Surface-Immobilized Bistable Polynucleotide Devices for Sensing and Quantifying Molecular Events

Abstract

A bistable device is constructed using a polynucleotide platform for sensing molecular events such as binding or conformational changes of a target molecule. Uses include measuring target concentration, measuring the effect of environmental conditions (e.g., heat, light, or pH) on the target, or screening libraries of molecules that bind to or modulate its biological function. The device comprises three regions: a top lid, a bottom lid, and a flexible joint or hinge therebetween. The device has an open configuration in which the top and bottom lids are separated and a closed configuration in which they are tightly bound together. A binding domain or variant of the target molecule is immobilized on the device such that when a molecular event occurs, the device switches from open to closed, or vice versa, which generates a signal. The optimal device design is determined by the signal mode (optical or electronic) used to measure the closing of the surface-immobilized device.

Description

[0001] Cross - Reference to Related Applications

[0002] This application is a partial continuation of U.S. application Ser. No. 16 / 350,115, entitled “Surface - Immobilized Bistable Polynucleotide Devices for Sensing and Quantifying Molecular Events,” filed on Sep. 25, 2018, the entire content of which is incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] This invention was made with government support under Grant No. CMMI1636364 awarded by the National Science Foundation and Grant No. N00014 - 14 - 1 - 0702 awarded by the Office of Naval Research. The government has certain rights in this invention. Technical Field

[0005] The present invention relates to structures for sensing and quantifying molecular events (e.g., binding events, conformational changes, chemical modifications, or enzymatic modifications) that may occur to target molecules, such as where quantification of such molecular events enables the measurement of the concentration of a single analyte or multiplexed detection and quantification of a set of analytes, where the analytes are molecular or particulate types in a fluid sample. Background Art

[0006] Multiple molecular entities exhibit selective affinities for one another, which results in the formation of multimolecular complexes, such as receptor - ligand, antibody - antigen, nanobody - antigen, or aptamer - target complexes. Such selective affinity of one molecule for another is of particular interest because the binding events that give rise to such affinities can be used to determine the presence of an analyte in a given sample solution and, in some settings, also the concentration of the analyte.

[0007] Over the past few decades, numerous detection methods have been developed based on the identification of specific complex formation, including direct or indirect strategies for detecting and / or amplifying signals associated with primary or secondary binding events, where the signals can be optical (spectroscopic, colorimetric, or fluorescent) or electrical (impedance, capacitance, inductance, or current). Due to the specificity, speed, and sensitivity of these methods (and related systems), they have become the cornerstone of modern analytical measurements and are used in academic as well as industrial research. Some specific application areas of such platforms include: environmental assessment, food safety, medical diagnosis, and the detection of chemical, biological, and / or radiological warfare agents.

[0008] However, existing high-sensitivity assays have technical details that make it difficult for them to perform multiplex analysis on multiple analytes. In addition, the procedures for quantification using such assays typically involve a series of dilution steps, which result in large amounts of sample. Therefore, there is a great need for methods that are simultaneously highly sensitive, modular, and multiplexable to quantitatively measure the presence of analytes in small sample volumes.

[0009] More generally, the detection of molecular events (such as conformational changes and enzymatic modifications) can be used to create powerful bioactivity assays. For example, transmembrane protein receptors bind endogenous ligands during their natural function, but artificial ligands for such receptors constitute one of the most important classes of drugs. Activation of a transmembrane receptor by a ligand on the outer side of the cell is typically accompanied by a conformational change or phosphorylation on the cytoplasmic side of the receptor, providing a direct indication of receptor activation and possible bioactivity. Currently, screening large molecular libraries for potential drugs is best performed on cells. Therefore, there is a need for in vitro sensors for arbitrary molecular events that can go beyond the simple binding of analytes to provide assays for some functional molecular events (such as phosphorylation or conformational changes). Similarly, screening molecules with various functional properties (such as activities that change according to heat, light, pH, or other environmental stimuli) would benefit from a sensitive, modular, and multiplexable platform for arbitrary molecular events. SUMMARY OF THE INVENTION

[0010] Aspects of embodiments of the present invention relate to a general platform for detecting possible molecular events (such as binding events, conformational changes, chemical modifications, or enzymatic modifications) of a target molecule. In some embodiments, the detection of a molecular event (such as binding) can be used to determine the concentration of a molecule or particle in a fluid sample. In some embodiments, the detection of a molecular event (such as a conformational change or enzymatic modification) can be used to screen a library of candidate drugs for activity against membrane receptors of medical interest.

[0011] In some embodiments of the present invention, a structure includes a bistable molecular sensor for optically or electronically detecting an external stimulus on a surface. The bistable molecular sensor has a polynucleotide platform, including: a first polynucleotide shape and a second polynucleotide shape having a flexible hinge or a flexible linker therebetween, one of the first polynucleotide shape or the second polynucleotide shape being fixed to the surface, resulting in a fixed polynucleotide shape and a tethered polynucleotide shape; and one or more functional molecules bound to at least one of the first polynucleotide shape and the second polynucleotide shape. The bistable molecular sensor has one of two states, the two states being a closed state and an open state, and wherein in the open state, the tethered polynucleotide shape is free to move relative to the second polynucleotide shape due to the constraint of the flexible hinge or the flexible linker; and in the closed state, the tethered polynucleotide shape is arranged adjacent to the fixed polynucleotide shape. In some embodiments, the polynucleotide platform is selected from scaffold deoxyribonucleic acid (DNA) origami, scaffold ribonucleic acid (RNA) origami, scaffold hybrid DNA:RNA origami, single-stranded DNA tiles, multi-stranded DNA tiles, single-stranded RNA origami, multi-stranded RNA tiles, or DNA or RNA origami having a hierarchical composition with multiple scaffolds.

[0012] In some embodiments of the present invention, the structure for optical detection as described above, wherein the surface is gold or graphene, and the tethered shape includes a luminescent body selected from an organic fluorophore, a quantum dot, a fluorescent bead, or a luminescent lanthanide compound, and the open state produces more light than the closed state.

[0013] In some embodiments of the present invention, as described above, the structure is for electrical detection, wherein the surface is a working electrode, including gold, platinum, graphene, indium oxide, or indium tin oxide, the tethered shape is labeled with one or more redox-active molecules, and a change in state results in an electron transfer between the one or more redox-active molecules and the working electrode.

[0014] In some embodiments of the present invention, the structure as described above further includes a solution above the surface and a working solution electrode, wherein the surface serves as a transistor, the surface is a gate material selected from carbon nanotubes, silicon nanowires, graphene, molybdenum disulfide, or indium oxide, the fixed shape is directly attached to the surface, and the solution above the surface serves as the gate electrode of the transistor.

[0015] In some embodiments of the present invention, the structure as described above is used for field effect sensing, and the structure further includes a solution and a working solution electrode above the surface, wherein the surface serves as a transistor, the surface includes a semiconductor gate under a covering layer selected from silicon dioxide, aluminum oxide or silicon nitride, the fixed shape is attached to the covering layer, and the solution above the surface serves as the gate electrode of the transistor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application document contains at least one color drawing. Copies of this patent or patent application publication with color drawings are provided by the Patent Office upon request and payment of the necessary fees.

[0017] The drawings, together with the description, illustrate exemplary embodiments of the present invention and, together with the description, are used to explain the principles of the present invention.

[0018] Figure 1A is a schematic diagram of a bistable molecular sensor designed to operate in solution, which includes an affinity tag and a DNA barcode.

[0019] Figure 1B is a schematic diagram of a bistable molecular sensor composed of two polynucleotide shapes, one of which is fixed on the surface and tethered by a flexible linker, suitable for optical or electronic detection.

[0020] Figure 2A is a schematic diagram of the operation of a bistable sensor in solution, which operates through a sandwich actuation mechanism and shows the hiding of the affinity tag when binding an analyte.

[0021] Figure 2B is a schematic diagram describing the extraction of a DNA barcode from a bistable sensor that has been closed by removing the opening sensor using an affinity bead.

[0022] Figures 3A - 3C depicts a bistable sensor suitable for sensing single-stranded nucleic acids using sandwich actuation, suitable for optical or electronic detection on a surface.

[0023] Figure 3D depicts a bistable sensor suitable for sensing double-stranded DNA, using sandwich actuation and using two CRISPR / dCas9 complexes.

[0024] Figure 3E depicts a bistable sensor suitable for sensing double-stranded DNA using sandwich actuation, using a single allosteric CRISPR / dCas9 complex, which reveals a hidden sequence when binding a double-stranded analyte. Figure 3FIncluding atomic force microscopy data showing that the dCas9 artificial guide sequence readily binds to short artificial targets along the edges of bistable sensors arranged on a substrate, and a schematic diagram of multiple bistable sensors arranged on the substrate.

[0025] Figures 4A - 4F Depicts three main actuation mechanisms of sandwich, competition, and function, including examples of these mechanisms using cooperativity, detecting phosphorylation of MAPK, or binding of a ligand to a transmembrane G-protein coupled receptor.

[0026] Figures 5A - 5D Depicts the functional actuation of bistable sensors based on riboswitches, cleavage reactions, and ligation reactions.

[0027] Figures 6A - 6C Depicts three different methods for the electronic detection of the actuation of bistable sensors immobilized on a surface.

[0028] Figures 7A - 7E Depicts three different methods for the optical detection of the actuation of bistable sensors immobilized on a surface, including cartoon sketches of signal traces for signal-on and signal-off implementations.

[0029] Figures 8A - 8D Depicts four different geometries of bistable sensors, each presenting different configurations of signal molecules or polynucleotide materials to the detector surface below when the bistable sensor is in its off state.

[0030] Figures 9A - 9C Depicts the problems and solutions for the surface placement of bistable sensors on optical or electronic surfaces.

[0031] Figure 10 Depicts the multiplex detection of multiple analytes by using an array of multiple bistable sensors, where each subarray is sensitive to a different target analyte. Detailed Description

[0032] In the following detailed description, only certain exemplary embodiments of the present invention are shown and described by way of illustration. As will be recognized by those skilled in the art, the present invention may be implemented in many different forms and should not be construed as limited to the embodiments set forth herein.

[0033] Aspects of embodiments of the present invention relate to bistable molecular sensors created on a substrate using a polynucleotide platform (e.g., a general structure for generating well-defined two-dimensional or three-dimensional shapes from polynucleotides). Polynucleotide platforms include, but are not limited to, scaffolded deoxyribonucleic acid (DNA) origami (Rothemund, Paul WK. "Folding DNA to create nanoscale shapes and patterns", Nature 440.7082 (2006): 297), scaffolded ribonucleic acid (RNA) origami (Torelli, Emanuela et al., "Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon", Scientific Reports 8 (2018): 6989), scaffolded hybrid DNA:RNA origami (Wang, Pengfei, et al "RNA–DNA hybrid origami: folding of a long RNA single strand into complex nanostructures using short DNA helper strands", Chemical Communications 49 (2013) 5462-5464), scaffold-free single-stranded DNA sheet (DNA brick) systems (Wei, Bryan, et al., "Complex shapes self-assembled from single-stranded DNA tiles", Nature 485 (2012): 623–626 and Ke, Yonggang, et al., "Three-Dimensional Structures Self-Assembled from DNA Bricks", Science 338 (2012): 1177-1183), scaffold-free multi-stranded DNA tile systems (Winfree, Erik, et al., "Design and self-assembly of two-dimensional DNA crystals", Nature 394 (1998) 539-44) or RNA tile systems (Chworos, Arkadiusz, et al., "Building programmable jigsaw puzzles with RNA.”Science 306(2004):2068-72), intramolecularly folded single-stranded RNA (Geary, Cody, et al., “A single-stranded architecture for cotranscriptional folding of RNAnanostructures”, Science 345(2014)799-804), or single-stranded DNA origami (Han, Dongran, et al., “Single-stranded DNA and RNA origami”, Science 358(2017):eaao2648), all of which are incorporated by reference in their entirety. For clarity, aspects of embodiments of the present invention will be described primarily in the context of scaffold DNA origami as a specific example of a “molecular shape”. However, embodiments of the present invention are not limited to scaffold DNA origami. Instead, embodiments of the present invention include molecular shapes made using other polynucleotide platforms, such as the platforms listed above, and some examples of applying embodiments of the present invention to other polynucleotide platforms are described in more detail below.

[0034] Some embodiments of the present invention have two parts: first, a bistable polynucleotide sensor that is designed to change its state upon application of an external stimulus (such as a solution of analyte molecules), and second, a set of protocols and methods that enable the state change of the device to be recorded as a DNA sequence, an optical signal, or an electronic signal.

[0035] Described herein are bistable polynucleotide sensors that can operate in solution ( Figure 1A ) or on a surface ( Figure 1B ). For the purpose of introducing the bistable polynucleotide sensor, the common features of the solution and surface forms are first described. In one embodiment, the bistable polynucleotide sensor consists of two polynucleotide shapes tethered together by one or more flexible linkers. For clarity, only a single linker is depicted in FIG. 1.

[0036] The bistable polynucleotide sensor represents a general platform for detecting molecular events. For clarity, as Figures 1A - 1B depicted sensors, where these sensors are adapted to detect binding events, a commonly used ability to measure the concentration of analyte molecules. In addition, Figures 1A - 1Bdepicts such sensors, particularly suitable for use with antibody pairs in a "sandwich actuation mode" to detect proteins, an ability commonly used in sandwich immunoassays (e.g., ELISA). Subsequently, Figure 3 depicts the sandwich actuation mode for detecting nucleic acids. Subsequently, Figures 4 and 5 depict multiple embodiments highlighting some other molecular events that can be detected and quantified using this platform, as well as details regarding the various molecular entities involved.

