Colorimetric sensing using reconfigurable chiral plasmonic structures and method for detecting an analyte in a sample using the same - Patents.com

Abstract

The present invention relates to a nanoscale construct comprising or consisting essentially of a nucleic acid structure having a rearrangeable or switchable configuration and at least two distinct metal nanoparticles bound to the nucleic acid structure, the two distinct nanoparticles being at interchangeable angles to one another, the nanoparticles providing high chirality and / or optical activity within the visible and near infrared (NIR) spectrum from 400 to 800 nm which produces the color of the construct, the construct having an absorption asymmetry factor (g-factor) of greater than 10%. A method of detecting an analyte in a sample is also provided, the method comprising contacting a nanoscale construct of the present disclosure specific for the analyte with a sample in a test assay, and detecting the analyte in the sample by optical means or the naked eye, the presence of the analyte in the sample causes a color change of the nanoscale structure, and the color change in the assay confirms the presence of the analyte in the sample.

Description

[Technical field]

[0001] The present invention relates to reconfigurable structures that generate colorimetric readouts for colorimetric sensing in chemical and biological detection assays. [Background technology]

[0002] Vision-based sensing has become an increasingly popular research field because the sensing response can be performed in a simple test tube without external components and a central laboratory, an advantage that is desirable and especially essential for on-site point-of-care testing in remote locations and mobile testing points.

[0003] Vision-based sensing includes chemical-based and plasmon-based color. Conventional approaches to vision-based and chemical-based sensing rely on chemical reactions that result in changes in color absorption or transmission, fluorescence or emission. Typical examples include ELISA and PCR, which use chemical dyes and chemical fluorescent probes to generate visual color.

[0004] Noble metal nanoparticles (NPs) have been widely used to develop colorimetric sensing or detection schemes. In NP solutions, the presence of analytes results in a significant change in color and / or intensity, which can often be detected by the naked eye. One of the traditional approaches to vision-based plasmonic-based sensing in solutions is NP-mediated sensing, such as quantum dots, which provide high-intensity luminescence in nanoscale spots and significantly improve the fluorescence signal. The use of gold nanospheres (AuNSs) in analyte-induced AuNS aggregation assays results in a change in visual absorption color, such as the color representation of lines in lateral flow assays. Meanwhile, gold nanorods (AuNRs) exhibit polarization-dependent plasmonic color responses. Since the plasmonic coupling of AuNRs exhibits two orthogonal plasmonic resonances, controlled plasmonic coupling of NR dimers with precise spatial arrangements can modulate the aggregation orientation to enhance localized surface plasmon resonance (LSPR) signals for biosensing. In addition, the specific geometric shape of gold nanorods induces an extrinsic anisotropic property, i.e., chirality.

[0005] Rearrangeable chiral plasmonic structures have emerged as promising candidates for developing novel sensing schemes. The sensing or detection mechanism of these structures is based on the strong correlation between the spatial arrangement of the chiral plasmonic structures and their chiroptical response. The geometric control in NR dimers enables polarization-dependent plasmonic resonance, resulting in quantifiable changes in chirality, i.e., circular dichroism (CD) signals. These changes or signals can then be detected by CD spectroscopy. Such chiral plasmonic sensors have several unique advantages: (i) high sensitivity, (ii) strong modulation of the optical response to analytes, and (iii) reliable optical detection in environments with strong optical attenuation.

[0006] Their operation under biologically relevant conditions is very complicated. Autofluorescence occurs naturally in biological samples, resulting in false positive signals. Visualization of fluorescent signals requires more complex optical setups and fluorescence excitation paths to observe the fluorescent emission colors. Moreover, traditional chemical dyes or fluorophores have several drawbacks, e.g. low stability, quenching effects and autofluorescence.

[0007] Alternatively, nanomaterials can be used, but in analyte-induced aggregation plasmonic sensing, impurities in biological samples may cause undesired aggregation. The aggregation of NPs may be induced or inhibited by environmental fluctuations (pH value, temperature, non-specific binding of molecules, etc.) rather than the presence of the analyte. This leads to high false positive / false negative rates for colorimetric sensors.

[0008] For the measurement of chirality, i.e., CD signal, the readout scheme relies heavily on CD spectroscopy, which requires expensive and bulky equipment with advanced optical designs. This has limited the usefulness of chiral plasmonic sensors outside the laboratory. In addition, conventional chirality-based sensing methods using nanomaterials often suffer from small modulation of optical response, i.e., weak intensity and / or limited color change of the solution due to low chirality, making them unsuitable for reliable detection by the naked eye.

[0009] Our approach relies on nanoscale precision controlled structural rearrangement of nanoparticle assemblies rather than the poorly controlled aggregation process. Rearrangement is the key step for tunable chirality in this current chiral-based sensing system for analytical function. Compared to nanoparticle aggregation-based paper strip analysis, the impact of environmental variations is minimal. Our rearrangeable nanostructures also show better accuracy than nanoparticle aggregation analysis. In addition, the modulation of optical response in our system is strong enough to be judged by the naked eye, and the visual response can be tuned to the spectral region most suitable for naked eye detection by a simple optical setup using a polarizer. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Liu B, et al. J. Am. Chem. Soc. 2017, Vol. 139, No. 28, 9471-9474 [Non-Patent Document 2] Kuzyk A, et al. Nat Mater. 2014, Vol. 13, No. 9, 862-866 Summary of the Invention

[0011] The aim of the present invention is the development of a repositionable nanoscale construct with a record high anisotropy factor (g-factor) of up to 16% and its tunable chirality with anisotropy factor reaching the range of 0-16%. Such a high dynamic g-factor allows colorimetric detection discernible by the naked eye. Analyte-dependent modulation of the chiroptical response with changing colorimetric signal supports optical readout independent of CD spectroscopy. The repositionable nanoscale construct can be fabricated using DNA origami techniques and recognition elements for sensing can be added to the ensemble to detect the presence of target analytes. Dynamic control of the nanoscale construct allows visual color change for sensing. The angle of the nanoscale construct has a significant impact on the chirality. This angle is controlled by the length of the origami bridge sequence. Chirality increases when the angle changes from an open configuration, e.g. 90°, where the two origami bundles are perpendicular or nearly perpendicular to each other, to a closed configuration, e.g. 45°.

