Aptamers to detect drugs, biomarkers and small molecules for ocular applications

Abstract

Described herein are various embodiments for the detection of ocular drugs, biomarkers, and small molecules using DNA or RNA aptamers. The DNA or RNA aptamer consists of a binding sequence to selectively and strongly bind to a compound of interest and can be linked to a readily detectable tag. The tag may be fluorescent, radioactive, or chromophore. The detection of the compound of interest can then be accomplished using a simple standard detection method, such as UV-Vis or fluorescence spectrophotometry, or a more complex method, such as mass spectrometry or electrochemical detection, preferably fluorescence detection or radioactivity detection with high sensitivity.

Description

Technical Field

[0001] Various embodiments described herein generally relate to the use of DNA or RNA aptamers to detect ophthalmic compounds, including drugs, biomarkers, tear film components, and small molecules used in the field of vision science. Background Art

[0002] The development of new ocular drug delivery methods requires sensitive and accurate detection of drugs and other small molecules. However, for many of these compounds, their detection using current methods such as HPLC (high performance liquid chromatography) and mass spectrometry is difficult, complex and expensive.

[0003] The detection of tear film biomarkers has attracted increasing attention for early screening and detection of ocular diseases. However, the concentrations of these biomarkers are usually very low, making them difficult to detect using current methods.

[0004] DNA or RNA aptamers are short single-stranded DNA or RNA molecules that can bind to specific target molecules with high affinity and specificity. Similar to antibodies, these aptamers can be developed to bind to specific targets.

[0005] There remains a great need to develop reagents and methods that can be used to detect drugs, biomarkers, and small molecules for ocular applications with high specificity, high accuracy, and low cost. Summary of the invention

[0006] According to the teachings herein, various embodiments are provided for developing DNA or RNA aptamers for detecting drugs, biomarkers and other small molecules.DNA or RNA aptamers include sequences designed to bind to target compounds with high affinity and specificity.

[0007] The optimal sequence of a DNA or RNA aptamer can be determined by various screening methods, including (but not limited to) Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and machine learning-guided aptamer searches.

[0008] Typically, the DNA or RNA aptamer is also linked to a detectable label (eg, a fluorescent, electrochemically active molecule, or a radioactive molecule or chromophore). Binding of the DNA or RNA aptamer-tag to the target enables sensitive quantification of the target.

[0009] In at least some embodiments, a DNA or RNA aptamer can have more than one binding site that binds to a single compound.

[0010] In at least some embodiments, a DNA or RNA aptamer may have binding affinity for more than one target compound.

[0011] In at least some embodiments, DNA or RNA aptamers can also bind to other small molecules that are not drugs or biomarkers.

[0012] In at least some embodiments, DNA or RNA aptamers can be immobilized on a surface, such as a test line, magnetic beads, microplate, nanoparticle, or microchip, as a high-throughput immobilized aptamer-based biosensor.

[0013] In at least some embodiments, the complementary strand of a DNA or RNA aptamer is also designed with a quencher for strand displacement-based detection.

[0014] In at least some embodiments, unlabeled DNA or RNA aptamers are also designed for target detection. The binding of DNA or RNA aptamers can also be detected by fluorescent dyes, such as ThT, SYBR green, EvaGreen or TaqMan probes.

[0015] DNA or RNA aptamers can also be used in combination with other detection methods, such as mass spectrometry or electrochemical detection, to enhance the sensitivity and selectivity of the assay.

[0016] The use of DNA or RNA aptamers for ocular applications may also involve optimization of assay conditions, such as pH, temperature, and incubation time, to maximize the binding affinity and specificity of the aptamer for the target molecule.

[0017] According to the following description and the appended claims, other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art. However, it should be understood that the following description, the appended claims and the specific examples, although indicating the preferred embodiments of the present application, are provided only as illustrations. By reading the following, various changes and modifications within the spirit and scope of the disclosed invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] For a better understanding of the various embodiments described herein, and to more clearly show how these various embodiments may be put into practice, reference will be made, by way of example, to the accompanying drawings which illustrate at least one exemplary embodiment and which will now be described.

[0019] Figure 1Examples of aptamers (A) binding to specific targets (B) including, but not limited to, biomarkers, small molecules (drugs, metabolites, toxins, environmental pollutants), proteins, nucleic acids, cells or tissues to (C) form various aptamer-target complexes are shown.

