascade circuit is described by a reaction graph, where two receptors R1 and R2 are serially connected to a floater F1. The circuit provides an ON/OFF level of 36 folds. As shown in
For the (1 AND 1) AND 1 condition, over 92% of F1 responds to the assembly input X3. This result indicates that the sequential disassembly-assembly cascade is highly efficient. Other two-input Disassembly gates, such as OR and INHIBIT gates, can be modularly rewired without optimization, resulting in the OR-AND cascade shown in
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In the two-layer OR-OR cascade circuit shown in
Since Disassembly gates support fan-out, any upstream Disassembly gates should be able to readily generate multiple floaters, each responsible for a distinct downstream computation. The released floaters can be subsequently “plugged” into another layer of Assembly gate through network-level AND wiring. This approach enables complex multi-layer cascades.
To demonstrate the modularity of circuit design on LNT, a multiplexer MUX 2-to-1 is implemented by wiring previously introduced logic gates via network programming.
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The nanoparticle surface ligand is designed to allow two different receptor-floater pairs to simultaneously process selector strands (Sel). The multiplexer selects one of two inputs X1 and X2 by using the selector strand (Sel) and translates the selected input into a single output.
The demonstration of the nanoparticle multiplexer shows that it is possible to design and operate nanoparticle circuits on the LNT platform in a modular and controllable manner.
Operating principles of LNT are unit in the following three aspects.
First, the computation is solely driven by SLB-tethered nanoparticles whose particle-by-particle interactions are programmable and readable in situ. A dynamic network of individual nanoparticles is equivalent to a logic circuit.
Second, since the cascading is exclusively driven by floaters, a process d does not require signal restoration or amplification. That is, floaters are “wires” carrying information of upstream gates into downstream gates via lateral diffusion that is robust to external conditions.
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There is no complementarity between the surface ligand of the floater G-NF and a dummy DNA 5′-GTTTAAGATTTATGGTTAAGCGTAGATTAAGTATTAAG-3′. AG-NF used in a single input Assembly YES gate is used in analysis of the G-NF (refer to Table 1). Analysis in each solution is repeated at three different positions in the lipid nanotablet.
Third, a spatial constraint is exploited to control the flow of molecular information in nanoparticle “signaling” networks. As shown in the multiplexer, an undesired interaction can be modularly controlled. Then, complex digital local operations can be implemented using a relatively small number of particles and ligand types. Through the integration with a lipid bilayer, nanoparticles are programmed, controlled, and visualized at the single-particle level, thereby designing and implementing a desired circuit according to digital principles. Such a function was not possible in the existing method, which relied only on nanoparticles “passively” clustered on a solution. The scope of molecular information that can be processed on LNT can be expanded several ways.
First, solution-phase molecular circuits that release single-stranded DNA as outputs can be synergistically interfaced with an LNT platform because only DNA molecules are needed to operate nanoparticle circuits on the NLT. In this case, a molecule circuit on the solution can additionally process molecular information. This approach can mediate communications across different nanoparticle circuit modules on a lipid bilayer as well as those with external environments.
Second, particle modifications based on diverse chemical ligands other than DNA can be readily introduced to process diverse chemical information. When introducing new surface chemistries on nanoparticles, the design constraints (that may arise from crosstalk between different surface ligands) can be reduced because particle-by-particle interactions are spatially or temporarily controlled on LNTs.
Third, integration of lipid bilayers with DNA nanostructures may provide a path toward a new type of molecular circuits. For example, by tethering DNA origami scaffolds that contain spatially localized DNA circuits to an SLB, a dynamic network of inter-origami interactions can be utilized. The principles used to construct or connect nanoparticle logic gates (interface programming and network programming) can be applied to construct a DNA origami scaffold circuit. This method may enable the implementation of more complex and even practical molecular computation.
Despite such potentials, further scaling up complexity of nanoparticle circuits on LNT will lead to a challenge because the input (DNA) and output (state-switching floaters) are of different forms. Currently, this intrinsic difference limits the construction of arbitrarily large circuits. This challenge can be potentially solved in two ways.