[0012] Dynamic configurations in response to analyte binding in biosensing can be obtained in rearrangeable plasmonic assemblies, i.e., nanoscale constructs, functioning as chiral plasmonic switches that exhibit dynamic response, i.e., switching between open and closed configurations, resulting in a significant change in chirality and thus in the colorimetric readout signal in terms of color and intensity. Colorimetric detection extends the utility of chiral plasmonic sensors beyond laboratory settings, making them suitable for diagnostic and sensing applications in point-of-care and / or remote settings.

[0013] Our approach allows for a wide variety of colors to be selected through tunable range of structural rearrangements and plasmonic colors within the visible wavelength spectrum through the selection of metal nanomaterials or nanoparticles. With the attachment of recognition elements, the rearrangeable or switchable configurations that tune the chirality exhibit responsive configurations in sensing, biosensing and analytical applications. Colorimetric chiral-based sensing exhibits significant advantages over existing colorimetric sensing approaches. A) Minimize the effects of environmental variation. B) The rate of false positives and false negatives is reduced. C) Direct modification of biorecognition elements to a broad range of targets. D) capable of operating in a physiologically relevant environment. E) The modulation of the optical response is strong enough to be observed by the naked eye. F) No sample purification or signal amplification is required, allowing rapid detection or sensing with conventional readout. G) There is the capability to perform reliable sensing or detection outside of the laboratory.

[0014] The invention is defined by the structure of the independent claims. Some particular embodiments are defined in the dependent claims.

[0015] According to a first aspect of the present invention, there is provided a nanoscale construct comprising or consisting essentially of a nucleic acid structure having a rearrangeable or switchable configuration and at least two separate metal nanoparticles associated with the nucleic acid structure, the two separate nanoparticles being at interchangeable angles to one another, the nanoparticles providing high chirality and / or optical activity within the visible and near infrared (NIR) spectrum of 400 to 800 nm which produces a colour for the construct, and the construct having an absorption asymmetry factor (g-factor) of greater than 10%.

[0016] According to a second aspect of the present invention, there is provided a method of detecting an analyte in a sample, the method comprising the steps of contacting a nanoscale structure of the present disclosure specific for the analyte with a sample in a test assay and detecting the analyte in the sample by optical means or with the naked eye, wherein the presence of the analyte in the sample causes a colour change of the nanoscale structure, and the colour change in the assay confirms the presence of the analyte in the sample.

[0017] According to a third aspect of the present invention, there is provided a use of the nanoscale constructs of the present disclosure for the detection of an analyte in a sample, wherein the presence of the analyte in the sample causes a colour change of the nanoscale structure when contacted with the sample, the colour change confirming the presence of the analyte in the sample. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing (a) the rearrangeable configuration of our nanoscale constructs during sample detection, and (b) a colorimetric detection scheme using our nanoscale constructs. [Diagram 2] FIG. 1 is a schematic diagram showing the construction of nanoscale constructs. [Diagram 3] (a) Schematic showing the configuration of adjustable angles that defined the cross-linked chain length for the obtained chiral signal with the corresponding color image, (b) spectrum of anisotropy factor with wavelength, and (c) polarization-resolved spectroscopy graph. [Figure 4](a) Schematic illustration, (b) spectra of anisotropy factors with wavelength, and (c) color images of tunable colorimetric signals of the same open and closed nanoscale constructs using different composition nanomaterials AuNRs (Au1 and Au2) and Au@AgNRs (AgAu1 and AgAu2). [Diagram 5] (a) Schematic illustration, (b) spectrum of anisotropy factor with wavelength, and (c) color image of an example of analyte-dependent configurational modulation of chiral plasmonic assemblies for protein sensing. [Figure 6] (a) Schematic illustration, (b) spectrum of anisotropy factor with wavelength, and (c) color image of an example of analyte-dependent configurational modulation of chiral plasmonic assemblies for nucleic acid sensing. [Figure 7] (a) Schematic illustration, (b) spectra of anisotropy factor with wavelength, and (c) color images of an example of analyte-dependent configurational modulation of chiral plasmonic assemblies for small molecule sensing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] As used herein, the term "functionalized" refers to the appearance of oligonucleotides or DNA on metal nanoparticles.

[0020] To calculate the g-factor as a percentage (%), take the CD value (in millidegrees) and divide it by the absorbance at that wavelength, then divide by 32980 and multiply by 100.

[0021] When the expression "g-factor" as a percentage (%) is used herein, it means the net value or magnitude of the g-factor, which may be positive or negative. In this disclosure, the inventors have utilized DNA origami techniques to create reconfigurable nanoscale constructs with record high anisotropy factors (g-factors), which JPEG2025505090000001.jpg3764, A L and A Rare its absorptivity for left- and right-circularly polarized light up to 16%, respectively, and its tunable chirality with anisotropy factor reaching the range of 0-16%. Such a high g-factor enabled colorimetric detection (simple detection by the naked eye) of the analyte-dependent modulation of the chiroptical response. Therefore, the developed chiral plasmonic sensing scheme with optical readout does not require CD spectroscopy.