[0020] Figure 2 Demonstration of the use of aptamers to detect a target includes, but is not limited to, strand displacement (A), fluorescence resonance energy transfer (B), aptamer beacons (C), or fluorescent dyes (D).

[0021] Figure 3 Demonstrated mechanism of detection of ocular drug (atropine) in tears using aptamer-based biosensor.

[0022] Figure 4 The binding affinity of the aptamers to timolol malate and atropine measured by ITC is shown.

[0023] Figure 5 Shown are the calibration curves and detection limits of atropine measured using UV-vis and aptamer-based fluorescence biosensors. DETAILED DESCRIPTION

[0024] Various methods will be described below to provide examples of at least one embodiment of the claimed subject matter. The embodiments described below do not limit any claimed subject matter, and any claimed subject matter may encompass methods or systems different from those described below. The claimed subject matter is not limited to a method or system having all the features of any method or system described below, or is not limited to features common to multiple or all methods or systems described below. The method or system described below may not be an embodiment of any claimed subject matter. Any subject matter disclosed in the method or system described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuation patent application, and the applicant, inventor, or owner does not intend to abandon, deny, or dedicate any such subject matter to the public through the disclosure in this document.

[0025] In addition, it will be understood that for simplicity and clarity of explanation, reference numerals may be reused between the drawings to indicate corresponding or similar elements where deemed appropriate. In addition, many specific details are described to provide a thorough understanding of the embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein may be practiced without these specific details. In other cases, well-known methods, procedures, and components are not described in detail to avoid confusing the embodiments described herein. In addition, the description should not be considered to limit the scope of the embodiments described herein.

[0026] It should be noted that terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term that does not significantly change the end result. These terms of degree can be construed as including the deviation of the modified term if the deviation would not negate the meaning of the modified term.

[0027] In addition, numerical ranges recited herein by endpoints include all numbers and fractions contained within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that, provided that all numbers and fractions thereof are modified by the term "about," this means that there is some degree of variation in the recited number which will not significantly change the end result.

[0028] As used herein, the term "and / or" is intended to mean an inclusive or. That is, for example, "X and / or Y" is intended to mean X or Y or both. As another example, "X, Y and / or Z" is intended to mean X or Y or Z or any combination thereof.

[0029] As used herein, the terms "a" or "an" are intended to mean "one or more" (i.e., at least one) of the grammatical object of the article. Unless otherwise defined in the context, singular expressions include plural expressions. As an example, "an element" means one element or more than one element.

[0030] As used herein, unless otherwise stated, the terms "comprise", "include" and "including" will be understood to imply the inclusion of the stated steps or elements or combinations of steps or elements, but not to exclude any other steps or elements or combinations of steps or elements that do not affect the end result. The terms "comprising", "comprises" and "comprised" may also include the terms "consisting essentially of" and "consisting of".

[0031] Described herein are various exemplary embodiments of DNA-aptamers or RNA-aptamers for detecting ocular drugs, biomarkers, and small molecules, as well as related methods of making and testing them in various applications.

[0032] DNA or RNA aptamers are short single-stranded DNA or RNA molecules that can bind to specific target molecules with high affinity and specificity. Figure 1As shown, aptamers can have different primary structures, such as aptamers 1-5 with different nucleotide sequences (A), and can be folded into different three-dimensional structures, i.e., have different tertiary structures (B). Targets of aptamers include (but are not limited to) biomarkers, small molecules (drugs, metabolites, toxins, environmental pollutants), proteins, nucleic acids, cells or tissues (e.g., targets 1' to 5'). When an aptamer is exposed to its corresponding specific target under appropriate conditions, it binds to the corresponding target in a three-dimensional shape with high affinity and specificity (C).

[0033] The aptamer selection process utilizes systematic evolution of ligands by exponential enrichment (SELEX) to select aptamers with high specificity and affinity for the target. SELEX has become the gold standard strategy for generating nucleic acid aptamers. Whether for DNA or RNA sequences, on proteins, at the cellular level, or in living animals, the selection cycle requires three key steps: (i) incubating the target with a library containing randomized sequences, (ii) separating bound sequences from unbound sequences, and (iii) recovering bound sequences and amplifying them by PCR. [1] Aptamers selected from a variety of materials can be used in the present application, as long as the selected material is relevant to the eye and can be used for detection related to ocular applications.