[0037] Thus, two illustrative examples of the sandwich actuation mechanism ( Figure 1A , "solution form" and Figure 1B "surface form"), each polynucleotide shape carrying a "binding molecule" (e.g., aptamer, antibody, or nanobody) that binds to unique non-overlapping regions of the analyte molecule or particle. In the absence of the target analyte, the two origamis move relative to each other via a flexible linker, a state referred to as "open". However, upon capturing the analyte, the two origamis bind to form a single semi-rigid unit, a state referred to as "closed". In some embodiments, at least one of the origamis carries a unique DNA barcode or a luminescent or electrochemically active signaling molecule that can be used to detect the change in state from open to closed. For clarity, in the description of one embodiment in solution form in Figure 1A , the possibility that one or both polynucleotide shapes carry a luminescent molecule has been omitted; however, in some embodiments, for the purpose of signal detection, the solution form carrying a luminescent molecule is described below.

[0038] Throughout this disclosure, the following terms are used interchangeably: bistable molecular sensor, bistable sensor, bistable detector, bistable device, bistable polynucleotide nanostructure, bistable polynucleotide device, bistable polynucleotide sensor, flytrap sensor, flytrap detector, or flytrap.

[0039] In this disclosure, reference is made to two at least semi-rigid polynucleotide shapes of the bistable sensor as "lids", and reference to the orientation of these lids in a diagram or relative to the bistable sensor on a surface refers to the "top lid" and "bottom lid". When these lids can be implemented using any of the polynucleotide platforms described above, they are "DNA origamis".

[0040] In the present disclosure, the flexible connection between the lids serves as a "joint" or "hinge". In some embodiments, there can be more than one joint between the lids, and in some embodiments, a single joint can be composed of single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, double-stranded DNA helical bundles, double-stranded RNA helical bundles, polynucleotide analogs, or non-polynucleotide polymers (such as polyethylene glycol (PEG)). According to the embodiment, the length of the joint can vary from a single covalent bond (about 2 angstroms) to a double-stranded joint of up to 10,000 nucleotides (about 3.5 micrometers). Embodiments using solution-based detection (such as but not limited to LRET or PCR-amplifiable DNA signals) typically use shorter joints (1 nm to 10 nm), whereas embodiments using surface-based optical or electronic detection typically use longer joints (10 nm to 4 micrometers).

[0041] While some embodiments of the bistable polynucleotide sensor comprise two independently folded DNA origami shapes self-assembled with independently synthesized joints, other embodiments comprise a single DNA origami, wherein both the polynucleotide shape and the joint are folded through a single long DNA scaffold strand. Yet other embodiments of the bistable sensor are generated by single-stranded DNA tiles, multi-stranded DNA tiles, single-stranded RNA, or DNA origami or any other suitable polynucleotide platform.

[0042] According to the embodiment, the state change induced by a molecular event (such as the binding of an analyte molecule) can be detected in solution or on a surface by one of several different methods, including but not limited to: (1) if the assay is performed in solution, the state change (open versus closed) can be detected by a unique DNA barcode encoding the identity of the analyte to be quantified or by luminescence resonance energy transfer, or (2) if the assay is performed on a surface, the state change can be recorded as a change in an optical or electrical signal, wherein the spatial position of the bistable origami device on the surface encodes the nature of the recorded molecular event (such as the identity of the analyte to be quantified).

[0043] For some "solution format" embodiments that provide detection through the output of a DNA barcode signal, the bistability of the device allows us to preferentially remove the devices that have not bound the target analyte (Figure 2). The open devices have a purification tag available ( Figure 2A , such as biotin), which can bind to an affinity column (such as a column containing streptavidin beads). The remaining closed devices, which have the analyte bound, have a hidden affinity tag, and thus they should pass through an affinity column with beads that bind the affinity tag ( Figure 2B ) and can be collected for analysis.

[0044] Once eluted from the affinity column, the device with the bound analyte in the OFF state can be detected by standard PCR or by various methods of quantitative PCR, droplet digital PCR, or next-generation DNA sequencing or deep DNA sequencing. Depending on the exact method used to detect and quantify the DNA barcode, the device can be used without further processing and purification, or the DNA barcode can be released from the bistable device and purified prior to reading. In some embodiments, the DNA barcode can be released from the bistable sensor by using a restriction enzyme that cleaves the barcode molecule from the sensor. In some embodiments, the DNA barcode can be released from the bistable sensor by strand displacement reactions as described in Zhang et al., “Dynamic DNA nanotechnology using strand-displacement reactions”, Nature Chemistry 3 (2011): 103-113, the entire content of which is incorporated herein by reference. In some embodiments, the DNA barcode is not released from the bistable sensor and it is read directly.

[0045] Advantages of some solution-based embodiments that use the DNA barcode signal as an output include: (1) they can be used with existing reagents (antibodies used for standard sandwich immunoassays can be conjugated to the bistable device), (2) they can be used without instrumentation other than that required for DNA amplification (e.g., PCR), (3) they enable high multiplexing, at least 8 analytes in the case of fluorescence-based qPCR and at least 1000 analytes in the case of next-generation sequencing, and (4) they can be highly quantitative by taking the DNA barcode output of the bistable sensor and using it as an input for droplet digital PCR (ddPCR). Thus, standard techniques for nucleic acid counting (ddPCR) can be used to count protein molecules by using a bistable sensor.

[0046] The sensitivity, false negative rate, and false positive rate of any particular solution-based embodiment will be set by: (1) the binding affinity of the analyte for the binding molecule within the device and the extent to which the bound analyte turns the device fully OFF, (2) the extent to which the purification tag is “hidden” in the bound state, and (3) the extent to which the “unhidden” purification tag allows the device to completely remove unbound analyte from the solution. For example, if the OFF device still allows a small molecule purification tag (such as biotin) to protrude slightly from the pores on the origami surface, some analyte molecules may be lost on the affinity column, resulting in false negatives or an underestimation of the protein concentration. Similarly, if the analyte binds relatively weakly to the bistable device and the device does not remain OFF continuously and it still dynamically opens and closes, it may be lost on the column.

[0047] Thus, in some embodiments, additional "weak locks", comprising a pair of short complementary single-stranded DNAs that are activated upon analyte binding, can reduce false negatives. The weak locks tend to shift the equilibrium of the device without analyte towards the closed configuration. If such a device spends sufficient time in the closed configuration, particularly the average time required for the device to pass through the column, it can avoid binding to the column and generating false positives.

[0048] Thus, in embodiments with weak locks, the strength of the weak locks must be adjusted such that the variation (between open and near-closed) of the bistable device without analyte binding creates sufficient opportunity for it to bind to the affinity column, with a high probability of it doing so.

[0049] The greater the chance that a device without analyte binding must bind to the column, the lower the chance that it passes through and generates false positives. Thus, in some embodiments, multiple sequential affinity column separations can be used to reduce false positives.

[0050] Regarding Figure 1A the appearance of the weak lock and affinity tag in, for clarity, the affinity tag is depicted as being disposed at the end of the single-stranded weak lock. Some embodiments have this configuration of the weak lock and affinity tag. In other embodiments, the weak lock and affinity tag are located on separate connectors on the inner surface of the bistable sensor. In still other embodiments, there are no weak lock and affinity tag on the connectors on the inner surface of the bistable sensor.

[0051] In the case of weak analyte binding, where the bistable device with bound analyte frequently toggles into the open configuration (where the analyte remains bound to one of the two caps), if additional binding sites are available, the binding of additional copies of the analyte can be used to reduce the frequency of the open configuration. Thus, in some embodiments of the solution-based bistable sensor, by including multiple copies of each binding partner on the top and bottom caps of the sensor in a manner similar to that shown for DNA detection on the surface in Figure 4B and labeled as the "cooperative sandwich mechanism", false negatives can also be reduced using cooperativity.

[0052] In some embodiments of solution-based bistable sensors, the use of time-resolved optical output enables the use of bistable sensors based on the widespread availability of commercial plate readers capable of measuring the time-resolved luminescence of long-lived emitters. In such embodiments, the solution-based sensors do not require DNA barcodes, affinity tags, or weak locks. The readout is based on luminescence resonance energy transfer between short-lived (nanoseconds to microseconds) emitters, such as organic fluorophores or quantum dots, and long-lived (milliseconds) luminescent compounds, such as europium and terbium chelates. In such embodiments, one lid of the bistable sensor is labeled with an organic fluorophore. In some embodiments, the organic fluorophore on one lid can be replaced with an organic quencher. In such embodiments, the second lid of the bistable sensor is labeled with a long-lived emitter, such as europium and terbium chelates.

[0053] Depending on the specific wavelengths used for the short-lived and long-lived emitters, energy transfer (from donor to acceptor) can be from the short-lived emitter to the long-lived emitter or vice versa. In either case, after the decay lifetime of the short-lived emitter has ended, pulsed excitation light is used and time-resolved measurements are made. In this way, all scattered excitation light has dissipated, and the only signal measured is the signal transferred between the short-lived and long-lived emitters, greatly improving the signal-to-noise ratio and thus the sensitivity of the assay. The general principle behind such measurements, luminescence resonance energy transfer (LRET), has been previously described in Selvin et al., “Luminescence Resonance Energy Transfer” Journal of the American Chemical Society, 116 (1994): 6029-6030, the entire contents of which are incorporated herein by reference.

[0054] On the other hand, if the bistable sensor is immobilized on a surface ( Figure 1B ), then the binding of the analyte can be recognized as a change in state measured electronically (Figure 6) or optically (Figure 7). Each embodiment of the surface-based method for detecting a change in state of the bistable device has a different set of false positive and false negative error mechanisms compared to the solution-based method, and thus in some embodiments, the detection may be more quantitative than in others. For example, as discussed subsequently for some embodiments, in the absence of the analyte, false positives can occur when the top origami shape binds nonspecifically to the surface ( Figure 9A ).

[0055] Like some solution embodiments, some surface-based embodiments achieve highly sensitive measurements. Some surface-based embodiments achieve high sensitivity by using TIRF microscopy (as described for single origamis in Gietl et al., “DNA origami as biocompatible surface to match single-molecule and ensemble experiments,” Nucleic Acids Res. 40 (2012): e110 and Tsukanov et al., “Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami,” Phys. Chem. B 117 (2013): 11932–11942) or electrochemical detection (as described for small nucleic acid reconfigurations in Lai, “Chapter Eight: Folding-and Dynamics-Based Electrochemical DNA Sensors,” Methods in Enzymology, 589 (2017): 221-252; Immoos et al., “DNA-PEG-DNA triblock macromolecules for reagentless DNA detection,” Journal of the American Chemical Society 126 (2004): 10814–10815; Wu et al., “Development of a “signal-on” electrochemical DNA sensor with an oligo-thymine spacer for point mutation detection,” Chemical Communications, 49 (2013): 3422–3424; and Wu et al., “Effects of DNA probe and target flexibility on the performance of a “signal-on” electrochemical DNA sensor” Analytical Chemistry 86 (2014) 8888–8895), the entire contents of all of which are incorporated herein by reference, where binding of an analyte nucleic acid to a nucleic acid on the surface brings an electrochemically active functional group (ferrocene or methylene blue) in proximity to the surface, where it can be electrically detected.

[0056] In some embodiments, surface-based optical and electronic measurements can be converted from analog measurements to digital measurements by using DNA origami placement techniques. Digital measurements can be achieved using DNA origami because individual DNA origami can be almost deterministically (>95% of sites have a single origami) placed into a grid on a surface using the lithographic techniques used to set them up, as described in Kershner et al., "Placement and orientation of individual DNA shapes on lithographically patterned surfaces", Nature Nanotechnology 4 (2009):557–561; Hung et al., "Large-area spatially ordered arrays of gold nanoparticles directed by lithographically confined DNA origami", Nature Nanotechnology 5 (2010):121-126; Gopinath et al., "Optimized Assembly and Covalent Coupling of Single-Molecule DNA Origami Nanoarrays", ACS Nano 8 (2014):12030–12040; and Gopinath et al., "Engineering and mapping nanocavity emission via precision placement of DNA origami", Nature 535 (2016):401-405, the entire contents of which are incorporated herein by reference. This previously described "DNA origami placement" technique enables individual bistable sensors to be placed on a single optical or electronic device with >95% probability, and thus >95% of the sensors can be used to sense target molecules. This is in contrast to droplet digital PCR, which relies on Poisson statistics to occupy droplets with individual analyte nucleic acids, achieving a single nucleic acid in no more than 37% of the droplets. Thus, some embodiments of surface-based methods for reading out bistable sensors can give more quantitative results than some solution-based methods that rely on droplet digital PCR for readout.

[0057] Some surface-based embodiments achieve high multiplexing by using widely available techniques to print spots of surface-immobilized devices that are specific for different analytes. Figure 10Devices are shown where each spot has thousands of the same binding molecule pairs, but different spots have different binding molecule pairs. Some embodiments use inkjet printing to achieve this spatial multiplexing, and other embodiments use microarray spotting to achieve multiplexing.

[0058] Similar to some solution-based embodiments, some surface-based embodiments can use existing sandwich ELISA reagents for binding target molecules, but such embodiments may require additional materials (electronic or optical chips) and instruments (electronic readers or TIRF / other optical readers or microscopes).