[0022] Several embodiments and examples of the present invention are described below.

[0023] To construct a characterization platform according to an embodiment of the present invention, the following materials can be used: scaffold DNA strands, staple strands including core and crosslink strands, test strands (NAL and its complementary strand), metal nanoparticles, thiol-modified DNA strands, nuclease-free water, buffer, analytical chemicals, sodium chloride, magnesium chloride, detergents (e.g., sodium dodecyl sulfate (SDS), Tween® 20).

[0024] In an embodiment of the invention, the following equipment can be used to use the platform: thermocycler, thermoshaker, centrifuge, and polarizer. For details, see the examples below.

[0025] One embodiment of the present invention is comprised of a reconfigurable chiral plasmonic structure that enables a colorimetric sensing method to detect samples of a particular chirality. Figure 1 is an overview of a colorimetric chiral-based sensing scheme, showing an exemplary embodiment of a nanoscale construct and its detection scheme. In this embodiment, the nanoscale construct exhibiting chiral colorimetric sensing by analyte-induced modulation of the chiroptical response in the present invention allows a readout independent of CD spectroscopy. The measurement method is visual observation by eye, digital camera or spectrometer.

[0026] FIG. 2 is a schematic diagram showing the construction of a nanoscale construct. The nanoscale construct is composed of DNA origami and metal nanoparticles. During the assembly of the DNA origami, bridging or sensing elements are inserted through an annealing process. Metal nanoparticles are assembled on the DNA origami to construct the nanoscale construct. The use of nanorods can broaden the colorimetric range on the visual spectrum. Also, compared with conventional chiral visual sensing, optical observation and identification can be relatively easy. The advantages of the present invention are (i) fewer steps in the fabrication process, and (ii) high efficiency of insertion of bridging or sensing elements into the nanoscale construct.

[0027] In our solution, we use fixed cross-linked strands of defined sequence at defined angles to reach a defined chirality. Figure 3 shows examples of rearrangeable angular configurations (i.e. interchangeable angles) in chirality, as well as the spectrum and color images of the anisotropy factor with wavelength. Different configurations with DNA origami linked by different cross-links correspond to their signals. The configurations include various cross-links of defined length to join two DNA bundles and regulate their angular rotation to specific angles, which results in different chiralities and therefore observable color differences.

[0028] Metal particles are immobilized on the DNA origami and generate optical signals through plasmonic coupling effects. Figure 4 shows examples of tunable visual colors when nanomaterials of different compositions, e.g. AuNR and Au@AgNR, are used in nanoscale constructs of similar chirality. The nanoscale constructs may be configured to interface with different plasmonic nanomaterials that change their absorption spectra. This leads to changes in the CD spectra as well as colorimetric readouts in terms of color and intensity.

[0029] In the present invention, different metal nanomaterials can be used in the nanoscale constructs with different sizes, shapes and compositions. The metal nanomaterials used in the nanoscale constructs can be gold nanorods or silver-coated gold nanorods.

[0030] The size of gold or silver coated nanorods that produce tunable g-factors can range between 60 and 90 nm in length, preferably between 65 and 80 nm, and between 20 and 50 nm in diameter, preferably between 25 and 40 nm. A preferred average size is about 70 nm in length and about 30 nm in diameter. In general, nanoscale constructs with increased nanorod size increase the g-factor in both open and closed configurations. For example, the g-factor of a nanoscale construct with gold nanorods ∼70 nm in length and ∼30 nm in diameter is higher than that with ∼65 nm in length and ∼25 nm in diameter. Preferably, the use of nanorods with increased uniformity increases the g-factor of the nanoscale construct by more than 10%.

[0031] Using gold nanorods of different shapes in nanoscale constructs can produce different colors with similar g-factors. For example, gold nanorods with rounded edges produce different colors than gold nanorods with sharp edges.

[0032] With respect to the size of the silver thick coating, different metal nanomaterials, for example AuNR with 5 nm silver coating, can produce different optical responses that shift the optical response and g-factor in the visual and near infrared (NIR) spectrum (Figure 4). Both AuNR (Au2 in this example) and Au@AgNR (AgAu2 in this example) are used in closed configuration nanoscale constructs, giving a g-factor of ~15%, but the g-factor peaks with Au2 are in the range of 575-625 nm and 650-700 nm, while the g-factor peaks with Au2 are in the range of 550-580 nm and 620-660 nm (Figure 4b). The optical responses are different. For example, the color of Au2 is orange, while AgAu2 is green (Figure 4c).

[0033] With respect to the size of the silver thick coating (from no coating = 0 nm to 8 nm coating), AuNRs of different metal nanomaterials can generate different optical responses that shift the optical response and g-factor in the range of 400-800 nm.

[0034] In the absence of thick silver coating of AuNR, the g-factor peaks of the nanoscale constructs are in the ranges of 575-625 nm and 650-700 nm.

[0035] When the silver thickness coating of AuNR is in the range of more than 0 nm and less than 3 nm, the g-factor peaks of the nanoscale constructs are in the range of 580-620 nm and 650-690 nm.

[0036] For silver thick coatings of AuNRs in the range of more than 3 nm and less than 6 nm, the g-factor peaks of nanoscale constructs are in the ranges of 560-600 nm and 620-660 nm.

[0037] For AuNRs with thick silver coatings in the range of more than 6 nm and less than 9 nm, the g-factor peaks of nanoscale constructs are in the ranges of 530-570 nm and 590-630 nm.