[0034] Aptamers can be selected by, but not limited to, a library immobilization method or a target immobilization method. [1,2] Compared with the traditional SELEX method, our library capture method provides enhanced robustness and improved success rate. The whole process involves several rounds of iterative selection and amplification, as outlined in the 7 steps below.

[0035] (1) Library synthesis: A library of randomized nucleic acid sequences, usually containing 10 13 -10 15 Different sequences and random libraries of 25-60 bases. Depending on the desired application, the library can be composed of DNA or RNA sequences (including chemically modified nucleic acids).

[0036] (2) Library immobilization: The single-stranded library is first bonded to the biotinylated capture strand in the selection buffer. The double-stranded library is immobilized on a solid support (e.g., a resin or magnetic beads coated with streptavidin) or a microplate. This can be accomplished by covalent attachment or non-covalent interaction.

[0037] (3) Selection: The target is incubated with the immobilized library under conditions that favor the formation of aptamer-target complexes. Unbound sequences are washed away, and bound sequences are eluted and amplified by polymerase chain reaction (PCR).

[0038] (4) Counter-selection: The amplified sequence pool is subjected to a counter-selection step to remove any sequences that bind to molecules other than the target molecule. This is usually performed by incubating the sequence pool with a non-specific target molecule that is similar to the target molecule but lacks the desired binding properties.

[0039] (5) The selected sequence is used as the starting point for the next round of selection and amplification. The number of rounds can vary depending on the complexity of the target molecule and the desired affinity and specificity of the aptamer.

[0040] (6) Aptamer characterization: The selected sequences are sequenced to identify aptamer candidates. The binding affinity and specificity of these candidates are further characterized using techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), electrophoretic mobility shift assay (EMSA), flow cytometry or enzyme-linked oligonucleotide assay (ELONA).

[0041] (7) Aptamer optimization: The identified aptamer candidates are then optimized through various modifications (including truncation, sequence modification, and chemical modification) to further enhance their affinity and specificity for the target molecule.

[0042] The success or failure of SELEX depends on a variety of different factors, including the structural diversity provided by the oligonucleotide library, efficient removal of non- and weak binders during the selection step, and selective enrichment of high affinity binders (ideally in the absence of byproducts) during the amplification step. The selection step should be designed to mimic the desired future application of the aptamer as closely as possible to avoid downstream difficulties. [2] Among them, the selection buffer should provide optimal conditions for the formation of the aptamer-target complex while minimizing non-specific binding. The selection buffer generally (but not limited to) contains: a buffer solution, such as phosphate buffered saline (PBS), Tris buffered saline (TBS) or HEPES buffered saline (HBS); a blocking agent, such as bovine serum albumin (BSA) or casein; and ions, such as magnesium ions, calcium ions, potassium ions, and sodium ions. One of the important novelties of the present application is the use of a selection method for real ocular drug testing. In some preferred embodiments, the selection buffer is modified from Tris to PBS to better replicate ocular conditions.

[0043] In some embodiments, the library is incubated directly with the target (eg, cells and tissues) without fixation.

[0044] In some embodiments, capillary electrophoresis-SELEX, random primer-primed polymerase chain reaction-SELEX, cell-SELEX, microfluidics-based SELEX, magnetic bead-based SELEX, and high-throughput sequencing-based SELEX can be used. [4]

[0045] In some embodiments, machine learning guided aptamer searches can also be used. [3] Computer-assisted prediction of aptamer sequences has been developed by considering conserved hairpins with highly variable sequences and three scores based on sequence abundance, stability, and structure, respectively, which focus on primary sequence alignment and motif comparison.

[0046] Compared with the traditional SELEX method, our library capture method provides enhanced robustness and improved success rate. In addition, when detecting small molecules, aptamer-based biosensors have been shown to have higher sensitivity and specificity than conventional UV-vis methods. In addition, compared with HPLC-MS, the cost-effectiveness, rapid detection capability and simplified sample preparation procedures of the present invention enhance its practicality and usability. Overall, these features make the present invention easy to implement and reproduce.

[0047] In some embodiments of the present disclosure, the improved methods of the present disclosure provide timolol maleate-specific aptamers with high binding affinity, specificity and sensitivity to timolol maleate, such as those shown by the nucleotide sequence of any one of SEQ ID NOs: 1-11 or having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 1-11. In some embodiments, the improved methods of the present disclosure provide atropine-specific aptamers with high binding affinity, specificity and sensitivity to atropine, such as those shown by the nucleotide sequence of any one of SEQ ID NOs: 12-21 or having at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity with any one of SEQ ID NOs: 12-21.

