Dnazymes for on-site portable detection of lithium

Abstract

A DNAzyme sensor for detecting Li+, kits including said sensor, and methods of using the same.

Description

Attorney Docket No. 10046-635W01DNAZYMES FOR ON-SITE PORTABLE DETECTION OF LITHIUMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 689,122, filed August 30, 2024, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. R35 GM141931 awarded by the National Institutes of Health. The Government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on August 29, 2025, as an .XML file entitled “10046-635W01_ST26.xml” created on August 5, 2025, and having a file size of 22,710 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] The past decade has witnessed an enormous expansion of applications for lithium- ion batteries (LIB), leading to a high demand in the search for additional lithium resources as well as the development of methods for lithium recycling [1,2]. There are two major mining resources from which this highly valuable metal can be extracted: high-lithium brines (natural brines or enriched industrial brines), which account for approximately 60% of global lithium, and pegmatite ores (spodumene and other high-lithium rocks and clays), which account for approximately 20% of global lithium [3]. Additionally, these resources are rare and notably geographically constrained, making the accurate and timely identification of commercially valuable lithium reserves a top priority in the LIB industry [4]. The third and last lithium resource comes from the opposing end of the LIB industrial process, battery recycling. However, not all recycled LIBs are cost-effective for lithium extraction, as the amount and quality of remaining and extractable lithium can vary between aged electrodes [5,6]. To meet the demands of improving both the search of more lithium resources and the efficiency of lithium recycling, an on-site portable method of detection for lithium will play a major role.

[0005] Thus, there is a need for improved methods of detecting lithium. This need and others are at least partially satisfied by the present disclosure.Attorney Docket No. 10046-635W01SUMMARY

[0006] In an aspect, provided is a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal.

[0007] In another aspect, provided is a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; wherein the substrate strand can include, from 5’ to 3’, a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid can include from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid can include from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid by at least one nucleotide.

[0008] In yet another aspect, provided is a DNAzyme sensor including: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; wherein the substrate strand can include SEQ ID NO: 1, SEQ ID NO: 2 or a variant thereof; and wherein the enzyme strand can include SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof.

[0009] In yet still another aspect, provided is a kit including: a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; and a leaching agent, a precipitating agent, and / or a chelating agent.

[0010] In yet still another aspect, provided is a method of detecting Li+, the method including: a) exposing a sample to any of the disclosed DNAzyme sensors; and b) identifying the detectable signal, thereby detecting Li+in the sample.Attorney Docket No. 10046-635W01

[0011] In yet still another aspect, provided is a method of detecting Li+in a sample, including: a) treating the sample with a leaching agent, a precipitating agent, and / or a chelating agent; and b) exposing the sample to a DNAzyme sensor, the DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; and c) identifying the detectable signal, thereby detecting Li+in the sample.

[0012] In yet still another aspect, provided is a method of determining usefulness of a test compound in modulating Li+, the method including: a) exposing the test compound to a sample; b) exposing the sample to any of the disclosed DNAzyme sensors; c) identifying the detectable signal, thereby detecting Li+in the sample; and d) using said detectable signal to determine an effect of the test compound on Li+.

[0013] In yet still another aspect, provided is a method of treating and / or preventing dysregulation of Li+in a subject in need thereof, the method including: a) administering to the subject any of the disclosed DNAzyme sensors; b) identifying the detectable signal, thereby detecting Li+in the subject; and c) using said detectable signal to trigger a therapeutic event.

[0014] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0015] FIGURES 1A-1D depict the performance of the original Li+sensor and comparisons with the new 20-4RT DNAzyme sensor. FIGS. 1A-1B show the sequences and Tm’s of the original Li+sensor (substrate strand: SEQ ID NO: 1) (enzyme strand: SEQ ID NO: 3) (FIG. 1A) and the 20-4RT DNAzyme sensor (substrate strand: SEQ ID NO: 2) (enzyme strand: SEQ ID NO: 4) (FIG. IB). FIG. 1C shows the activity of the original 20-4 (red) and the new 20-4RT (blue) DNAzyme sensors. FIG. ID shows the 12-hour endpoint fold intensity increase of Li+sensors in response to different types of metal ions (200 mM monovalent metal ions or 4 mM for divalent metal ions).

[0016] FIGURE 2 depicts 20-4RT sensor performance at higher temperatures. As in-the- field temperatures may be higher than room temperature, sensor performance was evaluated at 40°C.Attorney Docket No. 10046-635W01

[0017] FIGURES 3A-3D depict the effects of non-target metal ions on the activity of the 20-4RT DNAzyme sensor, specifically the 20-4RT DNAzyme sensor activity under 500 mM of Na+(FIG. 3A), 500 mM of K+(FIG. 3B), 10 mM of Mg2+(FIG. 3C), or 10 mM Ca2+(FIG. 3D) metal ions.

[0018] FIGURE 4A shows 20-4RT sensor performance under 500 mM Na+. FIGURE 4B shows 20-4RT sensor performance under 500 mM K+. FIGURE 4C shows 20-4RT sensor performance under 10 mM Mg2+. FIGURE 4D shows 20-4RT sensor performance under 10 mM Ca2+.