[0038] With regard to the change in wavelength intensity or shift in wavelength maximum, it produces a tunable color detectable by a light sensor with a spectrometer. In the above example, the g-factor peaks at 4 to 6% in the closed configuration, but the color can vary.

[0039] In one embodiment, the color of the nanoscale construct in a closed configuration using AuNR is pink. In another embodiment, the color of the nanoscale construct in a closed configuration with a silver thickness coating of AuNR of more than 0 nm and less than 3 nm is purple-cyan. In another embodiment, the color of the nanoscale construct in a closed configuration with a silver thickness coating of AuNR of more than 3 nm and less than 6 nm is blue-green. In another embodiment, the color of the nanoscale construct in a closed configuration with a silver thickness coating of AuNR of more than 6 nm and less than 9 nm is green.

[0040] The sensing response is based on the change in chirality of the structure. Visual readout with dynamic chiral response for sensing is shown in Figures 5 to 7. The sensing relies on analyte-dependent configuration modulation of the chiral plasmonic ensemble for sensing proteins, nucleic acids, and small molecules, respectively. Analyte interaction strongly modulates the plasmonic optical response, resulting in visual absorption or transmission color from one specific color to another. As shown in Figures 5 to 7, the presence of analyte increases the chirality with respect to the CD signal and changes the colorimetric signal from one color to another.

[0041] Similar to nucleic acid sensing, sensing of small molecules, such as adenosine triphosphate (ATP), is based on strand dissociation, i.e., the aptamer strand targeting the small molecule dissociates in the presence of ATP, which leads to hybridization of the two bridge sequences in the origami and thus changes the angle of the nanoscale construct.

[0042] Spatially reconfigurable chiral plasmonic structures have emerged as promising candidates for developing novel sensing schemes, where the sensing or detection mechanism is based on a strong correlation between the spatial configuration of the chiral plasmonic structures and their chiroptical response. Chiral plasmonic sensors have several unique advantages: (i) high sensitivity, (ii) strong modulation of the optical response in response to analytes, and (iii) reliable optical detection in environments with strong light attenuation. Chiral plasmonic sensors rely on analyte-dependent configurational modulation of chiral plasmonic ensembles in nanoscale constructs. Until now, readout has relied on CD spectroscopy. The present invention allows for a readout independent of CD spectroscopy, e.g., analyte-induced modulation of the chiroptical response can be observed with the naked eye. Such a high g-factor allows colorimetric detection (simple discrimination by the naked eye) of the analyte-dependent modulation of the chiroptical response. Colorimetric detection greatly extends the utility of chiral plasmonic sensors beyond laboratory settings, making them suitable for diagnostic and sensing applications in point-of-care and / or remote settings.

[0043] The nanoscale construct can be switched from an open state to a closed state for visual detection or vice versa, making the nanoscale construct useful for a wide range of applications in the field of chiral sensing, such as chiral plasmonic sensing in biomedical applications. Examples 3-5 below are visual detection using open and closed configurations of the nanoscale construct, which can be changed by direct binding of the target analyte or dissociation of a component, such as the sensing strand, from the nanoscale construct in the presence of the target analyte.

[0044] The embodiments and methods of the present invention can be implemented using specific hardware, software, or a combination thereof for colorimetric observation and analysis. In one embodiment, a development kit can be prepared that contains the materials to make the nanoscale constructs, and the user can add the desired recognition elements and bridging sequences, such as short nucleic acid sequences that hybridize with the structures, to sense the target analyte. The user can choose to optimize the bridging sequence until the desired color change is observed.

[0045] In one embodiment, the detection kit may contain the nanoscale constructs of the present disclosure in a cartridge or plastic strip to hold the sensing solution premix and allow sample injection. The sensing solution premix, which includes the present constructs with recognition elements, e.g., SARS-CoV2 antibodies, can target a specific analyte, in this case, the SARS-CoV2 antigen. In this way, the user only needs to add a defined amount of sample, e.g., saliva, to the cartridge or plastic strip with the aid of a dropper and observe the color change, e.g., from blue to red, with the naked eye or by placing the cartridge or plastic strip in an optical reader to read it out or record it with a camera.

[0046] Further embodiments of the present disclosure are set forth below.

[0047] 1. A nanoscale construct essentially comprising a nucleic acid structure having a rearrangeable or switchable configuration and a metal nanomaterial, which provides high chirality and / or optical activity, defined as having an absorption asymmetry factor (g-factor) of greater than 10%.

[0048] 2. The nanoscale construct of embodiment 1 comprises an assembly of a nucleic acid structure and a metal nanomaterial.

[0049] 3. The nanoscale construct of embodiment 1, wherein the range of chirality and / or optical activity is within the visible and near infrared (NIR) spectrum from 400 to 800 nm.

[0050] 4. The nanoscale construct of embodiment 3, wherein the range of chirality and / or optical activity is tunable or adjustable within the visible and NIR wavelength spectrum of 400 to 800 nm.

[0051] 5. The nanoscale construct of embodiment 1, wherein the chirality and / or optical activity is tunable or adjustable.

[0052] 6. The nanoscale construct of embodiment 5, wherein the chirality and / or optical activity is reversibly tunable or adjustable by rearrangeable or switchable actuation.

[0053] 7. The nanoscale construct of embodiment 6, wherein the rearrangeable or switchable configuration is reversible.

[0054] 8. The nanoscale construct of embodiment 6, wherein the chirality and / or optical activity is tunable or adjustable by changing the geometry and / or angle of the nanoscale construct.