[0059] Some embodiments of the present invention demonstrate a first example of an assay having one or more of the following characteristics: (1) Based on the principle of a hidden purification tag, a bistable DNA nanostructure device converts an analyte binding signal into a unique and amplifiable DNA signal (changing the state of the device from on to off). (2) An assay having a variety of surface-based methods for sensitively measuring the conversion of a large and flexible bistable DNA device into a small and compact rigid device upon binding a single analyte molecule. (3) An assay having the ability to perform multiplexed quantitative measurements of multiple analytes in solution by PCR / sequencing or by the spatial location of optical or electronic signals on a surface, based on the described bistable DNA nanostructure device. (4) An assay that can provide digital quantification of protein molecules on the same scale as digital droplet PCR.

[0060] Some embodiments of the present invention provide significant improvements over existing electrochemical assays. The described folding-based assays (Lai, “Chapter Eight: Folding-and Dynamics-Based Electrochemical DNA Sensors”, Methods in Enzymology, 589 (2017): 221-252; Immoos et al., “DNA-PEG-DNA triblock macromolecules for reagentless DNA detection”, Journal of the American Chemical Society 126 (2004): 10814–10815; Wu et al., “Development of a “signal-on” electrochemical DNA sensor with an oligo-thymine spacer for point mutation detection”, Chemical Communications, 49 (2013): 3422–3424; and Wu et al., “Effects of DNA probe and target flexibility on the performance of a “signal-on” electrochemical DNA sensor” Analytical Chemistry 86 (2014) 8888–8895) have been used to detect conformational changes due to the binding of nucleic acids to a detector on a surface via electrochemical sensing (using ferrocene or methylene blue signaling molecules). In this context, a single nucleic acid brings a single signaling molecule closer to the surface when the analyte nucleic acid binds and folds the entire structure such that the signaling molecule is closer to the surface.

[0061] Embodiments of the present disclosure differ in important respects: (i) the disclosed work enables proteins or any analyte to be examined, where previous work was limited to DNA or RNA. (ii) Previous work used a single signaling molecule for each binding event. The currently disclosed use of DNA origami or another large DNA nanostructure means that the currently disclosed signals may be stronger; at least 200 signaling molecules can be incorporated into an origami, providing an amplification that makes the assay potentially 200 times more sensitive in principle. (iii) Previous work only folded the detection molecule a few nanometers. This means that the signaling molecule does not move very far from the inactive (no analyte) state to the active (analyte bound) state. In turn, this means that the inactive and active states are not as different as they should be, and the assay is not as potentially sensitive as it should be. In the currently disclosed work, the tether can be up to several micrometers long (fully adjustable down to a few nanometers). This allows the origami carrying the signaling molecule to be set at an optimal height above the surface to minimize the signal in the inactive state, thus maximizing the sensitivity to the analyte-bound state. In previous work, short linkers limited the detection method to electrochemical sensing. In the currently disclosed work, because the linker can be long enough (e.g., 200 nm) to move the signaling molecule significantly out of the evanescent field of the TIRF substrate in the inactive state, TIRF microscopy can achieve higher sensitivity.

[0062] Embodiments of the present invention provide significant improvements over existing TIRF-based assays. TIRF has previously been used for surface-bound origami to detect molecular binding or conformational changes (Gietl et al., “DNA origami as biocompatible surface to match single-molecule and ensemble experiments,” Nucleic Acids Res. 40 (2012): e110 and Tsukanov et al., “Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami,” Phys. Chem. B 117 (2013): 11932–11942). These studies support that the currently disclosed methods can be used to quantify proteins in low-volume / low-concentration / single-molecule protocols. However, these previous studies: (i) studied nucleic acids rather than proteins and did not give the functionality of using two binding molecules to simultaneously bind an analyte (as in a sandwich assay). (i) Studied conformational changes in very simple hairpins or Holliday junctions using a single signaling molecule. The currently disclosed larger devices have over 200 signaling molecules for greater amplification and sensitivity of molecular events. (ii) Used short linkers / small conformational changes. Similarly, the currently disclosed work uses very large conformational changes, which gives higher sensitivity with the same number of signaling molecules.

[0063] The bistable origami detector has several advantages over other potential methods. In the terms of immunoassays (which are also applied to DNA and RNA detection but are not commonly used for nucleic acid detection), the bistable detector can be used as the basis for a “homogeneous assay,” where the sample to be analyzed can simply be added to the detector without any requirement to mix the components of the detection system together, and importantly without the need to wash away additional sample or add a secondary detection system. This means that the detection experiment can be carried out directly and quickly. Secondly, since origami domains are typically very large (several megadaltons), they are typically 100 - 1000X larger than the molecules to be detected (kilodaltons to tens of kilodaltons). This means that the origami can carry a large number of signaling molecules, which can give 200-fold amplification without the use of a secondary amplification system.

[0064] In FIGS. 1 and 2, embodiments using an antibody to detect a protein with a sandwich actuation mechanism are depicted. FIG. 3 depicts a different embodiment using sandwich actuation to detect nucleic acids. Figure 3AShows the basic geometry of the flytrap device on the surface. It consists of two DNA origami disks or "lids" with a diameter of 100 nm, and a double-stranded DNA linker between them with a length ranging from 10 nm to 4,000 nm. The detection of the single-stranded target nucleic acid sequence XY is mediated by a pair of probes X' and Y', each of which is complementary to half of the target sequence and binds to the inner sides of the top and bottom lids, respectively.

[0065] In the absence of XY, the lids of the flytrap diffuse independently, constrained by the tethering chain, which is referred to as the "open" state in the present disclosure. When both domains X and Y bind to the complementary probes on the flytrap (referred to as the "closed" state in the present disclosure), the two lids of the device co-localize, where the distance is set by the specific probe-target / device geometry selected. For example, if the probes are selected such that they are tethered to the lids at positions at the ends of the probe-target duplex, the lids remain at a distance approximately equal to twice the length of a single probe-target duplex (about 7 nm for a pair of 10-mer probes, about 14 nm for a pair of 20-mer probes). On the other hand, if the probes are selected such that they are tethered to the lids in a geometry that places the linker adjacent to the middle of the probe-target duplex, the lids can remain at a distance of approximately 2 - 4 nm (up to the width of two DNA helices) (independent of the length of the target sequence).

[0066] Figure 3C The flytrap described in [reference] is most effective for detecting single-stranded DNA, single-stranded RNA, or other single-stranded polynucleotide analogs. For specific DNA duplex sequences, i.e., those capable of forming DNA triple helices, Figure 3C the flytrap in [reference] can be used to detect double-stranded DNA, although the kinetics of DNA triple helix formation is slow and the target analyte is restricted to poly-purine:poly-pyrimidine triple helix-forming sequences.

[0067] On the other hand, by using the dCas9 / CRISPR complex, it is possible to generate flytraps capable of effectively detecting double-stranded DNA by two different methods. In the CRISPR system, the dCas9 protein is complexed with a gRNA, and each gRNA has a 20-nucleotide RNA "guide" sequence. With the help of dCas9, the guide sequence can strand-displace a suitable double-stranded DNA and bind to the complementary target essentially irreversibly.

[0068] Thus, in some embodiments ( Figure 3D)In this case, each lid has an attached gRNA which has one of two different guide sequences, chosen such that they bind adjacent 20-nucleotide target pairs in the target DNA sequence. Before introducing the analyte DNA, the dCas9 protein is introduced and assembled onto the gRNA. Thus, the simultaneous binding of two CRISPR / dCas9 complexes to a single double-stranded analyte DNA closes the trap.

[0069] In such an embodiment, it is stated beforehand that the target sequence must be adjacent to a so-called PAM site having a specific consensus sequence (e.g., NGG). Thus, in the case of natural DNA, the detection using conventional dCas9 is limited to DNA sequences that coincidentally have two appropriately spaced PAM sites (within about 50 nucleotides). The recently analyzed GFP construct was used as a control in CRISPR-based gene regulation experiments, and several pieces of DNA were found to have double-occurring PAM sites at distances suitable for Figure 3D the dual-target detection scheme depicted. Furthermore, in some embodiments, Cas proteins (or any suitable endonuclease) from organisms other than Streptococcus pyogenes and genetically engineered endonuclease proteins (e.g., Cas9) with different sequence specificities for their PAM sites enable the detection of a wider range of sequences. In cases where the target analyte is artificial DNA for barcode schemes or DNA storage, it is not difficult to add PAM site pairs when necessary.

[0070] An advantage of embodiments having such dual-target schemes is that they use standard gRNA sequences. Furthermore, atomic force microscopy (AFM) data ( Figure 3F ) indicate that dCas9 with artificial guide sequences readily binds short artificial targets at the edges of the origami. Thus, the CRISPR / dCas9 complex integrates well with the DNA origami at the desired positions. The dual-target scheme requires the DNA-binding portion of CRISPR / dCas9 to be free rather than bound to the origami, as Figure 3F shown. Thus, in some embodiments, the 3' extension of the gRNA is used to immobilize the CRISPR / dCas9 complex to the trap.

[0071] The limiting sequence constraints of such dual-targeting schemes are that they require the DNA analyte to have two target / PAM sites and a length of at least 46 nucleotides. Other embodiments are capable of detecting double-stranded DNA with fewer sequence constraints. Dynamic DNA and RNA nanotechnology utilize non-equilibrium DNA reactions to create cascades of ordered events (as described in Zhang et al., “Dynamic DNA nanotechnology using strand-displacement reactions”, Nature Chemistry 3 (2011): 103-113). A classical example is the so-called hairpin-strand reaction. This type of reaction enables a sequence to remain “hidden” by another protective sequence that forms a hairpin until a trigger sequence binds.

[0072] Thus, the principle of hiding a sequence to produce a so-called “allosteric” CRISPR / Cas9 complex can be used to implement a double-stranded DNA sensor ( Figure 3E ). The allosteric CRISPR / Cas9 on the bottom cover of the trap, when bound to its target double-stranded sequence X, reveals a new sequence hY that is complementary to the sequence hY' on the trap lid, causing the trap to close. For this purpose, the 5' end of the gRNA can be extended with a new sequence that forms a hairpin with the guide sequence. Thus, the guide sequence is used to hide and protect the sequence hY from hY' until the target double-stranded DNA (X) binds to the allosteric CRISPR / Cas9.

[0073] The use of the term allosteric here is consistent with the standard use of allostery in the literature, but somewhat unusual. By definition, allostery only involves the ability of one molecule (effector A) to alter the binding or activity of a second molecule B (usually a protein) towards a third molecule C. In the allosteric scheme presented here, the target double-stranded DNA acts as effector A, the CRISPR / dCas9 complex acts as B, and the sequence on the trap lid acts as C. Figure 3E The scheme in [] is unusual because allostery is usually defined in terms of the “normal” activity of a protein. Here, the “normal” activity of the protein is used as an allosteric trigger to turn on or off a new function of the protein - binding to the trap lid. Although standard allostery has been described, where a small molecule (4-hydroxytamoxifen) alters the activity of CRISPR / cas9, this type of scheme is not thought to have been reported in the CRISPR / cas9 literature (Oakes et al. “Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch”, Nature Biotechnology 34 (2016): 646-651).

[0074] Figure 3E The advantage of the allosteric scheme for detecting double-stranded DNA is that it only requires a single 20-nucleotide target plus its 3-nucleotide PAM site. Note that the XRN-1 5'-to-3' exonuclease or other activities inhibit the useful addition of functional sequences to the 5' end of the gRNA in yeast and mammalian cells in vivo (such additions are not protected by the protein envelope of Cas9). Here, since in vitro DNA detection is being performed, such extensions will not degrade. In some embodiments, small (<10nt stem) hairpins can be used to avoid interfering with the initiation of the CRISPR / Cas9 DNA complex at the first 10 important nucleotides of the guide (near the PAM site) and to maximize the sensitivity of the sensor.

[0075] In some embodiments, some target native sequences inevitably lack NGG, but in these cases, using different native or mutant CRISPR systems with different PAM sites (such as the Cas-protein Cpfl from Prevotella and Francisella bacteria, with its TTTN PAM site) greatly increases the chance of finding available target sequences. In addition, compared to dCas9, Cpf1 has a completely different gRNA structure and 3'-end guiding sequence, which can prove to be more compatible with some target sequences. Thus, in some embodiments, CRISPR / Cpf1 is used instead of CRISPR / dCas9.

[0076] In FIGS. 1-3, embodiments are described for detecting and quantifying molecular binding events using a sandwich actuation mechanism to quantify analytes such as proteins and nucleic acids. FIGS. 4 and 5 describe bistable molecular sensors in additional contexts. The basic bistable sensor design has several "actuation mechanisms" where the roles of the sensor top and bottom covers are different with respect to the detection of molecular events, and these roles are different based on whether the molecular event is a binding event, a conformational change, or other molecular events. As Figure 4A 、 4C and shown in 4D, the distinguishing sensing mechanisms are the sandwich mechanism "" ( Figure 4A , for binding events), the "competition mechanism" ( Figure 4C , for binding events) or the "functional mechanism" ( Figure 4D , for conformational changes caused by a molecular or physical environment, enzymatic or chemical cleavage, or enzymatic or chemical modification). Additionally, sensors described as operating in a sandwich, competition, or functional mechanism can also be described as operating in a non-cooperative or cooperative mechanism (e.g. Figure 4B ).