First, introducing new modes of nanoparticle reaction and ligand activation, such as dynamic reconfiguration, communication, DNA walker, and light-induced DNA release will be able to provide a much broader design space for circuit design, as this allows the nanoparticle interface and network to be controlled by more sophisticated mechanisms.
Second, increasing the number of different nanoparticle computing units per LNT will enhance the overall processing power of LNT. The different nanoparticle computing units can operate either in a parallel manner or assembled into a combinational circuit through network programming. This approach is similar to how an increase in integrated circuit density has led to the improvement in computing capacity of silicon-based computers. Ultimately, each nanoparticle independently performs a different computation on its own by using parallel processing.
As spatial constraints such as localization and encapsulation result in the modular execution of molecular circuits tethering nanoparticles to a lipid bilayer provides a systematic method to build complex nanoparticle circuits. The LNT-based approach according to the present disclosure may play a pivotal role in constructing highly functional “autonomous” nanostructures. Such devices may have broad impacts on molecular diagnostics and smart sensors. A nanosystem in the device should be able to exploit internal computational algorithms to sense multiple stimuli and trigger the most appropriate responses.
In addition, information-processing nanosystems on lipid bilayers can be applied to reconstituting artificial cell-cell junctions and used as tools for studying membrane-associated phenomenon in living cells. Unlike existing methods that rely on immutable materials such as patterned membranes, the LNT-based approach may allow networks of nanostructures on an SLB to autonomously form clusters or structural motifs and to be analyzed in response to signaling molecules from the cells. Allowing each nano- and cellular system to communicate with one another, such “active” SLB-cell junctions can also be employed to test how individual theranostic nanorobots navigate complex and dynamic environments.
A lipid solution (in chloroform) was mixed in a round-bottomed flask, a mixture containing diol-oil phosphatidylcholine (DOPC) at 97.2 mol %, biotinylated dioleoylphosphatidylethanolamine (DOPE) at 0.3 mol %, and poly(ethylene glycol) (1K)-DOPE at 2.5 mol % was obtained. The chloroform was removed by a rotary evaporator, and a lipid film formed inside the flask was completely dried for 15 minutes under a N2 stream. The dried mixture was resuspended in deionized water (DI water) so that the total concentration became 2 mg/mL. For the obtained lipid solution, a freeze-thaw cycle was repeated 3 times at a temperature between 78° C. and 40° C. The lipid solution obtained as described above can be stored in a liquid nitrogen tank for up to 2 weeks. The lipid solution was extruded 11 times at 30° C. through a polycarbonate membrane having 100 nm pores, and then subjected to ultrasonic wave treatment for 15 minutes to produce small unilamellar vesicles (SUVs) from the lipid solution. The SUV solution obtained as described above was stored at 4° C. until use.
SLBs were formed by a vesicle fusion method inside a glass chamber composed of upper and lower glass and parafilm spacers (4 mm×50 mm×200 μm).
The operating volume of the glass chamber is less than 40 uL (˜40 uL). After washing an upper slideglass (Paul Marienfeld GmbH) having an inflow port and an outflow port by ultrasonic wave treatment for 5 minutes in deionized water and piranha etching for 2 minutes, SLB formation was blocked by passivating with 10 mg/ml of bovine serum albumin (BSA) dissolved in 150 mM NaCl phosphoric acid buffer saline (1× PBS). The lower coverglass (Co. KG, Germany) was subjected to ultrasonic wave treatment in acetone and deionized water for 5 minutes, immersed in a piranha etching solution for 2 minutes, and washed, and then cleaned with deionized water. After that, a two-fold parafilm spacer was placed between two glass slides and heat-sealed at 100° C. The newly extruded SUV solution was diluted to 1 mg/mL in a 1× PBS solution, and then subjected to ultrasonic wave treatment for 15 minutes until use. SLB was formed by introducing a vesicle solution into a flow chamber at 30° C. After 60 minutes, the flow chamber was gently washed with deionized water (2 times) and 1× PBS. Then, 100 μg/mL BSA dissolved in 1× PBS was used to block defects on the SLB surface for 45 minutes. 17 nM streptavidin (STV) dissolved in 1× PBS was injected into the flow chamber to transform biotinylated lipids for 45 minutes. After BSA blocking and streptavidin transformation, the flow cell in the flow chamber was washed twice with 1× PBS. The flow chamber with STV-modified SLB can be stored in a humidified refrigerator at 4° C. for up to 3 days. Air-bubbles must be avoided in all processes related to the lipid chamber.