[0055] 9. The nanoscale construct of embodiment 6, wherein the chirality and / or optical activity is tunable or adjustable by using different metal nanomaterials.

[0056] 10. The nanoscale construct of embodiment 9, wherein the different metal nanomaterials differ in size, shape and composition.

[0057] 11. The nanoscale construct of embodiment 1, wherein the metal nanomaterial is a metal nanoparticle.

[0058] 12. The nanoscale construct of embodiment 11, wherein the metal nanoparticles are metal nanorods.

[0059] 13. The nanoscale construct of embodiment 11, wherein the metal nanomaterial is a gold nanorod or a nanorod made of gold and silver.

[0060] 14. The nanoscale construct of embodiment 6, wherein the chirality and / or optical activity is tunable or adjustable by the combined effect of changing the geometry and / or angle of the nanoscale construct and the use of different metal nanomaterials.

[0061] 15. The nanoscale construct of embodiment 1, wherein the color production is based on chirality and / or optical activity.

[0062] 16. The nanoscale construct of embodiment 15, wherein the color generation is based on a chiral optical response from the metal nanomaterial.

[0063] 17. The nanoscale construct of embodiment 4, wherein the tunable color generation is based on tunable chirality and / or optical activity.

[0064] 18. The nanoscale construct of embodiment 15, wherein the color production is optically detectable by eye and / or a light sensor.

[0065] 19. The nanoscale construct of embodiment 17, wherein the tunable color production is optically detectable by eye and / or a light sensor.

[0066] 20. The nanoscale construct of embodiment 15, wherein the color is optically detectable with the aid of a polarizer or multiple polarizers.

[0067] 21. The nanoscale construct of embodiment 15, wherein the color of the spectral composition can be quantitatively measured by a light sensor.

[0068] 22. The nanoscale construct of embodiment 1, which is capable of said responsiveness to an external stimulus.

[0069] 23. The nanoscale construct of embodiment 22, wherein the responsiveness corresponds to a change in chirality and / or optical activity, or a change in g-factor from 0 to more than 10%.

[0070] 24. The nanoscale construct of embodiment 22, wherein the external stimulus is a target analyte.

[0071] 25. The nanoscale construct of embodiment 22, wherein the nanoscale construct contains a peptide or oligonucleotide as a bioreceptor, sensing or recognition element.

[0072] It is to be understood that the disclosed embodiments of the invention are not limited to the particular structures, process steps, or materials disclosed herein, but extend to equivalents thereof that would be recognized by one of ordinary skill in the art. It is also to be understood that the terminology employed herein is used only for the purpose of describing particular embodiments, and is not intended to be limiting.

[0073] Throughout this specification, references to "one embodiment," "an embodiment," or "a preferred embodiment" mean that a particular configuration, structure, or feature described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearance of the phrases "in one embodiment," "in an embodiment," and "in a preferred embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment.

[0074] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as if each member of the list were individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as being de facto equivalents to other members of the same list solely based on their presentation in a common grouping, absent a contrary indication. In addition, various embodiments and examples of the invention may be referred to herein along with alternatives of the various components thereof. It is understood that such embodiments, examples, and alternatives should not be construed as de facto equivalents of each other, but should be considered as separate and autonomous representations of the invention.

[0075] Furthermore, the described configurations, structures, or features may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. However, one of ordinary skill in the art will recognize that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of the invention.

[0076] While the above-described embodiments illustrate the principles of the present invention in one or more particular applications, it will be apparent to those skilled in the art that numerous modifications in the form, use and details of implementation may be made without the exercise of inventive faculty and without departing from the principles and concepts of the present invention. Accordingly, the present invention is not intended to be limited, except as by the scope of the claims set forth below.

[0077] In this specification, the verbs "to comprise" and "to include" are used as open limitations that do not exclude or require the presence of unrecited features. Features recited in the dependent claims may be freely combined with each other, unless expressly stated otherwise. Furthermore, it is to be understood that throughout this specification, the use of "a" or "an", i.e., the singular, does not exclude the plural.

[0078] [Example] The materials used in the construction of the nanostructured molecules in the following examples are as follows:

[0079] DNA scaffold strand (p7650) purchased from tilibit nanosystems GmbH, Germany. Staple strand purchased from ThermoFisher®. Other DNA / RNA strand purchased from IDT®. Thiol-modified TTTTT TTTTT TTTTT T (SEQ ID NO: 20) DNA strand purchased from Biomers. Bridging and sensing strands are listed in Table 1.

[0080] [Table 1-1] [Table 1-2] [Table 1-3]

[0081] Designs 1-2 are composed of different cross-linking designs with different hybrid geometries, sequences and lengths that can adjust the angle and geometry between the two bundles of the origami structure in our nanoscale constructs. Examples of our nanoscale constructs made for various sensing applications use AbsSense (AbsSense-sequence 1, AbsSense-sequence 2) for protein sensing, NASense (NASense-sequence 1, NASense-sequence 2, NASense-sensing strand) for nucleic acid sensing of target sequences (NA), and SMSense (SMSense-sequence 1, SMSense-sequence 2, SMSense-sequence 3, SMSense-blocker, SMSense-aptamer, SMSense-activator) for small molecule sensing.

[0082] Examples of origami construction of nanoscale structures in embodiments are described below.