[0077] The sandwich actuation mechanism (Figure 4A ) has been described in a "sandwich" design, where a pair of binding partners (antibodies, RNA or DNA aptamers, natural binding proteins, etc.) for the target analyte are disposed on the top and bottom covers of the sensor, respectively. The sandwich mechanism facilitates the detection of the presence or absence of the analyte or the measurement of its concentration. In the case where antibodies are used as binding partners, the sandwich assay is comparable to a sandwich immunoassay (such as sandwich ELISA).

[0078] For a particular target molecule analyte, if a suitable pair of binding partners can be found, the sandwich mechanism ( Figure 4A ) is suitable for its detection. This is usually the case for larger target analytes (such as proteins) for which two different epitopes can be found, and one binding partner has an affinity for each epitope. For the case where the target analyte is single-stranded DNA or single-stranded RNA, the two binding partners can be single-stranded, where each binding partner itself is single-stranded DNA or RNA and is complementary to a different region or subsequence of the target analyte. In the case where the target analyte is double-stranded DNA, RNA:DNA hybrid, or double-stranded RNA, the binding partners can be (1) single-stranded DNA or RNA at two different regions or subsequences of the target analyte (in which case the sensor closes after the formation of a triple helix), or (2) the binding partners can be a nucleic acid / protein complex (such as CRISPR / dCas9) capable of specifically binding the target at two positions (as Figure 3D shown), (3) the binding partners can be an allosteric CRISPR / dCas9 complex (as Figure 3E shown) or (3) the binding partners can be zinc finger proteins or peptide molecules capable of sequence-specifically binding the target at two regions or subsequences, or (4) any pair of molecules capable of sequence-specifically binding the target at two different regions.

[0079] The competitive actuation mechanism ( Figure 4C)A description is made for the following situation: where the bottom cover of the sensor holds a single binding partner (antibody, RNA or DNA aptamer, natural binding protein, etc.) of the target analyte, and the top cover of the sensor holds a competing molecule that can bind to the binding partner on the bottom cover in a manner similar to the target analyte. The competition mechanism is conducive to detecting the presence or absence of the analyte or measuring the concentration. In the case where the binding partner is an antibody, the competition mechanism is similar to a competitive immunoassay. The competition mechanism is useful in cases where the target analyte is too small or the surface is too symmetric or chemically undifferentiated to find two different binding partners for the target. This is usually the case for small molecules (such as typical drugs or many hormones). The fact that the competition actuation mechanism only requires a single antibody or a single aptamer for a specific target analyte makes the number of potential analytes for which the competition actuation mechanism is applicable much larger than the number of potential analytes for which the sandwich actuation mechanism is applicable, which requires finding two antibodies or two aptamers for the target analyte.

[0080] In the competition actuation mechanism, depending on the potential of the binding and the competitor to non-specifically bind to the substrate surface adjacent to the sensor (which usually results in false negative signals), the roles of the top cover and the bottom cover can be interchanged; molecules with a lower non-specific affinity for the background surface are usually selected to be set on the cover.

[0081] The competing molecule can be the following examples: the target molecule, a related molecule of the target molecule that can bind to the binding partner, or any other molecule that can bind to the binding partner at the site where the target usually binds. This enables the competitor to block or otherwise inhibit the normal binding of the target when binding to the binding partner. Similarly, the target analyte has the ability to bind to the binding partner and inhibit the binding of the competitor. In the absence of the target, the competitor often binds to the binding partner, and the top cover of the sensor spends more time close to the bottom cover and the surface, generating a signal. In the presence of the target, the target molecule binds to some of the binding partners on the bottom cover of the sensor, and reduces the amount of time the competitor binds to the binding partner, and thus changes the signal. As the target concentration increases, the occupancy of the target on the binding partner is higher, the sensor opens more often, and the top cover spends more time away from the surface, enhancing the signal change.

[0082] The fact that the presence of an analyte increases the probability of the open state does not mean that all implementations of the competitive actuation mechanism must be "signal-off" detectors. Thus, depending on the particular sensing mode used to generate and measure the signal, the signal change in the competitive actuation mechanism can be positive or negative. For example, a competitive assay can be used with an optical sensing mode where the lid is labeled with a first fluorophore. In the case where the bottom lid is labeled with a second fluorophore, then addition of the target results in a decrease in FRET between the first fluorophore and the second fluorophore, reducing the signal from the second fluorophore, thus implementing a "signal-off" detector. In the case where the bottom lid is labeled with a fluorescence quencher, then addition of the target reduces the quenching between the first fluorophore and the quencher, thus implementing a "signal-on" detector. Thus, as for other actuation mechanisms, the competitive mechanism can result in signal-on and signal-off of the sensor as needed.

[0083] Functional actuation mechanisms ( Figure 4D ) enable the detection of molecular events more generally, including the binding, enzymatic or chemical activity (including cleavage or ligation), or enzymatic or chemical modification (such as phosphorylation, methylation or acetylation) of a class of molecules rather than a single target analyte. In the functional mechanism, the top lid and the bottom lid each carry a functional ligand (functional ligand 1 and functional ligand 2, respectively), which bind to each other or are released in the presence of an external stimulus, which can be a small molecule or a protein enzyme, but can also be a physical condition, such as a change in temperature, light, pH or ionic strength.

[0084] Typically, the roles of the top lid and the bottom lid can be interchanged, i.e., functional ligand 1 can be on the top lid and functional ligand 2 can be on the bottom lid, and vice versa. Thus, which functional ligand is chosen to be placed on the top lid generally depends on which functional ligand has the lowest non-specific binding to the background substrate. However, in some implementations, such as when one of the functional ligands is a transmembrane protein, the performance of the sensor can be improved when the transmembrane protein is attached to the top lid and the other functional ligand is attached to the bottom lid. In many implementations, the external stimulus causes a conformational change in functional ligand 1, which alters its affinity for functional ligand 2. In such implementations, functional ligand 2 is an antibody raised against functional ligand 1 such that it binds functional ligand 1 in a specific conformational state but not in another conformational state. In many implementations, the external stimulus causes a chemical modification (such as phosphorylation) of functional ligand 1, which alters its affinity for functional ligand 2. In such implementations, functional ligand 2 is an antibody raised against functional ligand 1 such that it binds functional ligand 1 in a specific modified state (such as phosphorylated) but not in another conformational state (such as unphosphorylated).

[0085] Thus, in one implementation of the functional actuation mechanism ( Figure 4E) that is capable of detecting phosphorylation of a mitogen-activated protein kinase (MAPK, such as P42 or P44) by a mitogen-activated protein kinase kinase (MAPKK) or any other reagent that phosphorylates MAPK. In this embodiment, functional ligand 1 is MAPK (on the bottom lid), and functional ligand 2 is an antibody that binds only to the phosphorylated form of MAPK (anti-phospho-MAPK). The sensor is on when MAPK is not phosphorylated and off when it is phosphorylated. Similar embodiments can be constructed by replacing functional ligand 1 with any protein that can be modified (by methylation, phosphorylation, or acetylation) and replacing functional ligand 2 with a binding ligand that binds only to the modified form of ligand 1.

[0086] Thus, in one embodiment of the functional actuation mechanism, it is capable of detecting a ligand, agonist, or antagonist of a protein receptor (such as any G-protein coupled receptor (GPCR)). In this embodiment ( Figure 4F), the functional partner 1 is a protein-based lipid nanodisc (as described in Bayburt et al., "Membrane Protein Assembly into Nanodiscs" FEBS Letters 584 (2010): 1721–1727), or a DNA-based lipid nanodisc, as described in Zhao et al., "DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry, Journal of the American Chemical Society, 140 (2018): 10639–10643 and Iric et al" "DNA-Encircled Lipid Bilayers" Nanoscale (2018) DOI: 10.1039 / C8NR06505E), wherein the transmembrane receptor protein (e.g., μ-opioid receptor (a prototype GPCR)) is loaded into the lipid portion of the nanodisc, the entire content of all of these is incorporated herein by reference. For the purpose of solubilizing membrane proteins, protein-based lipid nanodiscs have been well studied, and they can be linked to DNA strands, as taught in Zhao et al. above, thus providing a method for attaching the functional partner 1 to the top cap. Similarly, as disclosed in Zhao et al. and Iric et al. above, DNA-based lipid nanodiscs can be loaded with membrane proteins and can be loaded onto the top cap of the sensor, or can be directly used as the top cap of the sensor. The functional partner 2 is a protein, such as β-arrestin, whose affinity for the transmembrane protein changes when the receptor protein binds to the ligand. In the specific case of using β-arrestin as the functional partner 2, ligand binding and activation of the GPCR in the top cap cause the G-protein coupled receptor kinase (GRK) present in the solution to phosphorylate the GPCR, which results in β-arrestin binding to the GPCR and turning off the sensor. This embodiment provides a general method for screening drugs for GPCRs in an in vitro cell-free environment. In such an environment, the sensor does not simply bind and sense a specific target analyte, but responds to any molecule that affects the normal biological function of the receptor to be studied. In this case, the functional sensor is called a "detector-like".

[0087] The above detector is the most faithful mimic of the native β-arrestin pathway for detecting ligand binding of GPCRs. By changing the identity of β-arrestin from β-arrestin 1 (also known as "arrestin 2") to β-arrestin 2 (also known as "arrestin 3"), it is possible to detect and study different aspects of so-called biased agonism, where ligands of different GPCRs have slightly different effects and stimulate different downstream pathways, and it is possible to study the differences between class A and class B GPCRs that have different affinities for β-arrestin 1 and β-arrestin 2, as taught in Oakley et al., "Differential Affinities of Visual Arrestin, Arrestin1, and Arrestin2 for G Protein-coupled Receptors Delineate Two Major Classes of Receptors"

[0088] as taught in The Journal of Biological Chemistry 275(2000)17201-17210, the entire content of which is incorporated herein by reference. Similarly, different GRKs (GRK2 to GRK6) have different interactions with different GPCRs because they phosphorylate GPCRs at different residues depending on the GPCR type and specific ligand, as taught in Yang et al., "Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model" Molecular Pharmacology 92(2017)201-210, the entire content of which is incorporated herein by reference. Thus, in some embodiments, different combinations of two types of β-arrestin and five different GRKs are combined.

[0089] In some embodiments, to produce a sensor that does not require the use of GRK in solution, the desired GRK type is conjugated to DNA and placed on the bottom lid of the trap together with β-arrestin. In this way, all the necessary components of the signaling pathway are combined into a single bistable detector, and for sensing, only a ligand needs to be added. In such embodiments, when the ligand is bound and the GPCR is activated, the top lid of the trap first transiently interacts with the GRK on the bottom lid and the GPCR is phosphorylated and released. Then, the phosphorylated GPCR in the top lid interacts with the bottom lid a second time by binding to β-arrestin, and a sustained signal is detected.

[0090] Other embodiments of the present invention use antibodies to detect ligand binding of GPCRs without the need for β-arrestin. In one embodiment, the top lid has a GPCR attached (e.g., μ-opioid, as above), but the bottom lid does not have β-arrestin. Instead, it has an antibody raised against the phosphorylated state of the GPCR, as taught in Mouledous et al., "GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of mu-Opioid Receptors Induced by Neuropeptide FF(NPFF2)Receptors" The Journal of Biological Chemistry, 287(2012)12736-12749 and Just et al. "Differentiation of Opioid Drug Effects by Hierarchical Multi-Site Phosphorylation" Molecular Pharmacology 83(2013)633-639, the entire contents of which are incorporated herein by reference. Thus, when a ligand binds and GRK phosphorylates the receptor, the anti-phosphorylation-antibody binds to the GPCR, closing the trap and inducing a signal. Such embodiments allow for the study of ligand binding without the need for the specific features of β-arrestin interaction with the GPCR, and the use of antibodies against different phosphorylation patterns enables the study of the phosphorylation code of GPCRs (Yang et al., ibid.).

[0091] Yet other embodiments of the present invention use nanobodies to detect ligand binding of GPCRs without the need for β-arrestin or GRK. In one embodiment, the top lid has a GPCR attached (e.g., μ-opioid, as above), but the bottom lid does not have β-arrestin, and GRK is not present in the solution nor attached to the bottom lid. Instead, nanobodies raised against the active ligand-binding state of the GPCR are attached to the bottom lid. The generation of such nanobodies is taught in Huang et al., “Structural insights into mu-opioid receptor activation” Nature 524 (2015) 315-321, the entire content of which is incorporated herein by reference. In the presence of the ligand, the GPCR is activated, and the nanobody binds to the GPCR and closes the trap, and induces a signal, regardless of whether the GPCR is phosphorylated or non-phosphorylated. In such embodiments, which are simpler than the β-arrestin pathway mimics described above, the detector can be more stable for long-term storage and transportation, production can be cheaper, and thus it can be more useful in diagnostic settings. Particularly in the case of embodiments having the μ-opioid receptor, the trap detector can be used in law enforcement to detect the presence of opioids in an unknown sample or in a contaminated building.

[0092] Thus, in one embodiment of the functional actuation mechanism, detection and concentration measurement of small molecule targets can be achieved through conformational changes of a “riboswitch”. RNA and DNA aptamers can be selected by artificial molecular evolution (SELEX) to bind to a target small molecule target. However, (A) such targets are typically too small to be detected by a sandwich actuation mechanism, and (B) depending on the binding characteristics of the target and the aptamer, it may be difficult to construct a sensitive competitive actuation mechanism. In such cases, using a functional actuation mechanism with a riboswitch can directly detect small molecules without competition. In such embodiments, the aptamer is modified into a riboswitch such that upon binding to the small molecule target, it undergoes a conformational change to expose a DNA or RNA sequence ( Figure 5A ) or an RNA-protein ( Figure 5B ) or a DNA-protein binding domain. Thus, the riboswitch can serve as one functional partner, and a protein, DNA, or RNA molecule can serve as the second functional partner. In particular, some embodiments can use an RNA riboswitch that exposes a common MS2 aptamer upon binding to the target small molecule, where the MS2 aptamer then binds to the MS2-viral major capsid protein (MCP) attached to another lid ( Figure 5B ).