[0048] The detection of a target using an aptamer is described in detail below.

[0049] Aptamer-based biosensors are used as recognition elements in a variety of target detection methods.As used herein, a "target" can be any biomarker, small molecule (drug, metabolite, toxin, environmental pollutant), protein, nucleic acid, cell or tissue.

[0050] In some embodiments, the aptamer is labeled with a radioactive, fluorescent or chromophore tag.The short complementary strand of the aptamer can be labeled with a quencher.

[0051] The target can be detected by aptamer-based biosensors, where the transducers can be optical transducers (e.g., fluorescence readout, radioactivity intensity, colorimetric spectroscopy, or polarization), electrochemical transducers (e.g., potentiometry, amperometry, or impedance spectroscopy), and magnetic transducers (e.g., magnetic relaxation switches or magnetic resonance imaging). Figure 2 Embodiments of using aptamers to detect targets are depicted, including (but not limited to) strand displacement (A), fluorescence resonance energy transfer (B), aptamer beacons (C), or fluorescent dyes (D). After an aptamer binds to its specific target, (A) a quencher bound to the aptamer (which quenches the emission of a fluorophore also attached to the aptamer) is displaced by the target and enables quantification of target binding based on fluorescence; (B) a change in the dipole-dipole interaction between a donor and an acceptor both attached to the aptamer (e.g., due to a change in the distance between the donor and the acceptor), which enables FRET detection through the appearance of sensitized and excited fluorescence; (C) a donor-quencher pair (e.g., fluorescent dyes, quantum dots, carbon-based materials, and metal nanoparticles) that acts as an optical on / off switch for aptamer changes, enabling visualization of specific binding; and / or (D) a fluorescent dye can further bind to the aptamer-target complex to produce a detectable fluorescent signal. Other detection methods are known in the art and can be selected according to actual needs.

[0052] The target to be detected can be in collected tears, extracted animal tissues, and extracted cell lysates or culture medium. Tears form the outermost layer of the ocular surface, and their characteristics and composition are associated with various ocular surface diseases. Because tear proteomics enables the non-invasive study of protein levels in tears, it has become an increasingly popular method in the study of ocular surface and systemic diseases. [5] Therefore, tears are an important target for the aptamers of the present disclosure for ocular applications.

[0053] Figure 3 The mechanism of detecting ocular drugs (atropine) in tears using an aptamer-based biosensor is demonstrated. The atropine-specific aptamer can be added to and mixed with a sample (preferably a tear sample) containing or suspected of containing atropine to be detected. After the aptamer specifically binds to atropine, the quencher-labeled chain leaves the FAM-labeled atropine-specific aptamer and generates a fluorescent signal.

[0054] Possible targets for aptamers for ocular applications include, but are not limited to, those listed in Table 1. [5,6] In some embodiments, the target of the aptamer is an ophthalmic drug, such as selected from drugs used for glaucoma care, corticosteroids, antibiotics, combination drugs, drugs used for ocular surface care, allergy drugs, drugs used for shingles therapy, and drugs used for visual insight. Table 1. Examples of DNA aptamer targets for ocular applications

[0055] A noteworthy innovation introduced by the present invention is the development of aptamer biosensors designed specifically for ocular applications that utilize fluorescent or radioactive signals. For example, conventional UV-vis analysis may encounter interference from various molecules or hydrogel materials that absorb at 257nm, potentially compromising the accuracy of atropine detection. Aptamer-based methods offer enhanced specificity because aptamers can selectively bind to their targets, resulting in lower limits of detection compared to UV-vis. In addition, aptamer-based detection is generally less complex and more cost-effective in terms of material and equipment use when compared to HPLC-MS.

[0056] In addition, Wang Z. et al. disclosed an aptamer-based graphene affinity nanobiosensor for detecting inflammatory markers in the eye. The nanobiosensor is a graphene field-effect transistor in which nucleic acid aptamers and biomolecule-permeable polyethylene glycol (PEG) nanolayers are immobilized on the graphene surface. [7] Wang Y et al. disclosed aptamer-based liquid crystal films on glass supports for the detection of kanamycin. [8] However, detection in both preparations requires aptamer attachment on the surface. In contrast, the simple detection method of the present disclosure can be performed with or without aptamer attachment to the surface.