[0083] To prepare the staple solution, 156 core strands, bridge strands with or without sensing element were mixed in a 1:1.5 ratio. 10 μL of staple (0.64, 0.96, 1.3 μM, core, bridge and sensing strand) solution was mixed with 5 μL of TE (10X), 5 μL of scaffold p7560 (100 nM), 10 μL of MgCl2 (100 mM), 2.5 μL of NaCl (100 mM), 17.5 μL of H2O. The mixture was annealed from 80 °C to room temperature for about 28 hours to synthesize the DNA origami structure. The DNA origami was purified by agarose gel purification. The concentration of the DNA origami was measured by measuring the absorbance at 260 nm in a spectrometer (approximately 1.3 × 10 8 M -1 cm -1 The DNA origami samples were stored in DNA Robin tubes at 4°C.

[0084] In an embodiment, an example of constructing gold nanorods (AuNR) and defining the thickness of the silver coating on the AuNR to obtain silver-coated AuNR (Au@AgNR) in a nanoscale construct is described below.

[0085] Before synthesizing small AuNRs and growing them into AuNRs of the desired size and aspect ratio, gold seed formation was performed. First, a gold seed solution was prepared by mixing 20 μL of HAuCl4 (50 mM) and 10 μL of L-ascorbic acid (100 mM) with 2 mL of a mixture of 1-decanol (13.5 mM) and hexadecyltrimethylammonium bromide (CTAB, 50 mM) at 25 °C. 80 μL of NaBH4 (20 mM) was quickly injected into the solution under vigorous stirring. The seed solution was incubated at 25 °C for 1 h before use. Small AuNRs were synthesized by gently mixing 240 μL of AgNO3 (10 mM), 300 μL of HAuCl4 (50 mM), 390 μL of L-ascorbic acid (100 mM), 2.1 mL of hydrochloric acid (1 M), and 1.8 mL of gold seeds with 30 mL of a mixture of 1-decanol (13.5 mM) and CTAB (50 mM) at 25 °C. After incubation at 25 °C for 4 h, the small AuNRs were purified and analyzed by OD analysis with 10 mM CTAB. 400nm The AuNR was then prepared by gently mixing 600 μL of AgNO3 (10 mM), 200 μL of HAuCl4 (50 mM), 160 μL of L-ascorbic acid (100 mM), 100 μL of hydrochloric acid (1 M), and 130 μL of small AuNR with 20 mL of a mixture of 1-decanol (11 mM) and CTAB (50 mM). After incubation at 28 °C for 4 hours, the AuNR was purified and redispersed in cetyltrimethylammonium chloride (CTAC, 25 mM). For silver coating of AuNR, 1 mL of AuNR (average size diameter 70 nm × 30 nm, 12 nM in this example) and 444 μL of AgNO3 (10 mM) were mixed with 11 mL of CTAC (25 mM), followed by dropwise injection of 444 μL of L-ascorbic acid (100 mM). After incubation at 60 °C for 4 h, the Au@AgNR was purified, redispersed in CTAB, and stored at 4 °C before use.

[0086] To assemble the nanoscale constructs, first, poly-T DNA strands were attached to the nanorods via thiol groups. The free thiol-DNA was washed away by centrifugation. The poly-T DNA strands on the AuNR or Au@AgNR were hybridized with the poly-A strands extending from the staple strands of the origami to immobilize the AuNR. The AuNR-DNA and origami were mixed in a ratio of 5:1 to 20:1 and annealed from 40°C to room temperature. The assembled nanoscale constructs may contain dimer assemblies, i.e., two nanorods joined into one origami structure, or polymer assemblies, e.g., trimers or tetramers, or combinations thereof. To purify nanoscale constructs of specific assemblies, the nanoscale constructs were loaded into a 0.7% agarose gel containing 11 mM MgCl2. After gel electrophoresis at 80 V for 3 hours, the gel bands corresponding to specific fractions of origami-AuNR, e.g., dimers, could be extracted through an agarose extraction filter.

[0087] Examples of controlled assembly ratios of metal nanoparticles on templates to prepare nanoscale constructs that provide high g-factors (>10%) or that change g-factors in response to external stimuli are shown in Table 2.

[0088] [Table 2] key: The AuNRs and Au@AgNRs are cylindrical nanorods unless otherwise noted. Design 1 (Design 1-sequence 1, Design 1-sequence 2-21nt), Design 2.1 (Design 2-sequence 1, Design 2-sequence 2-20nt, Design 2-sequence 3, Design 2-sequence 4), Design 2.2 (Design 2-sequence 1, Design 2-sequence 2-14nt, Design 2-sequence 3, Design 2-sequence 4) AbsSense (AbsSense-sequence 1, AbsSense-sequence 2) NASense (NASense-sequence 1, NASense-sequence 2, NASense-sensing strand) SMSense (SMSense-sequence 1, SMSense-sequence 2, SMSense-sequence 3, SMSense-blocker, SMSense-aptamer)

[0089] Example 1: Assembly of nanoscale structures with g-factors >10% To assemble the nanoscale constructs, the nanorods are functionalized with poly-T DNA strands attached to them. This attachment can be done, for example, with thiol-modified TTTTT TTTTT TTTTT T (SEQ ID NO: 20) DNA strands. The nanorods can be, for example, gold nanorods with a size of 70 nm in length and 30 nm in diameter. The molar ratio of functionalization between poly-T DNA strands and nanorods is 5000:1 to 40000:1. Preferably, the molar ratio of functionalization between poly-T DNA strands and gold nanorods is 10000:1. The molar ratio of functionalization between poly-T DNA strands and silver-coated nanorods is 40000:1. Before the functionalization method, a surfactant is added to the mixture. For example, a final concentration of 0.05% SDS is added to the mixture of poly-T DNA strands and gold nanorods, and a final concentration of 0.2% SDS and a final concentration of 0.05% Tween 20 are added to the mixture of poly-T DNA strands and silver-coated gold nanorods. The functionalization method proposed by Liu et al. (see, for example, Liu et al., 2017) is to place the mixture at a lowered temperature, e.g., −20° C., for 2 hours. The free unbound DNA is washed away by centrifugation with a buffer, e.g., 0.5X TBE containing 0.1% SDS. DNA origami (see, for example, Kuzyk et al., 2014) and our strands, e.g., Design 2-sequence 1, Design 1-sequence 2-14nt, Design 2-sequence 3, and Design 2-sequence 4 in Table 1, are assembled by functionalized nanorods. AuNR-DNA and origami were mixed in a ratio of 5:1 to 20:1, e.g., 7:1, and annealed from 40° C. to room temperature. The assembled nanoscale constructs, which may include dimeric assemblies, i.e., two nanorods joined into one origami structure, or polymeric assemblies, e.g., trimers or tetramers, or combinations thereof, as well as unlinked nanorods, give g-factors >10%, e.g., 16%.