[0093] Thus, in one embodiment of the functional actuation mechanism, the chemical or enzymatic ligation (joining or coupling) of two proteins, two nucleic acids, or their hybrids is detected ( Figure 5C)。In such an embodiment, functional partner 1 and functional partner 2 are two molecules whose connection is to be measured. The introduction of a chemical or enzymatic linker causes functional partners 1 and 2 to covalently bind together, resulting in the sensor turning off and generating a signal. In such embodiments, the presence or absence, activity strength, or concentration of the linker is being measured.

[0094] In a related embodiment of the functional actuation mechanism, chemical or enzymatic cleavage (cutting) of a protein or DNA is detected ( Figure 5D )。Such embodiments present the opposite scenario to those involving ligation. In this embodiment, functional partners 1 and 2 are prepared in such a state that they exist as a single binding entity or are pre-ligated prior to measurement. The introduction of a chemical or enzymatic cleavage agent separates functional partners 1 and 2 from each other, resulting in the sensor turning on and generating a signal. In such embodiments, the presence or absence, activity strength, or concentration of the cleavage agent is being measured.

[0095] Some embodiments of the present invention can be enhanced by using cooperativity. Cooperativity can be imparted to sandwich ( Figure 4D ), competitive, or functional actuation sensors by increasing the number of binding partners, competitors, or functional partners present on the sensor top and bottom covers.

[0096] Embodiments in which the bistable molecular sensor is immobilized on a surface can be read out electronically or optically using one of several well-known detection methods. Figure 6 shows three different device architectures that can be used to electronically read out embodiments on the surface of a bistable molecular sensor.

[0097] In Figure 6A , the bistable molecular sensor is immobilized on top of the gate region of a standard planar semiconductor transistor. Here, the sensor in the "on" or "off" state affects the local ionic environment around the transistor gate in a manner that can be quantified by transistor characteristics. For example, the transistor can be biased such that the sensor being in the "on" or "off" state directly causes the transistor to be "turned on" or "turned off".

[0098] Biosensing FETs constructed from classical semiconductor materials have been previously described (Veigas et al., "FieldEffect Sensors for Nucleic Acid Detection: Recent Advances and FuturePerspectives" Sensors 15 (2015): 10380 - 10398).

[0099] In Figure 6BIn this case, the bistable molecular sensor is immobilized on the channel region of a field-effect transistor (FET), which is constructed from low-dimensional materials (e.g., one-dimensional (1D) materials (carbon nanotubes or silicon nanowires) or two-dimensional (2D) materials (graphene, molybdenum disulfide [MoS2], or indium oxide thin films)). Here, the FET consists of a channel made of 1D or 2D materials, located between two electrodes, with a gate contact (in solution) to modulate the electronic response of the channel. The sensor in the "on" or "off" state affects the local ionic environment around the transistor gate, which can be quantified by transistor characteristics. For example, the transistor can be biased such that the sensor being in the "on" or "off" state directly causes the transistor to be "turned on" or "turned off".

[0100] Biorecognition FETs constructed from low-dimensional materials have been previously described: carbon nanotubes (Allen et al., "Carbon Nanotube Field-Effect-Transistor-Based Biosensors" Advanced Materials 19 (2007) 1439 - 1451); silicon nanowires (Chen et al., "Silicon nanowire field-effect transistor-based biosensors for biomedical diagnosis and cellular recording investigation" Nanotoday 6 (2011) 131 - 154); graphene (Afsahi et al., "Towards Novel Graphene-Enabled Diagnostic Assays with Improved Signal-to-Noise Ratio" MRS Advances 60 (2017) 3733 - 3739); molybdenum disulfide (Sarkar et al., "MoS2 Field-Effect Transistor for Next-Generation Label-Free Biosensors", ACS Nano 8 (2014) 3992 - 4003) and indium oxide (Nakatsuka et al., "Aptamer–field-effect transistors overcome Debye length limitations for small-molecule sensing", Science 6 (2018) eaao6750).

[0101] In Figure 6CIn it, the bistable molecular sensor is fixed on top of a planar electrode, which is composed of a material with appropriate conductivity (such as a metal (such as gold or platinum), graphene, indium tin oxide, or indium oxide). Here, the lid of the bistable molecular sensor carries a redox-active molecule, whose proximity to the electrode causes electron transfer that can be detected as a current within the metal electrode in the "on" or "off" state. Electrochemical detection of bistable sensor actuation is performed using one of several well-known methods, including but not limited to: square wave voltammetry, cyclic voltammetry, electrochemical impedance spectroscopy, or chronoamperometry.

[0102] In one embodiment, electrochemical detection is performed on a gold electrode, where the gold surface has been prepared by electron beam deposition or templated stripping from a super-flat template (such as mica or a silicon wafer). The redox-active molecule on the top lid of the bistable sensor is a methylene blue reporter molecule. The bottom lid of the bistable sensor is fixed to the gold surface by thiol modification, phosphorothioate modification of the polynucleotide backbone, or polyadenosine extension. And the gold electrode is covered with a self-assembled monolayer of mercaptohexanol (or a similar alkanethiol), which prevents unwanted electrochemical reactions from masking the desired signal of the methylene blue molecule. In such an embodiment, the closing of the bistable sensor results in an increased rate of electron transfer from methylene blue to the surface, producing a "signal-on" behavior for the system. In some embodiments, the change in the electron transfer rate is measured by square wave voltammetry.

[0103] The combination of the gold electrode, the methylene blue redox reporter molecule, and the alkanethiol passivation layer (read out by square wave voltammetry) is common in the literature, as previously described (Ricci et al., "Linear, redox modified DNA probes as electrochemical DNA sensors" Chemical Communications 36 (2007): 3768-3770).

[0104] Figure 7 shows three different device configurations that can be used for optical readout of embodiments on the surface of the bistable molecular sensor.

[0105] In Figure 7AIn this case, the bistable molecular sensor is fixed on a transparent optical substrate (glass, quartz, silica), and total internal reflection illumination (TIRF illumination, where light below the critical angle is confined to propagate within the substrate) is used to generate an evanescent field at the surface. In such embodiments, an optical reporter molecule (such as a lumophore (such as an organic fluorophore or quantum dot) or a light scatterer (such as 25 - 50 nm plasmonic particles, or 500 nm to 1 µm dielectric particles)) is attached to the top cover. In the open state, the optical reporter molecule is far enough from the surface such that a small fluorescence or scattering signal is observed. In some embodiments, the plasmonic nanoparticles are gold or silver nanoparticles. In some embodiments, the dielectric particles are silica or polystyrene nanospheres. In the closed state, the optical reporter molecule is in the strong part of the evanescent field such that a large fluorescence or scattering signal is observed. The distance-dependent decay of the evanescent field is related to the wavelength λ of the light generated by the emitter or scattered by the particles, and the critical distance for the strong signal is typically λ / 10. The use of single DNA origami for TIRF optical measurements has been previously described (Gietl et al., “DNA origami as a biocompatible surface to match single-molecule and ensemble experiments,” Nucleic Acids Res. 40 (2012): e110 and Tsukanov et al., “Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami,” Phys. Chem. B 117 (2013): 11932–11942).

[0106] In Figure 7CIn this case, the bistable sensor is fixed on a substrate (gold or graphene), which strongly quenches the fluorescence of the emitter, as described for gold (Dulkeith et al., "Gold Nanoparticles Quench Fluorescence by Phase Induced Radiative Rate Suppression" Nano Letters 5 (2005): 585–589) and graphene (Kasry et al., "Highly Efficient Fluorescence Quenching with Graphene" J. Phys. Chem. C 116 (2012): 2858–2862). Thus, in the open state, the optical signal from the top lid of the bistable sensor is large, and in the closed state, the optical signal from the top lid of the bistable sensor is much smaller. In such embodiments, the strongest quenching effect is observed when the emitter is within a few nanometers of the surface, and thus such embodiments can use a bistable device geometry where the signal molecules on the top lid are rigidly positioned and in close contact (less than a few nanometers) with the surface. In Figure 7C One such potential geometry is illustrated, in particular a top lid with rigid arms extending beyond the bottom lid region.

[0107] In Figure 7D this case, using the well-known DNA origami placement technique, the bistable sensor is fixed on a microfabricated ring resonator (as described in Sarkaleh et al., "Optical Ring Resonators: A Platform for Biological Sensing Applications" J. Med. Signals. Sens. 7 (2017): 185–191), where the microfabricated ring resonator is strongly coupled to an optical waveguide. In such embodiments, both the emitter or the light scatterer are compatible optical reporters of the bistable sensor state change. The excitation light input at one end of the waveguide enters the ring resonator and may either be emitted as fluorescence or scattered by the reporter molecule on the lid of the bistable device, or it may not. If the bistable device is open, the light circulating in the ring simply returns to the waveguide and is observed as a transmission signal at the output. On the other hand, if the bistable device is closed, the light circulating in the ring resonator is converted to emitted light of a longer wavelength (in the case where the reporter molecule is an emitter) or scattered away (in the case where the reporter molecule is a light scatterer). Thus, when the device is closed, the amount of light returning from the ring to the waveguide is reduced, and a reduction in signal transmission is measured at the waveguide output. For such embodiments, the position of the lid in the closed state can be up to 50 nanometers away from the surface of the ring resonator.

[0108] For embodiments, such as Figure 7A those illustrated in Figure 7B , the measured optical signal increases when the bistable sensor is off, resulting in a so-called "signal-on" detection mode ( Figure 7C and 7D ). For embodiments, such as Figure 7E those illustrated in

[0109] , the measured optical signal decreases when the bistable sensor is off, resulting in a so-called "signal-off" detection ( ).

[0109] In other embodiments read out on an optical surface, the bistable sensor is immobilized on other types of microfabricated optical devices using the well-known DNA origami placement technique. In some embodiments, the bistable sensor with a light emitter is placed at the center of a metallic (e.g., gold) optical bowtie antenna. The strong electric field at the center of such bowtie antennas is known to enhance the fluorescence of the light emitter (as described in Kinkhabwala et al., "Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna", Nature Photonics 3 (2009): 654-657). Thus, in such embodiments, the off state of the bistable sensor exhibits enhanced light emission, resulting in a system with "signal-on" behavior. For such embodiments, for maximum optical signal, the position of the lid in the off state must be within a few nanometers of the bowtie center.

[0110] In other embodiments read out on an optical surface, the bistable sensor is immobilized within a photonic crystal cavity (PCC) using the well-known DNA origami placement technique. As previously described (Gopinath et al., "Engineering and mapping nanocavity emission via precision placement of DNA origami", Nature 535 (2016): 401-405), the interaction of emitters on the DNA origami with the PCC strongly depends on the nanoscale setting relative to the nodes within the PCC resonance mode. At some positions, as can be accurately predicted by finite-difference time-domain (FDTD) analysis, the coupling between the emitter and the cavity can be weak, and at other positions it can be strong. For a bistable device properly placed at a peak within the PCC optical resonance mode, the optical signal is enhanced when the bistable device is off, resulting in a system with "signal-on" behavior.

[0111] For some embodiments of surface-based optical detection, readout of the bistable sensor is achieved by measuring polarization in an epi-fluorescence microscope. For such embodiments, anisotropic gold rods are used as optical reporters on the lid of the origami. Thus, when the bistable sensor is off and the lid is bound, the gold rods transition from a freely rotating state to being fixed in a specific orientation. This change in the rotational diffusion of the gold rods is easily detected with an epi-fluorescence microscope by examining light scattered from the rods with two different polarizations and calculating the ratio between them. A ratio close to 1 indicates that the bistable sensor is in the on state, and a ratio far from 1 indicates that the bistable sensor is in the off state. Embodiments using linear polarization include a single anisotropic nanorod on the lid of the origami. Embodiments using circular polarization include a pair of nanorods, one on the lid of the origami and one on the bottom lid of the origami. A dual-nanorod system using circular polarization has been described (Zhou et al., “A plasmonic nanorod that walks on DNA origami,” Nature Communications 6 (2015): 8102).

[0112] In some embodiments, the detection mechanism can be a potentially label-free optical technique such as surface plasmon resonance (SPR) or reflectometric interference spectroscopy (RI). The general principles of SPR have been previously described in Tiang et al., “Surface Plasmon Resonance: An Introduction to a Surface Spectroscopy Technique,” Journal of Chemical Education 87 (2010): 742–746, the entire content of which is incorporated herein by reference. The general principles behind RI have been previously described in Kussrow et al., “Interferometric Methods for Label-Free Molecular Interaction Studies,” Analytical Chemistry 84 (2012): 779–792, the entire content of which is incorporated herein by reference.

[0113] For some embodiments where the detection method is SPR or RI, the top lid of the bistable detector is unlabeled, and it is the movement of the substance of the detector top lid, from free diffusion in the open state to surface binding in the closed state, which results in a change in the refractive index near the surface. Here, the amplification of each binding event achieved by the bistable detector depends on the molecular weight of the analyte to be measured relative to the polynucleotide lid. For a small molecule analyte with a molecular weight of 500 relative to a 5-megadalton lid, the amplification factor is as high as 10,000. For a 50kD protein analyte or a 150kD antibody, the amplification factor for each binding event ranges from 30 to 100 times.