[0057] As a specific embodiment, the present invention can be used to detect atropine in tears to monitor drug delivery efficiency. As another specific embodiment, the present invention can be used to detect timolol maleate in tears to monitor drug delivery efficiency or drug maintenance in the target. These embodiments are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way. Example

[0058] The present invention is further illustrated by the following examples. These examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the present invention in any way.

[0059] The publications cited herein and the materials cited therein are incorporated herein by reference in their entirety. Unless otherwise stated, all reagents are commercially available. Unless otherwise stated, all parts and percentages are by weight. Unless otherwise stated, the average value of the results is provided. Unless otherwise defined, the abbreviations used herein are conventional.

[0060] Materials and methods

[0061] 1. Materials

[0062] DNA sequences used for selection and sensing experiments were synthesized by commercial companies such as Integrated DNA Technologies. Agarose resin coated with streptavidin was purchased from Thermo Scientific. 3k and 10k Ultra-0.5 centrifugal filter units were purchased from Millipore-Sigma. Micro bio-spin columns and SsoFast EvaGreen supermix were from Bio-Rad. dNTP mix and Taq DNA polymerase and ThermoPol buffer were from New England Biolabs. Atropine and timolol maleate were purchased from Sigma-Aldrich.

[0063] 2. Methods

[0064] 2.1 Capture-SELEX for selection of aptamers

[0065] 2.1.1 Preparation of DNA library The DNA library contains a randomized region of 30 nucleotides flanked by primer binding sequences. The length of the randomized nucleotides can vary between 10 and 60.

[0066] 2.1.2 Preparation of target solution, selection buffer and separation buffer Prepare target solution by dissolving target in selection buffer. Two buffers are used. The first contains 1× PBS, 1 mM MgCl 2 , 5 mM KCl and 1 mM CaCl 2 A selection buffer of pH 7.5 is used for binding of DNA to the target. Another buffer is a separation buffer of pH 7.5 containing 1× PBS and 5 mM KCl for strand separation. 8 mM EDTA is used to dissociate the bound DNA from the target. The concentrations of salt and EDTA can be optimized for different targets.

[0067] 2.1.3 Capture-SELEX Procedure Before starting aptamer selection, a target stock solution was prepared using selection buffer. First, the DNA library was bonded to the biotinylated capture strand in selection buffer and then cooled to room temperature and subsequently stored at -20°C. The agarose resin coated with streptavidin was then introduced into a microbiospin column and six washing cycles were performed with selection buffer to eliminate any residual preservatives. The prepared biotin-DNA complex was then introduced into the agarose resin and subjected to multiple loading cycles of more than six times. This was followed by 12 washing cycles with selection buffer to remove unbound or weakly bound sequences. The target working solution was applied to the library at room temperature. After the aptamer binds to the target, the aptamer-target complex is released from the resin and eluted with the target solution. The eluted DNA was collected by gravity flow. The eluate was then concentrated and further purified using a 3k filter for PCR, where the PCR product was additionally concentrated and purified using a 10k filter. The purified PCR product was loaded onto a clean agarose resin and another wash was performed with separation buffer. The agarose resin bound to the DNA is treated with 0.2 M NaOH, and the eluate is concentrated and purified again using a 3k filter. Finally, the concentration of the collected single-stranded DNA is confirmed and used for a subsequent round of selection, which is usually repeated at least 12 times. The sequences collected during the last round of selection are sequenced.

[0068] 2.2 Isothermal titration calorimetry (ITC) ITC was performed using MicroCal VP-ITC. All aptamers and target molecules were prepared in selection buffer. The DNA was bonded, cooled to room temperature, and degassed for 5 minutes before loading. The target solution was then loaded into the syringe and the aptamer was injected into the cell chamber. In addition to the initial injection of 0.5 μL, 10 μL of target solution was titrated into the cells each time over a duration of 20 seconds at 25 °C, for a total of 28 injections. The interval between injections was set to 360 seconds. The syringe stirring speed was maintained at 90 rpm (low speed) to prevent bubble formation, which is critical to the success of the ITC experiment. The binding constant was determined by fitting the titration curve to a single-site binding model using Origin software.