Gold nanorods are synthesized with silver nanoshells, gold nanospheres and silver nanospheres on gold seeds that mainly exhibit red R, green G, and blue B scattering signals, and they were used throughout the following example. First, a gold nano-rod with an aspect ratio of 4 was synthesized by a seed-mediated growth mechanism. Seeds were mixed with a HAuCl4·3H2O solution (5 mL, 0.5 mM) with a cetyltrimethylammonium bromide (CTAB) solution (5 mL, 0.2 M), and then an ice-cooled NaBH4 solution (600 μl, 10 mM) was rapidly injected. The seed solution was left for 2 hours after the reduction step. The HAuCl4·3H2O solution (5 mL, 1 mM) was mixed with the CTAB solution (5 mL, 0.2 M), and then an L-ascorbic acid solution (70 μl, 78 mM) was added after adding a AgNO3 solution (250 μl, 4 mM). 12 μl of the seed solution was added thereto and gently mixed. The obtained solution was incubated at 60° C. for 4 hours, centrifuged, and re-dispersed in deionized water 3 times. A gold nanorod solution (1 mL, 100 nM) was mixed with a cetyltrimethylammonium chloride (CTAC) solution (1 mL, 10 mM), AgNO3 (1 mL, 0.2 mM), and ascorbic acid (1 mL, 50 mM), and then the gold nanorod was coated with about 5 nm silver shells. After incubation at 60° C. for 4 hours, the solution was washed by centrifugation, the supernatant was removed, and then re-dispersed three times in deionized water to obtain red (R) nanoparticles. Gold nanoparticles (50 nm) of a spherical shape were purchased from BBI Solutions (Cardiff, UK) and used as green (G) nanoparticles. Blue (B) nanoparticles were prepared by growing a 17 nm silver shell on a 20 nm spherically shaped gold seed. A sodium ascorbate solution (100 μl, 50 mM) was rapidly injected to a mixture containing 150 pM 20-nm gold nanoparticles, 0.2% polyvinylpyrrolidone (PVP), and 0.24 mM nitric acid to form a silver shell on the gold seed.
Synthetic DNA oligonucleotides (Bioneer, Daejeon, Korea) having a thiol functional group were reduced with a solution of 100 mM dithiothreitol (DTT) dissolved in 100 mM pH 8.0 phosphate buffers (PB) for 1 hour, and then separated by using a NAP-5 column (GE Healthcare, Buckinghamshire, UK). The sequence, modification, and density of the surface DNA ligand used in the following example are summarized in Table 2 to Table 12. Table 2 to Table 12 show the sequence of thiolated DNA strands used in functionalizing nanoparticles (r: a spacer used in a ligand and a linker having a surface density of 5′ thiol group of a thiolated ENA strand: a space used in a ligand and a linker having 5′-A15 to EG6-3′3′ thiol group: and 5′-EG to A15-3′ sequence numbers disclosed in the following tables correspond to sequences of ligands and linkers excluding spacers).