[0090] [Example 2] Fabrication of nanoscale structures with tunable g-factors The geometry of the nanoscale constructs can change the g-factor. The geometry of the nanoscale constructs can be changed by the presence, design and length of the crosslinking chains. Nanoscale constructs in an open configuration can be made without any modification by crosslinking chains. Nanoscale constructs in a closed configuration can be made with crosslinking chain design 1 (design 1-sequence 1, design 1-sequence 2-21nt), crosslinking chain design 2.1 (design 2-sequence 1, design 1-design 1-sequence 2-20nt, design 2-sequence 3, design 2-sequence 4), crosslinking chain design 2.2 (design 2-sequence 1, design 1-sequence 2-14nt, design 2-sequence 3, design 2-sequence 4) (Figure 3a) to give tunable g-factors ranging from 0 to 16% (Figure 3b). For example, nanoscale constructs in an open configuration give a g-factor of .about.2%, nanoscale constructs in a closed configuration using Design 1 give a g-factor of .about.9%, nanoscale constructs in a closed configuration using Design 2.1 give a g-factor of .about.12%, and nanoscale constructs in a closed configuration using Design 2.2 give a g-factor of .about.16%.

[0091] Exemplary buffers for the sensing reaction are 1×TBE or 1×PBS containing 5-12 mM MgCl 2 , and may optionally contain 0.02-0.2% sodium dodecyl sulfate (SDS), 0.02-0.2% Tween-20.

[0092] [Example 3] Protein sensing using nanoscale structures The nanoscale construct contains DNA-conjugated digoxigenin (AbsSense in Table 1) that binds to a protein antibody that interacts with digoxigenin. The angle of our nanoscale construct changes, and therefore the chirality, due to the interaction between the antibody and the attached digoxigenin in the nanoscale construct. The change in chirality causes the colorimetric signal to change from one color to another, or the transmitted intensity of polarized light to change (Figure 5).

[0093] Details of Example 3 of the responsiveness of antibody detection to external stimuli are described. The nanoscale construct incorporates cross-linked AbsSense-sequence 1 and AbsSense-sequence 2 listed in Table 1. The nanoscale construct is placed in a sensing reaction buffer 1X PBS containing 5 mM MgCI2 and 0.02% SDS at a concentration of ∼1 nM. Anti-digoxigenin antibody is added to the nanoscale construct at a final concentration of ∼200 pM. The geometry of the nanoscale construct changes. The peak g-factor changes from ∼3% to ∼9% (Figure 5b). When gold nanorods are used in the nanoscale construct, the color changes from blue to pink (Figure 5c). When silver-coated gold nanorods are used in the nanoscale construct, the color changes from blue to green.

[0094] [Example 4] Nucleic acid sensing using nanoscale structures The nanoscale construct contains a specific DNA sequence (NASense in Table 1) that hybridizes with it, but is blocked by the sensing strand DNA (NASense-sensing strand in Table 1). In the presence of the cancer gene marker EGFR (NA in Table 1), the sensing strand DNA is displaced from the nanoscale construct, so that the nanoscale construct switches from one angle (unhybridized) to another angle (hybridized), and thus the chirality changes accordingly. The change in chirality causes the colorimetric signal to change from one color to another, or the transmission intensity of polarized light to change (Figure 6).

[0095] Details of Example 4 of responsiveness to external stimuli for nucleic acid detection are described. The nanoscale constructs incorporate the crosslinked strand design 1-sequence 1, NASense-sequence 1, NASense-sequence 2 and NASense-sensing strands listed in Table 1. The nanoscale constructs at a concentration of ∼1 nM are placed in sensing reaction buffer 1X PBS containing 5 mM MgCI2 and 0.02% SDS. Target (NA) at a final concentration of ∼25 nM is added to the nanoscale constructs. The geometry of the nanoscale constructs changes. The peak g-factor changes from ∼3% to ∼6% (Figure 6b). When gold nanorods are used in the nanoscale constructs, the color changes from blue to pink (Figure 6c). When silver-coated gold nanorods are used in the nanoscale constructs, the color changes from blue to green.

[0096] An example of the responsiveness corresponds to a change in g-factor from 0 to more than 10%. Nanoscale constructs of DNA origami in an open configuration can be modified and fabricated with crosslinked strand design 1 (design 2-sequence 1, design 2-sequence 3, design 2-sequence 4) with or without the presence of target nucleic acid (design 1-sequence 2-14nt) before being assembled with functionalized nanorods. In the absence of target nucleic acid in the DNA origami, the peak g-factor can range from 0 to 3%. In the presence of target nucleic acid at a final concentration of 500 nM in the DNA origami, the peak g-factor changes to a range from 12 to 16%.