[0019] FIGURE 5 depicts sensor performance on two saline solutions with or without spiked Li+. Solution A is a saline combining high concentrations of Na+, K+, Mg2+, and Ca2+as tested in FIGS. 3A-3D. Solution B is the brine saline.

[0020] FIGURES 6A-6C depict on-site portable detection of Li+in brine samples. FIG. 6A shows sensor performance on samples prepared by cation reductions at pH 11 and further Ca2+reduction by EDTA or CO32" (PPT = precipitate). FIG. 6B is a scheme showing the steps of on-site Li+detection in brine water. FIG. 6C shows the detection of Li+in brine water samples using the portable 20-4RT DNAzyme sensor.

[0021] FIGURE 7A shows broad-range titration of EDTA concentrations for Ca2+reduction on brine samples. Sensor performance on brine solutions with or without spiked Li+with Ca2+reduction by the EDTA method. EDTA concentrations ranging from 0 to 10 mM were tested. FIGURE 7B shows narrow-range titration of EDTA concentrations for Ca2+reduction on brine samples. Sensor performance on brine solutions with or without spiked Li+with Ca2+reduction by the EDTA method. EDTA concentrations ranging from 3 to 6 mM were tested.

[0022] FIGURE 8 shows sensor concentration data.

[0023] FIGURES 9A-9E depict on-site portable detection of Li+in rock samples. FIG. 9A shows Li+leaching efficiency from spodumene and other materials by ICP-MS. FIG. 9B shows a comparison of signal increase using different neuralization methods. FIG. 9C shows a comparison of sensor performance on neutralized samples with or without EDTA treatment. FIG. 9D is a scheme showing the steps of on-site Li+detection in rock samples. FIG. 9E detection of Li+in rock samples using the portable 20-4RT DNAzyme sensor. Samples with different percentage of Li2O content were prepared by premixing pulverized spodumene (8% LizO content) with pulverized limestone (0% LizO content) at different ratios.

[0024] FIGURE 10 shows titration of EDTA concentrations for Ca2+reduction on neutralized samples. Sensor performance on solutions mimicking neutralized rock leachingAttorney Docket No. 10046-635W01 samples and method control samples with EDTA-based Ca2+reduction. EDTA concentrations ranging from 0 to 5 mM were tested.

[0025] FIGURES 11A-11B depict on-site portable detection of Li+in battery electrodes. FIG. 11A is a scheme showing the steps of on-site Li+detection in battery electrodes. FIG. 11B shows differentiating different levels of Li+in electrode samples from new and aged batteries using the portable 20-4RT DNAzyme sensor.

[0026] FIGURE 12 shows that the new 20-4RT sensor can detect and differentiate Li+in live cells at lower levels.

[0027] FIGURE 13 shows that the statistical results of live cell imaging showed the new 20-4RT sensor can detect and differentiate Li+in live cells at 0.5 mM from the background.

[0028] FIGURE 14 shows performance of the 20-4RT DNAzyme sensor under 5 mM Mg2+.

[0029] FIGURE 15 shows performance of the 20-4RT DNAzyme sensor under 5 mM Ca2+.

[0030] FIGURE 16 depicts titration of EDTA concentrations for Ca2+reduction on neutralized samples. Sensor performance on solutions mimicking neutralized rock leaching samples and method control samples with EDTA-based Ca2+reduction. EDTA concentrations ranging from 0 to 5 mM were tested.DETAILED DESCRIPTION

[0031] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0032] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0033] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof.Attorney Docket No. 10046-635W01Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0034] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0035] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0036] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y ’ as well as the range greater than x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0037] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numericalAttorney Docket No. 10046-635W01 values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0. 1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0038] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can he the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0039] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0040] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, bodyAttorney Docket No. 10046-635W01 weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0041] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0042] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.Attorney Docket No. 10046-635W01

[0043] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0044] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0045] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0046] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0047] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.Attorney Docket No. 10046-635W01

[0048] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0049] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0050] Reference is made herein to nucleic acid and nucleic acid sequences. The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be singlestranded or double- stranded and may represent the sense or the antisense strand).

[0051] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0052] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5 '-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.Attorney Docket No. 10046-635W01

[0053] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0054] Fusion polynucleotides also are contemplated herein. A “fusion polynucleotide” refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5' end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0055] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.

[0056] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).Attorney Docket No. 10046-635W01

[0057] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0058] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0059] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide. A variant polynucleotide may have substantially the same functional activity as a reference polynucleotide.

[0060] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0061] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0062] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segmentsAttorney Docket No. 10046-635W01 of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0063] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0064] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0065] The term “mismatched” or “mismatched target sequence” refers to an off-target sequence that is not perfectly complementary to the first DNA sequence or the second DNA sequence of the chimeric deoxyribonucleic acid described herein. The dual retargeted DNA may have at least one mismatch, but can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides to the off-target sequence.