The mixture of thiolated DNA was incubated with nanoparticles (final concentration: 15 PM) for 1 hour at room temperature. The entire concentration of thiolated DNA was used so as to exceed 14,400 times and 19,200 times with respect to R nanoparticles and G nanoparticles, respectively. The ratios of biotinylated DNA linker to the entire surface DNA ligand were 0.5% (w/v), 0.5% (w/v), 0.5% (w/v), 35% (w/v), and 50% (w/v), respectively with respect to a red nano-floater (R-NF), a green nano-floater (G-NF), a blue nano-floater (B-NF), a green nano-receptor (G-NR), and a blue nano-receptor (B-NR). After that, in the case of gold nanorods (R nanoparticles), the solution was adjusted to 0.1% (w/v) PVP in 10 mM PB, and in the case of spherically shaped gold nanoparticles (G nanoparticles), the solution was adjusted to 0.1% (w/v) sodium dodecyl sulfate (SDS) in 10 mM PB, and the spherical shape was adjusted in a solution of 10 mM PB for nanoparticles (B nanoparticles). Three aliquots of a 1M NaCl, 0.1% SDS, and 10 mM PB salt solution were added at intervals of 1 hour so that the final concentration of NaCl was 0.3M. After each salt was added, the mixture was heated at 50° C. for 10 minutes and incubated at room temperature. Two hours after reaching the final concentration, the nanorod solution was centrifuge-washed and re-dispersed in 1× PBS. Another nanoparticle solution was incubated for 12 hours, centrifuge-washed, and re-dispersed in deionized water (spherical-shaped gold nanoparticles) or 1× PBS (spherical-shaped nanoparticles). Features of modified nanoparticles were observed by using a transmission electron microscope (JEM-2100, JEOL Ltd, Japan), a UV-Vis spectrophotometer (Agilent 8453, Agilent Technologies, USA), and a dark field microscope (Axiovert 200M, Carl Zeiss, Gottingen, Germany) (
In order to completely assemble a lipid nanotablet, a solution containing DNA-modified nanoparticles with a biotinylated linker (1 to 10 pM) was introduced into a flow chamber of which a lower glass substrate was coated with a streptavidin-modified lipid bilayer surface. The solution was incubated for 1 to 5 minutes to obtain a desired particle density. Under these conditions, the particle density was linearly proportional to the incubation time (
During dark-field imaging, 500 μl of an input solution was injected into the flow chamber to test the performance of each nanoparticle circuit at 25° C. Dark-field imaging was performed using a 40× objective lens (NA 0.6) and a dark-field microscope (Dail Systems, South Korea) equipped with an AxiCam HRC color camera on an optical table. Before the injection of the input solution, 31 images were acquired with a 200 ms imaging time step to confirm receptor nanoparticles. Circuit performance was recorded during and after the input solution injection at 2.5 s imaging intervals. Two image sequences were acquired in a fixed position.
Image data obtained from time-lapse dark field images were processed and analyzed to quantify an output of the nanoparticle circuit. The quantification of the logic gate output is based on three steps: signal identification, tracking, and classification.
After time-lapse imaging with a dark field microscope, an optical scattering signal from the nanoparticle logic gate is verified and traced with ImageJ software and custom MATLAB code. In ImageJ, the image is first registered by the StackReg plug-in to correct transverse drift during imaging. The drift-corrected image sequence is processed by an image analysis algorithm capable of detecting and tracking particles. This procedure is described in
Receptor signal tracking provided sufficient information for quantitative logic gates, as changes in the receptor signal depend on the signal of the floater with which the receptor interacts. (4) Finally, a scattering signal from 3×3 pixels around the local position of each receptor was sampled and averaged, and one average scattered signal was assigned to the receptor at a given time. This process is repeated for all receptors and frames, and thus traces of scattered signals were obtained for all receptors identified in the field of view. The stepwise change of the receptor signal due to the increase by the floater assembly and the increase by the floater disassembly was confirmed as assembly and disassembly events, respectively. Transient interactions (short and transient increase or decrease in receptor signal) were eliminated. Homebuilt code excludes intensity changes that do not last until imaging is complete. That is, only the receptors showing signal traces similar to the step function were identified as output produce particles.