[0097] [Example 5] Small molecule sensing using nanoscale structures The nanoscale construct contains a specific DNA sequence (SMSense in Table 1) that hybridizes together, but is blocked by aptamer and blocker DNA (SMSense-Blocker and SMSense-Aptamer in Table 1). In the presence of small molecule ATP, the ATP-bound aptamer from the nanoscale construct optionally binds with an auxiliary (SMSense activator in Table 1) that has an activating DNA strand that lowers the kinetic energy required for the dissociation of the aptamer, so that the nanoscale construct switches from one angle (unhybridized) to another angle (hybridized). Thus, the chirality changes accordingly. The change in chirality causes the colorimetric signal to change from one color to another, or the transmission intensity of polarized light to change (Figure 7).

Claims

1. A nanoscale construct, the construct comprising or consisting essentially of a nucleic acid structure having a rearrangeable or switchable configuration and at least two separate metal nanoparticles bound to the nucleic acid structure, the two separate nanoparticles being at interchangeable angles to each other, the nanoparticles providing high chirality and / or optical activity within the visible and near-infrared (NIR) spectrum from 400 to 800 nm that produces the color of the construct, and the construct having an absorption asymmetry factor (g-factor) of greater than 10%.

2. The nanoscale construct of claim 1, wherein the nucleic acid structure comprises a switchable bridge structure, the angle being adjusted by the open and closed positions of the bridge structure, and adjustment of the bridge structure between the open and closed positions changes the color-generating properties of the construct, and the length of the bridge structure preferably affects the color generated by the construct in the closed position of the bridge structure.

3. The nanoscale construct of claim 2, wherein the bridged structure comprises a recognition element, preferably an oligonucleotide or a peptide, specific to a target analyte, and the open and closed positions of the bridged structure are switched due to the presence or absence of the target analyte.

4. The nanoscale construct of any one of claims 1 to 3, wherein the nucleic acid structure is DNA origami.

5. The nanoscale construct of any one of claims 2 to 3, wherein the bridge structure comprises at least two oligonucleotides bound to the nucleic acid structure, the two oligonucleotides comprising sequences complementary to each other, and the length of the bridge structure preferably affects the color produced by the construct in the closed position of the bridge structure.

6. 10. The nanoscale construct of claim 1, wherein the chirality and / or optical activity is reversibly tunable or adjustable by rearrangeable or switchable operation.

7. The nanoscale construct of claim 6 , wherein the rearrangeable or switchable configuration is reversible.

8. The nanoscale construct of any one of claims 1 to 3, wherein the metal nanoparticles vary in size, shape and composition.

9. The nanoscale construct of claim 8 , wherein the metal nanoparticles are metal nanorods.

10. 9. The nanoscale structure of claim 8, wherein the metal nanomaterial is a gold nanorod or a nanorod made of gold and silver.

11. 11. The nanoscale construct of claim 10, wherein the length of the nanorods is in the range of 60-90 nm and the diameter is in the range of 20-50 nm, preferably with an average length of 70 nm and a diameter of 30 nm.

12. 12. The nanoscale construct of claim 11, wherein preparing the nanoscale construct comprises first attaching the nanorods and DNA strands, preferably poly-T DNA strands, to the nucleic acid structure, preferably DNA origami, by attaching the DNA strands to the nanorods in a molar ratio of about 10,000:1 to 40,000:1 to obtain a nanorod-DNA strand assembly, wherein the DNA strands comprise a sequence complementary to that of the nucleic acid structure.

13. The nanoscale construct of claim 12, wherein the nanorod-DNA strand assemblies are annealed to the nucleic acid structure, preferably DNA origami, in a molar ratio of about 5:1-20:1 to obtain a macromolecular assembly comprising nanorods bound to the nucleic acid structure, preferably DNA origami.

14. 14. The nanoscale construct of claim 13, wherein a dimeric assembly comprising two nanorods joining one nucleic acid structure, preferably DNA origami, is purified from the macromolecular assembly.

15. The nanoscale construct of claim 1 , wherein the color generation is based on a chiral optical response from metal nanoparticles.

16. 10. The nanoscale construct of claim 1, wherein the color production is optically detectable by the eye and / or a light sensor.

17. 10. The nanoscale construct of claim 1, wherein the color is optically detectable with the aid of a polarizer or multiple polarizers.

18. The nanoscale construct of claim 1 , wherein the color can be quantitatively measured by an optical sensor.

19. The nanoscale construct of claim 1 , wherein the color change is based on responsiveness to an external stimulus.

20. 20. The nanoscale construct of claim 19, wherein the response corresponds to a change in chirality and / or optical activity or a change in g-factor from 0 to greater than 10%.

21. 20. The nanoscale construct of claim 19, wherein the external stimulus is a target analyte.

22. 20. The nanoscale construct of claim 19, wherein the nanoscale construct contains a recognition element, preferably a peptide or an oligonucleotide, as a biological receptor.

23. A method for detecting an analyte in a sample, the method comprising the steps of contacting a nanoscale structure according to any one of claims 1 to 3 specific for the analyte with a sample in a test analysis, and detecting the analyte in the sample by optical means or the naked eye, wherein the presence of the analyte in the sample causes a color change of the nanoscale structure, and the color change in the analysis confirms the presence of the analyte in the sample.

24. 4. Use of the nanoscale construct of any one of claims 1 to 3 for the detection of an analyte in a sample, wherein the presence of the analyte in the sample causes the nanoscale structure to change color when contacted with the sample, and the color change confirms the presence of the analyte in the sample.

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