[0069] 2.3 Aptamer-based biosensor assay In the standard procedure, prepare 500 μL of 5 mM MgCl 2PBS and a mixture of 1 μM aptamer and 10 μM thioflavin T (ThT). This mixture was then transferred to a quartz cuvette. Subsequently, atropine with an initial concentration of 6000 ppm or 20.7 mM was gradually added to the solution until a final concentration of 400 μM was reached in the reaction. Throughout the titration, fluorescence measurements were performed with an excitation wavelength set at 400 nm, while emission was monitored between 460 nm and 550 nm. The recorded fluorescence value at 490 nm was used exclusively for subsequent analysis. ThT shows fluorescence when bound to DNA. When atropine was introduced into the solution, ThT was displaced from the DNA binding site and the fluorescence value decreased. Therefore, the decrease in fluorescence was used as an indication of the binding interaction between the aptamer and atropine. The limit of detection (LOD) of the experiment was determined using the formula [LOD = 3.3σ / S], where σ represents the standard deviation of the blank and S represents the slope of the calibration curve.

[0070] 2.4 Ultraviolet-visible (UV-vis) spectroscopy First, a 6000 ppm (6 mg / mL or 20.7 mM) atropine solution was diluted with PBS to different concentrations of 0.6, 0.3, 0.24, 0.2, and 0.17 mg / mL. Subsequently, a 100 μL aliquot of each solution was transferred to a separate cuvette for UV-vis analysis. UV absorbance readings were recorded in the spectral range of 200 to 600 nm, with particular attention paid to the absorbance at 257 nm for the calibration curve ( Figure 5 (A)). Finally, the limit of detection (LOD) was calculated using the formula [LOD = 3.3σ / S], where σ represents the standard deviation and S represents the slope.

[0071] Example 1. Aptamer selection for timolol maleate and atropine

[0072] Aptamer selection against timolol maleate and atropine was performed by Capture-SELEX following the procedure disclosed in Section 2.1 above.

[0073] Tables 2 and 3 show the sequencing results of the analysis. The most abundant sequences underwent ITC analysis to confirm their binding to their respective targets ( Figure 4 ). Table 2. The top 11 sequences from timolol maleate-SELEX in the sequencing results of the enrichment pool Note: The sequences are designated as SEQ ID NO: 1-11 from top to bottom. A read refers to a DNA sequence from a fragment (a small portion of DNA). The percentage is determined by calculating (number of reads corresponding to a specified sequence / total number of reads obtained in sequencing). This calculation estimates the relative abundance of the targeted sequence within the sample. Table 3. Top 10 sequences from atropine-SELEX in the sequencing results of the enrichment pool Note: The sequences are designated as SEQ ID NO: 12-21 from top to bottom. A read refers to a DNA sequence from a fragment (a small portion of DNA). The percentage is determined by calculating (number of reads corresponding to a specified sequence / total number of reads obtained in sequencing). This calculation assesses the relative abundance of the targeted sequence within the sample.

[0074] The results demonstrate that the library capture SELEX method of the present application can be used for the efficient selection of aptamers and provides enhanced robustness and improved success rate.

[0075] Example 2. Aptamer-based biosensor for atropine

[0076] To evaluate the detection efficiency of the aptamer for atropine, we used ThT as a probe to demonstrate the binding of the aptamer to atropine according to the procedure disclosed in Section 2.3 above. In the absence of a target, ThT binds to DNA, producing green fluorescence detectable by fluorescence spectroscopy. We titrated atropine into the aptamer. The binding of atropine to the aptamer caused a decrease in fluorescence due to the displacement of ThT. The detection limit was then determined. This limit was then compared with the limit obtained by the traditional UV-vis method according to the procedure disclosed in Section 2.4 above.

[0077] like Figure 5 As shown, the detection limit of UV-vis was 0.1 mM (see (A)), while the aptamer-based ThT biosensor exhibited a detection limit of 0.00018 mM (see (B)).

[0078] The results show that the aptamer-based biosensor has higher sensitivity and specificity than conventional UV-vis methods for detecting small molecules. These features make the present invention easy to implement and reproduce.