Each assembly/disassembly event corresponding to the desired logic gate was classified according to how the receptor signal changes over time. Signal profiles of R, G, and B nanoparticles were used for classification (
In
For each circuit, the number of receptors that generate a correct output was calculated over time. Recording continued until the kinetic plot became constant. The final number of events was normalized for each type of logical gate to minimize the effect of the variability of the particle population. For the assembly gate, the event count was standardized by the number of floaters detected in the initial 31 images. In the case of the disassembly gate, the disassembly event count was standardized by the number of dimers formed in the pre-dimerization. The ON/OFF level is calculated by dividing the lowest output count obtained in the TRUE condition by the highest output count obtained in the FALSE condition (when the output count in the FALSE condition is 0, the output count is set to 1).
The diffusion behavior of the nanoparticle logic gate, particularly, diffusion behavior of the floater particle, was analyzed as follows. (1) The particle density (˜200 particles per total field of view) of the floater particles loaded on the SLB is sufficiently low, and thus long-term tracking is provided without orbital overlap. (2) A signal is detected from each frame, and a position of each floater particle is localized based on the same algorithm described above. (3) The determined position is used to generate a trajectory of each floater, and then used in calculation of diffusion coefficients. For each particle, the mean squared displacement (MSD) value is obtained as a function of time. The MSD plot of this trajectory is fitted to the equation <r2>=4Dt, where <r2> is the MSD, D is the diffusion coefficient, and t is the time interval.
An assembly reaction of SLB-tethered nanoparticles was modeled and simulated by using MATLAB. Such a calculation method was developed to evaluate how an NR/NF ratio affects a degree of dimer formation. In this model, given numbers of NR and NF were randomly dispersed in an area of 128×128 μm2 with periodic boundary conditions. A diffusion constant of NF was specified to have a normal distribution with an average of 0.9 μm2/s and a standard deviation of 0.3 μm2/s. This approximation is shown in
Nanoparticles were functionalized with single-stranded DNA strands containing thiol modifications at the 3′ end or the 5′ end. The DNA sequence was designed at domain-level by using DomainDesign (“D. Y. Zhang, A. J. Turberfield, B. Yurke, E. Winfree, Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA. Science. 318, 1121-1125 2007”, and “D. Y. Zhang, in DNA Computing and Molecular Programming, Y. Sakakibara, Y. Mi, Eds. (Springer Berlin Heidelberg, 2010. Lecture Notes in Computer Science, pp. 162-175”), which is a free source code. The thiolated DNA strand includes (1) a biotinylated “linker” DNA strand for use in tethering a nanoparticle to a streptavidin-modified SLB surface, and (2) a “ligand” DNA strand that is directly involved in nanoparticle computation through hybridization with surface ligands of other nanoparticles on SLBs and an input DNA strand in a solution.
A linker strand having a 5′-thiol modification includes (i) a 15-based polyA domain after the 5′-thiol group, (ii) 6 ethylene glycol EG units (PEG moiety), and (iii) a 34-base linker domain followed by biotin modification
A linker strand with 3′-thiol modification (with biotin modification at the 5′-end) is followed by (i) a 34-base linker domain, (ii) a PEG moiety, and (iii) 3′-thiol modification followed by a 15-based polyA domain.
A surface ligand DNA strand used in the following example is classified into the following two types: (1) a “normal” single-stranded DNA strand that does not form a hairpin loop; and (2) a hairpin-type DNA ligand used in an Assembly AND gate.
Normal ligand types are further classified into two groups: a group with 3′-thiol modification; and a group with 5′-thiol modification.
The 5′-thiol ligand includes (i) a PEG moiety (after 5′-thiol), (ii) a 10-base spacer domain, and (iii) a 14-base binding domain. The 3′-thiol ligand includes (i) a 14-base binding domain, (ii) a 10-base spacer domain, and (iii) a PEG moiety (which later included 3′-thiol modification). Unless otherwise stated, the 3′-thiol ligand and the 5′-thiol ligand were used for receptors and floaters, respectively. The hairpin-type DNA ligand was thiolated at the 5′ end. In the following example, the 10-base toehold domain was used.
While this invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. On the contrary, it is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0061345 | May 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/006215 | 5/23/2019 | WO |