[0114] In some other embodiments where the detection method is SPR or RI, optically active particles (such as gold particles or silica particles) can be attached to the top lid of the origami. In such embodiments, the optically active particles provide a greater refractive index change and greater amplification compared to what can be achieved with a top lid constructed entirely of DNA.

[0115] Embodiments of the present invention provide advantages over prior bistable molecular detectors. One advantage of the currently disclosed structure is that, as disclosed herein, the structure includes a well-defined shape that confers increased sensitivity to surface-based optical and electronic detection methods. The fully flexible bistable detector works well when the actuation detection method is gel electrophoresis and a large gel displacement is observed between the open and closed states. However, the fully flexible bistable detector is not suitable for the optical or electronic detection methods described herein, where the ability to control the geometry of the polynucleotide shape enables high signal amplification, typically 200-fold amplification for each binding event.

[0116] For the electronic detection method, it is important that the tethered shape moving from the solution to the surface upon actuation has a geometry that brings a sufficient portion of the shape mass (e.g., up to 5 megadaltons) close to the surface (in label-free field-effect biosensing) or a sufficient number (e.g., up to at least 200 methylene blue labels) of electroactive molecules (in electrochemical sensing) within a surface of only a few nanometers. The currently disclosed bistable detector cannot confine a sufficient mass or a sufficient number of electroactive molecules within a 2nm surface layer. The ability to reach the 2nm surface layer is achieved through the rigidity of the polynucleotide shape and their ability to present a specific geometry (such that it aligns with the window formed in the fixed shape as in Figure 8B or the arm as in Figure 8C or the dome as in Figure 8D (which can extend beyond the region of the fixed shape)). This is necessary in cases where the height of the functional molecule combination used for detection exceeds 2nm. For example, a combination of two 12-nanometer antibodies plus a protein antigen (e.g., with a diameter of 1 to 6 nanometers) results in a stack with a height of 24 to 30 nanometers.

[0117] Similarly, for optical detection methods (such as TIRF, SPR, or RI), using a fully flexible bistable detector does not maximize the number of fluorophores or amount of material within the critical distance of the technology to achieve high signal amplification. For technologies that rely on fluorophores or other emitters (such as TIRF), the bistable detectors described herein can bring at least 200 emitter tags to the critical distance from the surface, where a fully flexible detector brings at most a few emitters. For optical technologies that rely on bringing a large number of unlabeled molecules to the surface to create a refractive index change (SPR or reflectometric interference spectroscopy), the bistable detectors described herein can bring at least 5 megadaltons to the surface, where a fully flexible detector can bring at most a few hundred kilodaltons to the surface (e.g., the molecular weight of an antibody is 150 kilodaltons).

[0118] Embodiments of the surface-based readout of the bistable sensor can produce analog or digital signals. In some embodiments, optical or electronic measurements are made over a larger area that includes a large number of biosensors, and thus such measurements provide the sum of the signals of a large number of sensors. In such embodiments, the behavior of individual biosensors is averaged, and the readout is effectively analog.

[0119] However, in some embodiments, the discrete nature of the bistable sensor and the signal amplification potentially provided by the polynucleotide lid and the large number of signaling molecules enable the measurement of discrete single-molecule events. In some embodiments, such single-molecule measurements are also enabled by the ability to position individual bistable sensors into a grid using DNA origami placement. In such embodiments, the readout is effectively digital, as depicted by the time / signal traces in Figure 7B 、 Figure 7E and Figure 10 Some optical embodiments enable the simultaneous digital measurement of thousands of bistable sensors, such as over an entire microscope field in the context of TIRF microscopy, as commonly used in single-molecule biophysics. Some electronic embodiments can achieve digital single-molecule electronic measurements, such as in the case of using DNA origami placement to position a bistable sensor between two electrodes to utilize single-molecule redox cycling (as described in Lemay, "Single-Molecule Electrochemistry: Present Status and Outlook" Acc. Chem. Res. 46 (2013): 369-377).

[0120] Embodiments that achieve single-molecule digital measurements of the bistable sensor are capable of observing fluctuations in the bistable sensor state, as early shown in Figure 7B 、 Figure 7E andFigure 10 as depicted in the time / signal trace in Figure 10 . The bistable sensor in the open state toggles between the case where the top lid is away from the surface and the case where the top lid is close to the surface. Depending on the length of the linker and the diffusion constant of the top lid, this toggling has a characteristic time constant T1, which determines the transition of the signal trace between the "on" and "off" states. When detecting molecular events, whether it is a binding event or other events (such as modification), the top lid and the bottom lid of the bistable sensor have at least a greater affinity for each other, so that the toggling of the bistable sensor has a different characteristic time constant T2, where T2 is greater than T1.

[0121] The greater the difference between T2 and T1, the easier the molecular event can be detected. In the limit where the dissociation rate of the top lid due to the molecular event is negligible (because of its extremely high affinity for the bottom lid), the off state is stable and irreversible. In this limit, the binding event or other molecules cause a continuous change in the time / signal trace of a single molecule measurement, as depicted later in Figure 7B , Figure 7E and Figure 10 as depicted. For clarity, this limit of strong binding and irreversible change of the bistable sensor is illustrated, but it applies to many embodiments. In many embodiments, the binding of the target molecule within the sensor or the modification of the functional molecule does not result in irreversible changes, the time / signal trace changes its toggling rate, and the detection of the molecular event has to be inferred from this rate change.

[0122] Different embodiments of the bistable sensor employ lids with different shapes ( Figures 8A - 8D ), as determined by the requirements to maximize the performance of a particular surface-based readout mechanism and the characteristics of the functional molecules used (such as antibodies, aptamers). Among many possible geometries, Figure 8A four are illustrated in Figure 8A : a simple form where the bottom lid and the top lid have the same shape; ( Figure 8B ) a form where the bottom lid has a window such that the signal-conducting molecule on the top lid can contact the surface, thus ensuring maximum signal when forming a stable "off" state; Origami with such a window has been previously described (Rothemund, Paul WK. "Folding DNA to create nanoscale shapes and patterns", Nature 440.7082 (2006): 297 and patent application 161284 / CIT-7845); ( Figure 8C ) a form where the top lid is designed to be asymmetric, having rigid arms that ensure tight and stable contact of the top lid and any signal molecules it carries with the substrate; ( Figure 8D)A form in which the top lid is designed as a 3D hemisphere or dome, the radius of its edge extending beyond the radius of the bottom lid, which both ensures a tight contact of the top lid and signal molecules with the surface and further allows the bistable sensor to accommodate large-sized binding molecules (e.g., two antibody pairs with large antigens). DNA with such a hemisphere or dome shape has been previously described (Han et al., “DNA origami with complex curvatures in three-dimensional space”, Science 332 (2011): 342-346).

[0123] The performance as a function of the geometry of the bistable device depends on the specific implementation. Figure 8A The geometry illustrated in Figure 7A ) is suitable for implementations that utilize TIRF microscopy ( Figure 7D ), for which the luminescent or scattering signal molecules do not need to be very close to the surface to produce a large signal. In such implementations, strong signals can be observed for a top lid to surface distance of λ / 10, where λ is the wavelength of the light used. Thus, for green light with λ equal to 532 nm, a strong signal is obtained within 50 nm of the surface, where the top lid is within the strong part of the evanescent field. For a top lid to surface distance of λ / 10, the coupling of the luminophore on the top lid of the bistable device to the microfabricated optical cavity ( Figure 8A ) is also strong. Thus, implementations that use a microfabricated cavity to enhance optical detection by scattering or fluorescence can achieve high performance using the simple geometry illustrated in

[0124] Maximum quenching on metals is typically observed within 2 nanometers of the surface ( Figure 7C ), maximum interference with the gate capacitance ( Figure 6A and Figure 6B ), and maximum electron transfer rate in an electrochemical environment ( Figure 6C ). Thus, implementations that use quenching-based optical sensing ( Figure 7C ), as well as implementations that use field-effect sensing ( Figure 6A and Figure 6B ), and implementations that use electrochemical sensing ( Figure 6C ) can all benefit from a device geometry that can bring the top lid and the signal molecules it can carry into closer contact with the surface, such as the bistable device geometry illustrated in Figure 8B , Figure 8C and Figure 8D .

[0125] The performance of the surface flytrap is limited by many potential problems that do not exist in solution. In different embodiments, these problems are addressed by adjusting the surface chemistry of the substrate and the different components of the bistable sensor, as shown in FIG. 9.

[0126] For example, one lid of the flytrap must be fixed to the surface (bottom lid), and the other (top lid) must float freely in solution. If the top lid has too high an affinity for the surface, it sticks next to the bottom lid, and it is possible that the flytrap binds and detects the target molecule (false positive). Such problems occur on unpatterned surfaces, as well as on surfaces patterned with DNA origami binding sites ( Figure 9A ). Empty sites, double binding, and trapped closed sensors are all problems that can result from improper adhesion of the bistable sensor to the surface.

[0127] The ability to control the adhesion of the origami to the surface has been best developed on silicon nitride and silica substrates (such as Kershner et al., "Placement and orientation of individual DNA shapes on lithographically patterned surfaces", Nature Nanotechnology 4(2009):557–561; Hung et al., "Large-area spatially ordered arrays of gold nanoparticles directed by lithographically confined DNA origami", Nature Nanotechnology 5(2010):121-126; Gopinath et al., "Optimized Assembly and Covalent Coupling of Single-Molecule DNA Origami Nanoarrays", ACS Nano 8(2014):12030–12040; and Gopinath et al., "Engineering and mapping nanocavity emission via precision placement of DNA origami", Nature 535(2016):401-405), the entire contents of all of which are incorporated herein. Figure 9CIllustrated is an embodiment for properly attaching a flytrap substrate having a suitable surface oxide (such as silica, quartz, and silicon nitride). In a side view, the flytrap has five different regions that must have appropriate stickiness or non-stickiness to the surface for the flytrap to be properly oriented: the two surfaces of the top lid must not stick to the negatively charged silanol / carboxysilane binding sites or the surrounding trimethylsilyl background (produced by hexamethyldisilazane [HMDS] vapor deposition), the joint between the lids must not stick to the binding sites or the background, one surface of the bottom lid must not stick, and one surface of the bottom lid must stick to the binding sites. Under common experimental conditions (with 10 mM Mg 2+ ions), a flat disc-shaped origami strongly adheres to the binding sites because a layer of Mg 2+ ions provides a bridge between the negatively charged surface sites and the negatively charged origami surface. On the other hand, linear double-stranded DNA (such as some embodiments of the linker) does not, as shown experimentally by their movement under atomic force microscopy. Other work (as described in patent application 161284 / CIT-7845) teaches how to make one side of the DNA origami non-sticky to the negatively charged binding sites by adding a layer of 20-mer poly-T single-stranded DNA hair. This modification is highly effective for silica: when depositing an origami with a flat side and a hairy side, more than 98% of the origami binds with the flat side facing the surface. Thus, in some embodiments, three faces of the flytrap disc can be functionalized with DNA hair to provide proper orientation.

[0128] For some embodiments of using DNA origami placement, specific surface treatments and specific solution conditions are used to adhere the bottom lid of the flytrap to the surface, as taught by Gopinath et al ACS Nano 8 supra vida, and Gopinathet al Nature 535 above. For quartz, silica with a natural or thermal oxide coating, silicon nitride, indium oxide, or any surface where negatively charged groups can be introduced to the surface by oxygen plasma treatment, positively charged divalent magnesium ions can be used to form an adhesion bridge between the negatively charged surface groups and the negatively charged bottom lid of the flytrap detector. In embodiments where the negative surface groups are ionized silanols, a magnesium concentration of 30 to 40 millimoles of magnesium can be used. In some embodiments, the surface is silanized by introducing carboxysilane treated with a negatively charged carboxylic acid. Similarly, magnesium ions can be used to form an adhesion bridge between the negatively charged surface groups and the negatively charged bottom lid of the flytrap detector. In such embodiments where the negative surface groups are carboxylic acids, a magnesium concentration of less than 5 millimoles can be used.

[0129] In some embodiments, on different substrate materials, other solutions may be needed to prevent the problem of the top lid sticking to the background. The bottom lid constrains the top lid to be permanently adjacent to the surface through a joint. This gives a higher local concentration of the top lid, which changes the balance of weak interactions and / or may allow sufficient time for alternative binding mechanisms to occur. For some embodiments in which the top lid of the flytrap sticks to the surface, the stickiness of the top lid can be reduced by changing its shape and reducing its surface area (e.g., by implementing them as 6-helix bundles, as in Figure 9B ). This method has a significant effect on mica, where 6-helix bundles and other 3D origamis are less likely to adhere to the surface than flat origamis with a higher surface area. For some embodiments in which electronic sensing is used, this solution may come at the cost of reduced sensitivity, as it reduces the mass of the origami and / or the number of signal-conducting molecules close to the sensor surface.