[0079] The scope of the present invention is not limited by the embodiments disclosed herein, which are intended to be a single illustration of various aspects of the present invention, and any functionally equivalent embodiments are within the scope of the present invention. In addition to the compositions and methods described herein, various modifications to the compositions and methods of the present invention will become apparent to those skilled in the art from the foregoing description and teachings, and are also intended to fall within the scope of the present invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of the present invention. References 1. Ni, S. et al., Recent Progress in Aptamer Discoveries and Modifications for Therapeutic Applications. ACS Appl Mater Interfaces, 2021.13(8): 9500-9519. 2. Kohlberger, M. and Gadermaier, G. SELEX: Critical factors and optimization strategies for successful aptamer selection. Biotechnol Appl Biochem, 2022. 69(5): 1771-1792. 3. Perez Tobia, J. et al., Machine Learning Directed Aptamer Search from Conserved Primary Sequences and Secondary Structures. ACS Synthetic Biology, 2023.12(1): 186-195. 4. Zhuo, Z. et al., Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine. International Journal of Molecular Sciences, 2017.18(10):2142. 5. Patrick, R., Thomas, R., and Vollmer, P. 2019 Ophthalmic Drug Guide. Review of Optometry, 2019. 6. Nattinen, J. et al., Clinical Tear Fluid Proteomics-A Novel Tool in Glaucoma Research. Int J Mol Sci, 2022. 23(15). 7. Wang Z, Dai W, Yu S, et al. Towards detection of biomarkers in the eye using an aptamer-based graphene affinity nanobiosensor. Talanta 2022;250:123697. 8. Wang Y, Wang B, Shen J, et al. Aptamer based bare eye detection of kanamycin by using a liquid crystal film on a glass support. Microchimica Acta (1966) 2017; 184: 3765-3771.

Claims

1. A DNA or RNA aptamer for detecting an ocular target compound, wherein the aptamer comprises: A DNA or RNA sequence that selectively binds to the ocular target compound; and attached or not attached to a detectable tag; And wherein the detection is fluorescence detection or radioactivity detection.

2. The DNA or RNA aptamer according to claim 1, wherein the aptamer is capable of binding to the following target compounds: drugs, biomarkers and other small molecules for ocular applications. 3 . The DNA or RNA aptamer according to claim 1 , wherein the aptamer is capable of binding to the target compound at multiple sites. The DNA or RNA aptamer according to claim 1 , wherein the aptamer is capable of binding to a plurality of identical target compounds. 5 . The DNA or RNA aptamer according to claim 1 , wherein the aptamer is capable of binding to two or more different target compounds simultaneously.

6. The DNA or RNA aptamer according to claim 1, wherein the tag is radioactive, fluorescent or chromophore; and / or The detection is based on one or more of strand displacement, fluorescence resonance energy transfer, aptamer beacons and fluorescent dyes.

7. The DNA or RNA aptamer according to claim 1, wherein the aptamer is attached to or immobilized on a surface; or, is not attached to or immobilized on a surface.

8. The DNA or RNA aptamer according to claim 1, which is used for: detecting drug release on the eye or from a biological material; detecting biomarkers from the tear film; and / or detecting a target on the surface of the eye. 9 . The DNA or RNA aptamer according to claim 1 , wherein the aptamer is a timolol maleate-specific aptamer or an atropine-specific aptamer.

10. The DNA or RNA aptamer according to claim 9, wherein the timolol maleate-specific aptamer is selected from any one of SEQ ID NOs: 1-11 or an aptamer represented by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1-11; and / or wherein the atropine-specific aptamer is selected from any one of SEQ ID NOs: 12-21 or an aptamer represented by a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 12-21.

11. The DNA or RNA aptamer according to claim 1, which is immobilized on a surface, such as a test line, magnetic beads, microplate, nanoparticles or microchip, as a high-throughput immobilized aptamer-based biosensor.

12. The DNA or RNA aptamer according to claim 1, wherein the aptamer is used for chromophore detection of an ocular target compound.

13. A method for detecting an ocular target compound in a sample, wherein the method comprises contacting the sample with a DNA or RNA aptamer targeting the ocular target compound, wherein the aptamer comprises: a DNA or RNA sequence that selectively binds to the ocular target compound of interest; and is or is not linked to a detectable label; and wherein the detection is fluorescent detection or radioactive detection.

14. The method of claim 13, wherein the sample is an in vivo, in vitro or ex vivo sample; and / or wherein the sample is an ocular sample, such as from the eye or an ocular region in the eye, tears, tear film, ocular discharge; and / or wherein the target compound is one or more of a biomarker, a drug, a metabolite, a toxin, an environmental pollutant, a protein, a nucleic acid, a cell or a tissue; and / or The detection is selected from: drug detection in tears, animal tissues and human samples for monitoring drug delivery efficacy; biomarker detection in the eye for disease diagnosis and disease progression; biomarker detection for eye imaging.

15. The method of claim 13, wherein the detection of the target compound is based on detection of strand displacement, fluorescence resonance energy transfer, aptamer beacons or fluorescent dyes; or by chromophore detection.

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