[0130] For some embodiments that employ gold electrodes ( Figure 9C ), it is possible to closely mimic the silica system, in which the adhesion force can be regulated by the Mg 2+ concentration. Ultra-flat template-stripped gold with an RMS roughness of 3.6 Å (comparable to a silica wafer) can be used as the substrate. To generate negatively charged binding sites similar to those available on silica, carboxylated thiols (e.g., 11-mercaptoundecanoic acid) are used to create self-assembled monolayers. Such monolayers have previously been used to adhere origami to gold in the presence of Mg 2+ ions, as described previously (Gerdon et al., "Controlled Delivery of DNA Origami on Patterned Surfaces", Small 5(2009):1942-1946). The non-adhesive background can be implemented using a selected alkanethiol to obtain a self-assembled monolayer with a contact angle similar to that generated on silica by HMDS.

[0131] Some other embodiments using gold electrodes use polyadenosine (polyA) strand extensions, thiol labeling, or phosphorothioate backbones on DNA origami to provide adhesion of the flytrap bottom lid to gold. DNA adhesion based on thiols, phosphorothioates, and polyadenosine strands has been previously described (Zhou et al., “Tandem phosphorothioate modifications for DNA adsorption strength and polarity control on gold nanoparticles.” ACS Applied Materials Interfaces 6 (2014): 14795-147800). Anti-adhesion between the top lid and the background surface can be provided by using polyethylene glycol or dextran modifications of the top lid, and polyethylene glycol-thiol modifications of the background on the gold substrate.

[0132] For some embodiments employing a graphene FET surface, a silica system can be mimicked to provide Mg 2+ -driven adhesion ( Figure 9C ). For some such embodiments, carboxylic acid-modified graphene can non-specifically bind DNA origami, and PEG can be added to the non-adhesive flytrap surface.

[0133] However, since double-stranded DNA does not strongly adhere to the graphene surface, and the exposed hydrophobic bases of single-stranded DNA do strongly adhere to graphene, other options for managing adhesion on graphene are opened. Thus, in some embodiments, unpatterned, unmodified graphene can be used. In such embodiments, single-stranded DNA (e.g., poly thymine [polyT]) can be added to the adhesive flytrap surface on the bottom lid ( Figure 9C ); in such embodiments, the adhesion of the other surfaces of the flytrap to the unmodified graphene is very low. A similar single-stranded linker strategy has been used to attach carbon nanotubes to DNA origami (Maune et al., "Self-assembly of carbon nanotubes into two-dimensional geometries using DNA origami templates" Nature Nanotechnology (2010) 61-66) to form field effect transistors.

[0134] For surface-based embodiments, multiplexing of different bistable sensors can be achieved by independently synthesizing sensors that are specific for different target molecules or sensitive to different functions in a single test tube and spatially localizing the different sensors into an array onto a surface suitable for optical or electrical detection. Spatial localization can be achieved at the microscale using a variety of techniques, including inkjet printing and microarray printing (as described in Barbulovic-Nad et al., "Bio-microarray fabrication techniques--a review." Critical Reviews in Biotechnology. 26(2006):237-59). Thus, for surface-based embodiments, microscale spotting produces arrays suitable for analog measurements of the aggregate bistable device behavior, where each spot contains multiple randomly arranged bistable devices; in some embodiments, each spot contains at least 10 bistable devices.

[0135] For surface-based embodiments, single-molecule arrays suitable for single-molecule optical or electronic detection can be lithographically constructed using DNA origami placement techniques, as described in the above references. Construction of 65,536 optical devices (Gopinath et al., "Engineering and mapping nanocavity emission via precision placement of DNA origami", Nature 535(2016):401-405), where each device is a 5-micron x 5-micron area that contains a photonic crystal cavity, and where each device has a defined number of single DNA origamis set therein, where the numbers arranged programmatically from 0 to 7 are of particular relevance. Thus, in some embodiments ( Figure 10 ), microscale spotting can be combined with DNA origami placement to achieve a perfectly regular single-molecule single-bistable sensor array, where each of the N arrays is specific for a particular analyte, and within each of the N arrays, there are M binding sites for a single bistable device that exactly fills a bistable sensor, with a probability greater than 95%. Based on the number of single binding sites generated, some embodiments have arrays that contain the total number of spots N multiplied by M (equal to up to 100,000). Some embodiments have up to 1000 arrays, each with at least 10 binding sites for bistable devices.

[0136] Multiplexed electrical detection has been implemented for more than 4,000 CMOS electrochemical sensors (as described in Sun et al., "A scalable high-density electrochemical biosensor array for parallelized point-of-care diagnostics", 2015 IEEE Biomedical Circuits and Systems Conference, IEEE Journal of Solid-State Circuits 53 (2018) 2054-2064). Accordingly, some embodiments combine microarray spotting with an electronic device to implement a multiplexed array of up to 4,000 different types of bistable devices, where each spot is printed on a microelectronic device, each spot contains a plurality of randomly arranged bistable sensors, and the readout from each microelectronic device is the sum response of the plurality of bistable sensors; in some embodiments, each spot contains at least 10 bistable devices.

Claims

1. A structure of a bistable molecular sensor for optically or electronically detecting an external stimulant on the surface of a substrate, the bistable molecular sensor comprising a nucleic acid structure, comprising: A first polynucleotide shape and a second polynucleotide shape with a flexible hinge or a flexible linker therebetween, one of the first polynucleotide shape or the second polynucleotide shape being fixed on the surface of the substrate, resulting in a fixed polynucleotide shape and a tethered polynucleotide shape, the tethered polynucleotide shape comprising a first shape inner surface and a first shape outer surface and the fixed polynucleotide shape comprising a second shape inner surface and a second shape outer surface, wherein the tethered polynucleotide shape forms a rigid two-dimensional (2D) or rigid three-dimensional (3D) shape, wherein the second shape outer surface adheres to the surface of the substrate, and Wherein the inner surface of the first shape can face the inner surface of the second shape; and One or more functional molecules bound to at least one of the first polynucleotide shape and the second polynucleotide shape, The bistable molecular sensor has one of two states, the two states being a closed state and an open state, wherein: In the open state, the tethered polynucleotide shape moves freely relative to the second polynucleotide shape due to being constrained by the flexible hinge or the flexible linker; and In the closed state, the tethered polynucleotide shape is arranged to be adjacent to the fixed polynucleotide shape, wherein the one or more functional molecules further comprise: A first braking molecule; and A second braking molecule, wherein the braking mechanism of the bistable molecular sensor is configured such that a molecular event involving the first and second braking molecules moves the equilibrium conformation of the bistable molecular sensor towards the closed state.

2. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a first capture molecule and a second capture molecule, the first capture molecule being capable of binding to a different region of the target molecule with the second capture molecule, the first capture molecule and the second capture molecule being selected from a first antibody and a second antibody, a first nanobody and a second nanobody, or a first aptamer and a second aptamer, One or more copies of the first capture molecule are attached to the first polynucleotide shape, One or more copies of the second capture molecule are attached to the second polynucleotide shape, and In the presence of the target molecule, the first capture molecule and the second capture molecule bind to the target molecule, thereby moving the equilibrium conformation of the bistable molecular sensor towards the closed state.

3. The structure according to claim 1, wherein the one or more functional molecules bind to the first shape inner surface and / or the second shape inner surface.

4. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a first single-stranded nucleic acid and a second single-stranded nucleic acid, the first single-stranded nucleic acid and the second single-stranded nucleic acid being different from each other and complementary to a target single-stranded nucleic acid, One or more copies of the first single-stranded nucleic acid are attached to the first polynucleotide shape, One or more copies of the second single-stranded nucleic acid are attached to the second polynucleotide shape, and In the presence of the target single-stranded nucleic acid, the first single-stranded nucleic acid and the second single-stranded nucleic acid bind to the target single-stranded nucleic acid, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

5. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a first CRISPR-inactive enzyme guide RNA complex and a second CRISPR-inactive enzyme guide RNA complex, the first CRISPR-inactive enzyme guide RNA complex and the second CRISPR-inactive enzyme guide RNA complex are different from each other and complementary to the target double-stranded nucleic acid, One or more copies of the first CRISPR-inactive enzyme guide RNA complex are attached to the first polynucleotide shape, One or more copies of the second CRISPR-inactive enzyme guide RNA complex are attached to the second polynucleotide shape, and In the presence of the target double-stranded nucleic acid, the first CRISPR-inactive enzyme guide RNA complex and the second CRISPR-inactive enzyme guide RNA bind to the target double-stranded nucleic acid, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

6. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise an allosteric CRISPR-inactive enzyme guide RNA complex and a complementary allosteric nucleic acid sequence, the allosteric CRISPR-inactive enzyme guide RNA complex having a conditionally hidden allosteric nucleic acid sequence, The allosteric CRISPR-inactive enzyme guide RNA complex is capable of binding to the target double-stranded nucleic acid, thereby exposing the conditionally hidden allosteric nucleic acid sequence, One or more copies of the allosteric CRISPR-inactive enzyme guide RNA complex are attached to the first polynucleotide shape, One or more copies of the complementary allosteric nucleic acid sequence are attached to the second polynucleotide shape, and In the presence of the target double-stranded nucleic acid, the allosteric CRISPR-inactive enzyme guide RNA complex binds to the target double-stranded nucleic acid, and the complementary allosteric nucleic acid sequence binds to the exposed conditionally hidden allosteric nucleic acid sequence, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

7. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a capture molecule capable of binding to a target molecule and a competing molecule capable of binding to the capture molecule in the absence of the target molecule, the capture molecule being selected from an antibody, a nanobody or an aptamer, One or more copies of the competing molecule are attached to the first polynucleotide shape, and one or more copies of the capture molecule are attached to the second polynucleotide shape, In the absence of the target molecule, the competitor molecule binds to the capture molecule, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state, and in the presence of the target molecule, the competitor molecule is displaced by the target molecule, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the open state.

8. The structure according to claim 1, wherein: the first and second braking molecules of the one or more functional molecules comprise a first protein capable of being chemically or enzymatically modified by a chemical or enzymatic reagent to produce a modified first protein and a second protein capable of binding to the modified first protein, one or more copies of the first protein are attached to the first polynucleotide shape, one or more copies of the second protein are attached to the second polynucleotide shape, and the molecular event chemically or enzymatically modifies the first protein and enhances the affinity between the first and second proteins, such that the equilibrium conformation of the bistable molecular sensor is shifted towards the closed state.

9. The structure according to claim 8, wherein the first protein is capable of being modified by at least one of phosphorylation, acetylation, ubiquitination, isoprenylation, adenylation or glycosylation.

10. The structure according to claim 9, wherein the second protein is a naturally occurring protein capable of binding to the modified first protein.

11. The structure according to claim 9, wherein the second protein is an antibody capable of binding to phosphorylation, acetylation, ubiquitination, isoprenylation, adenylation or glycosylation.

12. The structure according to claim 1, wherein: the first and second braking molecules of the one or more functional molecules comprise a capture nucleic acid and a capture molecule, the capture nucleic acid being capable of being chemically or enzymatically modified by a chemical or enzymatic reagent to produce a modified capture nucleic acid, the capture molecule being capable of binding to the modified capture nucleic acid, one or more copies of the capture nucleic acid are attached to the first polynucleotide shape, one or more copies of the capture molecule are attached to the second polynucleotide shape, and in the presence of the chemical or enzymatic reagent, the capture nucleic acid is modified to produce a modified capture nucleic acid, and the capture molecule binds to the modified capture nucleic acid, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

13. The structure according to claim 12, wherein the capture nucleic acid is capable of being modified by at least one of cytosine methylation, cytosine hydroxymethylation, cytosine formylation, cytosine carboxylation, adenine methylation, alkylation or thymine dimerization.

14. The structure according to claim 13, wherein the capture molecule is a naturally occurring molecule capable of binding to the capture nucleic acid.

15. The structure according to claim 12, wherein the capture molecule is an antibody capable of binding to the modified capture nucleic acid.

16. The structure according to claim 12, wherein the capture molecule is an antibody capable of binding cytosine methylation, cytosine hydroxymethylation, cytosine formylation, cytosine carboxylation, adenosine methylation, alkylation, or thymine dimerization.

17. The structure according to claim 1, wherein: the first and second braking molecules of the one or more functional molecules comprise a first protein and a second protein, the first protein being a transmembrane receptor protein capable of binding at least one type of ligand, and when the transmembrane receptor protein is bound by the at least one type of ligand, the second protein is capable of binding the first protein, one or more copies of the first protein are attached to the first polynucleotide shape, one or more copies of the second protein are attached to the second polynucleotide shape, and in the presence of the at least one type of ligand, the second protein binds to the first protein, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

18. The structure according to claim 17, wherein the one or more copies of the first protein attached to the first polynucleotide shape are attached by: a direct linker molecule between the first protein and the first polynucleotide shape, inserting the first protein into a protein-lipid nanodisc capable of attaching to the first polynucleotide shape, inserting the first protein into a DNA-lipid nanodisc capable of attaching to the first polynucleotide shape, or inserting the first protein into a DNA-lipid nanodisc formed as part of the first polynucleotide shape.

19. The structure according to claim 17, wherein: the bistable molecular sensor further comprises a G-protein receptor kinase (GRK), the G-protein receptor kinase being in solution or attached to the nucleic acid structure, the first protein comprises a G-protein coupled receptor (GPCR), and the second protein comprises β-arrestin or an antibody capable of binding phosphorylated GPCR, and in the presence of at least one type of receptor ligand of the GPCR, the GPCR is phosphorylated by GRK, and thus β-arrestin or the antibody binds to the phosphorylated GPCR, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

20. The structure according to claim 17, wherein: the first protein comprises a G-protein coupled receptor (GPCR), the second protein is an antibody, nanobody, or aptamer, and in the presence of a GPCR ligand, the second protein binds to the first protein, thereby bringing the bistable molecular sensor into the closed state.

21. A method for determining a receptor ligand that binds to a transmembrane receptor, the method comprising: providing a candidate receptor ligand to the structure according to claim 17, wherein the surface is a chip.

22. The method according to claim 21, wherein the transmembrane receptor is a G-protein coupled receptor (GPCR).

23. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a first molecule and a second molecule, the first molecule comprising a DNA riboswitch or an RNA riboswitch capable of binding a riboswitch ligand, the binding of the riboswitch ligand inducing the exposure of a nucleotide sequence or an aptamer, and the second molecule comprising a DNA sequence, an RNA sequence, or a protein capable of binding the nucleotide sequence or aptamer exposed on the DNA riboswitch or RNA riboswitch. One or more copies of the first molecule are attached to the first polynucleotide shape. One or more copies of the second molecule are attached to the second polynucleotide shape, and in the presence of the riboswitch ligand, the second molecule binds to the first molecule, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

24. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a capture molecule capable of being modified by a chemical reagent or an enzyme reagent to form a modified capture molecule, the capture molecule capable of binding the first polynucleotide shape and the second polynucleotide shape, the modified capture molecule not being capable of binding the first polynucleotide shape and the second polynucleotide shape, the capture molecule being selected from a protein or a nucleic acid, and one or more copies of the capture molecule are attached to the first polynucleotide shape and the second polynucleotide shape, and in the presence of a chemical reagent or an enzyme reagent, the capture molecule is modified, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the open state.

25. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a first molecule and a second molecule, at least one of the first molecule and the second molecule being capable of being modified by a chemical reagent or an enzyme reagent, resulting in the binding of the first molecule to the second molecule, the first molecule and the second molecule being selected from a nucleic acid or a protein, one or more copies of the first molecule are attached to a first polynucleotide shape, one or more copies of the second molecule are attached to a second polynucleotide shape, and in the presence of a chemical reagent or an enzyme reagent, the first molecule and the second molecule bind together, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

26. The structure according to claim 1, wherein: The first and second braking molecules of the one or more functional molecules comprise a capture molecule and a probe molecule, the capture molecule being capable of being modified by one of temperature, light, pH, or ionic conditions to produce a modified capture molecule, the probe molecule being capable of binding the modified capture molecule, the capture molecule and the probe molecule each independently being a nucleic acid or a protein, one or more copies of the capture molecule are attached to the first polynucleotide shape, one or more copies of the probe molecule are attached to the second polynucleotide shape, and In the presence of one of the temperature, light, pH, or ionic conditions, the capture molecule is modified, and the probe molecule binds to the modified capture molecule, thereby shifting the equilibrium conformation of the bistable molecular sensor towards the closed state.

27. The structure according to claim 1, for optical detection, wherein: The surface is gold or graphene, The tethered polynucleotide shape includes a luminescent body selected from organic fluorophores, quantum dots, fluorescent beads, or luminescent lanthanide compounds, and The open state produces more light than the closed state.

28. A method for optically detecting an external stimulant, comprising: Determining an external stimulant using the structure according to claim 1, wherein: The surface is gold or graphene, The tethered polynucleotide shape includes a luminescent body selected from organic fluorophores, quantum dots, fluorescent beads, or luminescent lanthanide compounds, and The structure is located in a microfabricated device capable of enhancing the light generated by the luminescent body.

29. The method according to claim 28, wherein the microfabricated device is selected from a photonic crystal cavity, a ring resonator, or an optical bowtie.

30. The structure according to claim 1, for optical detection using total internal reflection (TIRF) microscopy, wherein: The surface is transparent, and The tethered polynucleotide shape is fluorescently labeled, luminescently labeled, or labeled with light-scattering particles.

31. The structure according to claim 1, for optical detection using surface plasmon resonance (SPR), wherein: The surface is gold, and The tethered polynucleotide shape is unlabeled or labeled with optically active particles.

32. The structure according to claim 1, for optical detection using surface reflection interferometry (RI), wherein: The surface is transparent or opaque, and The tethered polynucleotide shape is unlabeled or labeled with optically active particles.

33. The structure according to claim 1, further comprising a substrate comprising one or more bistable molecular sensors, wherein the substrate is the surface of each of the one or more bistable molecular sensors.

34. The structure according to claim 33, wherein the one or more bistable molecular sensors are disposed on the substrate by directed self-assembly or lithography.

35. The structure according to claim 34, wherein when the one or more bistable molecular sensors are lithographically disposed on the substrate, the substrate includes lithographically patterned binding sites that are adhesive for immobilizing polynucleotide shapes and non-adhesive for tethered polynucleotide shapes.

36. The structure according to claim 1, for electrical detection, wherein: The surface is a working electrode, comprising: gold, platinum, graphene, indium oxide, or indium tin oxide, The tethered polynucleotide shape is labeled with one or more redox-active molecules, and A change in state results in electron transfer between the one or more redox-active molecules and the working electrode.

37. The structure according to claim 36, wherein the one or more redox-active molecules are selected from methylene blue, ferrocene, 1,3-diaza-2-oxophenothiazine, or tricyclic cytosine analogs.

38. The structure according to claim 36, for electrical detection by square wave voltammetry, the structure further comprising a silver / silver chloride reference electrode and a platinum wire counter electrode disposed above the surface, wherein: the surface of the working electrode is a gold surface, the gold being electron beam deposited or template-stripped gold, the one or more redox-active molecules are methylene blue, and the positions on the surface where the fixed polynucleotide shapes are attached are coated with an alkanethiol self-assembled monolayer.

39. The structure according to claim 38, wherein the fixed polynucleotide shape comprises a thiol modification for attachment to the gold surface.

40. The structure according to claim 38, wherein the fixed polynucleotide shape comprises a single-stranded polyadenosine chain for attachment to the gold surface.

41. The structure according to claim 38, wherein the fixed polynucleotide shape comprises a thiophosphate modification for attachment to the gold surface.

42. The structure according to claim 38, wherein the tethered polynucleotide shape comprises a polyethylene glycol modification to inhibit attachment to the gold surface.

43. The structure according to claim 38, wherein the tethered polynucleotide shape comprises a dextran modification to inhibit attachment to the gold surface.

44. The structure according to claim 38, wherein the gold surface comprises thiolated polyethylene glycol molecules.

45. The structure according to claim 36, wherein: the tethered polynucleotide shape forms a rigid two-dimensional (2D) plate, the one or more redox-active molecules are distributed on the tethered polynucleotide shape, and the fixed polynucleotide shape is selectively disposed between the surface and one of the one or more redox-active molecules.

46. The structure according to claim 36, wherein: the tethered polynucleotide shape forms a rigid 2D plate, the fixed polynucleotide shape forms a plate with a window or pore that provides a direct channel to the surface, and in the closed state, the tethered polynucleotide shape having the one or more redox-active molecules thereon is disposed above the window or pore.

47. The structure according to claim 36, wherein: the tethered polynucleotide shape forms a rigid three-dimensional (3D) shape with rigid arms, wherein the one or more redox-active molecules are attached to the ends of the rigid arms.

48. The structure according to claim 36, wherein: the tethered polynucleotide shape forms a rigid hemisphere or rigid dome, wherein the one or more redox-active molecules are attached along the peripheral edge of the rigid hemisphere or rigid dome, and in the closed state, the redox-active molecules on the peripheral edge are disposed adjacent to the fixed polynucleotide shape.

49. The structure according to claim 1, for field effect sensing, the structure further comprising a solution above the surface and a working solution electrode, wherein: The surface function is a transistor, The surface is a gate material selected from carbon nanotubes, silicon nanowires, graphene, molybdenum disulfide, or indium oxide, The immobilized polynucleotide shape is directly attached to the surface, and The solution function above the surface is the gate electrode of the transistor.

50. The structure according to claim 49, wherein the surface is graphene and the immobilized polynucleotide shape is attached to the graphene by a single-stranded DNA extension.

51. The structure according to claim 49, further comprising magnesium ions, wherein: The surface is graphene coated with pyrene carboxylic acid, and The immobilized polynucleotide shape is attached to the coated graphene surface by electrostatic interaction between the magnesium ions and the pyrene carboxylic acid.

52. The structure according to claim 49, wherein: The surface is graphene coated with polylysine, and The immobilized polynucleotide shape is attached to the surface by electrostatic interaction.

53. The structure according to claim 49, wherein: The surface is graphene, and The tethered polynucleotide shape comprises polyethylene glycol.

54. The structure according to claim 49, wherein: The surface is graphene, and The tethered polynucleotide shape comprises a polylysine-grafted-polyethylene glycol polymer.

55. The structure according to claim 49, further comprising magnesium ions, wherein: The surface is indium oxide treated with oxygen plasma or indium oxide coated with carboxysilane, and The magnesium ions bridge the immobilized polynucleotide shape to the surface.

56. The structure according to claim 49, wherein: The surface is indium oxide, and The tethered polynucleotide shape comprises trimethylsilyl and / or polyethylene glycol (PEG) silane.

57. The structure according to claim 49, wherein: The tethered polynucleotide shape forms a rigid 2D plate capable of maintaining its position in the closed state.

58. The structure according to claim 57, wherein: The immobilized polynucleotide shape is a plate with a window or a hole, and In the closed state, the window or hole in the immobilized polynucleotide shape creates a space between the surface and the tethered polynucleotide shape without any of the immobilized polynucleotide shape therebetween.

59. The structure according to claim 49, wherein: The tethered polynucleotide shape forms a rigid three-dimensional (3D) shape with rigid arms, and In the closed state, the rigid arms are disposed above the surface without any of the immobilized polynucleotide shape therebetween.

60. The structure according to claim 49, wherein: The tethered polynucleotide shape forms a rigid hemisphere or a rigid dome with a peripheral edge, and In the closed state, the peripheral edge is disposed adjacent to the surface and at the periphery of the immobilized polynucleotide shape without any of the immobilized polynucleotide shape between the peripheral edge and the surface.

61. The structure according to claim 1, for field-effect sensing, the structure further comprises a solution above the surface and a working solution electrode, wherein: The surface function is a transistor, and the surface includes a semiconductor gate under a capping layer selected from silicon dioxide, aluminum oxide, or silicon nitride. The immobilized polynucleotide shape is attached to the capping layer, and The solution function above the surface is the gate electrode of the transistor.

62. The structure according to claim 61, further comprising magnesium ions, wherein: The capping layer is treated by oxygen plasma or coated with carboxyl silane, and The magnesium ions bridge the immobilized polynucleotide shape to the capping layer.

63. The structure according to claim 61, wherein the tethered polynucleotide shape comprises trimethylsilyl and / or polyethylene glycol (PEG) silane.

64. The structure according to claim 61, wherein the tethered polynucleotide shape forms a rigid 2D plate capable of maintaining its position in the closed state.

65. The structure according to claim 61, wherein: The immobilized polynucleotide shape is a plate with a window or a pore, and In the closed state, the window or pore in the immobilized polynucleotide shape creates a space between the capping layer and the tethered polynucleotide shape, with no intervening immobilized polynucleotide shape.

66. The structure according to claim 61, wherein: The tethered polynucleotide shape forms a rigid three-dimensional (3D) shape with rigid arms, and In the closed state, the rigid arms are disposed above the capping layer, with no intervening immobilized polynucleotide shape.

67. The structure according to claim 61, wherein: The tethered polynucleotide shape forms a rigid hemisphere or a rigid dome with a peripheral edge, and In the closed state, the peripheral edge is set adjacent to the capping layer and at the periphery of the immobilized polynucleotide shape, with no intervening immobilized polynucleotide shape between the peripheral edge and the capping layer.

68. A detection system for optical detection, the detection system comprising: Multiple structures according to claim 1, the multiple structures comprising up to 1,000 different bistable molecular sensors, each bistable molecular sensor capable of detecting a different external stimulant or an analyte that interacts with an external stimulant, Each of the multiple structures disposed on one of up to 1,000 corresponding different regions on a substrate surface using inkjet printing or microarray printing, wherein multiple copies of each different bistable molecular sensor are disposed at each corresponding different region on the substrate.

69. The detection system according to claim 68, wherein the up to 1,000 corresponding different regions are lithographically patterned, and each corresponding different region has multiple single-molecule binding sites for nucleic acid origami placement.

70. The detection system according to claim 69, wherein each of the multiple single-molecule binding sites comprises no more than one bistable sensor.

71. The detection system according to claim 68, capable of detecting an external stimulant by one of the following: Total internal reflection spectroscopy on a transparent substrate, Quenched fluorescence on a gold surface, Quenched fluorescence on a graphene substrate, or Enhanced fluorescence using an optical bowtie.

72. A detection system for electrical detection, the detection system comprising: a plurality of structures according to claim 1, the plurality comprising up to 4,000 different bistable molecular sensors, each bistable molecular sensor being capable of detecting a different external stimulant or an analyte that interacts with an external stimulant, each of the plurality of structures disposed on one of up to 4,000 corresponding different regions on a substrate surface using inkjet printing or microarray printing, wherein a plurality of copies of each different bistable molecular sensor are disposed at each corresponding different region on the substrate.

73. The detection system according to claim 72, wherein: the surface is selected from gold, graphene, platinum, graphene, indium oxide, molybdenum disulfide, carbon nanotubes, silicon nanowires, or silicon.

74. The structure according to claim 36, wherein: the DNA origami is placed for positioning the bistable molecular sensor at a position between electrodes spaced less than 100 nanometers apart, and single molecule measurements are obtained by redox cycling.

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