[0066] As used herein, the term “at least partially complementary” refers to two nucleic acid sequences which are partially or fully complementary. For example, two sequences may be “at least partially complementary” if at least about 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least aboutAttorney Docket No. 10046-635W0198%, at least about 99%, about 100%) of one sequence is complementary to at least about 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%) of the other sequence.

[0067] As used herein, the term “detecting” used in context of detecting a signal from a detectable label or detectable signal to indicate the presence of a target nucleic acid (such as a signaling nucleic acid) in the sample does not require the method to provide 100% sensitivity and / or 100% specificity. As is well known, “sensitivity” is the probability that a test is positive, given that the sample has a target nucleic acid sequence, while “specificity” is the probability that a test is negative, given that the sample does not have the target nucleic acid sequence. A sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. A specificity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. Detecting also encompasses assays with false positives and false negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% or even higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or even higher. The term “detecting” is also used in the context of detecting the amplified target nucleic acid by its melting temperature using melting curve analysis, as is known in the art.

[0068] As used herein, “detectable labels” or “detectable signals” are chemical or biochemical moi eties useful for labeling a nucleic acid (including a single nucleotide), amino acid, or antibody. “Detectable labels” or “detectable signals” include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionuclides, enzymes, substrates, cofactors, inhibitors, magnetic particles, quantum dots, and other moieties known in the art. “Detectable labels” or “detectable signals” are capable of generating a measurable signal, or can be used to capture nucleic acids, and may be covalently or noncovalently joined to an oligonucleotide or nucleotide (e.g., a non-natural nucleotide).

[0069] A detectable label or detectable signal is said to be “activated” when it is completely or partially activated (i.e., detectable). For example, a detectable label or detectable signal can be said to be “activated” when the intensity of its measurable signal is greater than about 50% (e.g., greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than aboutAttorney Docket No. 10046-635W0197%, greater than about 98%, greater than about 99%, about 100%) of the expected intensity of its measurable signal based on a reasonable assumption by one of skill in the art. It is understood that the threshold to classify a detectable label or detectable signal as “activated” can be reasonably ascertained by one of skill in the art depending on the application and properties (e.g., sensitivity, specificity, etc.) of the detectable label or detectable signal.

[0070] A detectable label or detectable signal is said to be “deactivated” when it is completely or partially deactivated (i.e., not detectable). For example, a detectable label or detectable signal can be said to be “deactivated” when the intensity of its measurable signal is less than about 50% (e.g., less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, about 0%) of the expected intensity of its measurable signal based on a reasonable assumption by one of skill in the art. It is understood that the threshold to classify a detectable label or detectable signal as “deactivated” can be reasonably ascertained by one of skill in the art depending on the application and properties (e.g., sensitivity, specificity, etc.) of the detectable label or detectable signal.

[0071] As used herein, the term “DNAzymes,” also called “deoxyribozymes,” are DNA molecules that display enzymatic activities, such as protein enzymes and ribozymes, in the presence of a cofactor such as metal ions or another target molecule.DNAZYMES

[0072] In recent years, the global demand for lithium has soared, surpassing the development of relevant technologies related to its sourcing and recycling. The identification of additional commercially valuable lithium reserves in the environment and more effective lithium recycling methods are thus in urgent need. To meet this need in lithium-driven industries, disclosed herein is a highly Li+selective DNAzyme for the detection of lithium in brine water, spodumene rock, and lithium-ion battery electrodes down to 1.4 mM (10 ppm) using portable devices. The methods developed and demonstrated in this work can allow highly selective, on-site, portable detection of lithium in both environmental samples to identify new lithium resources and in battery electrodes to guide recycling strategies in order to meet the global demand for lithium.

[0073] In an aspect, provided is a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to theAttorney Docket No. 10046-635W01 substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal.

[0074] In another aspect, provided is a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; wherein the substrate strand can include, from 5’ to 3’, a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid can include from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid can include from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid by at least one nucleotide.

[0075] In yet another aspect, provided is a DNAzyme sensor including: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; wherein the substrate strand can include SEQ ID NO: 1, SEQ ID NO: 2 or a variant thereof; and wherein the enzyme strand can include SEQ ID NO: 3, SEQ ID NO: 4 or a variant thereof.TABLE 1. DNAzyme sensors.Attorney Docket No. 10046-635W01

[0076] In some aspects, the cleavage site can be interspersed between two segments of nucleic acid. In some aspects, the two segments of nucleic acid can have a same length. In other aspects, the two segments of nucleic acid can have different lengths.

[0077] In some aspects, each of the two segments of nucleic acid can include about 3 or more nucleotides (e.g., about 4 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more). In some aspects, each of the two segments of nucleic acid can include about 90 or less nucleotides (e.g., about 80 or less, about 70 or less, about 60 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less, about 5 or less, about 4 or less, about 3 or less).

[0078] It is considered that each of the two segments of nucleic acid can include a number of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, each of the two segments of nucleic acid can include from about 3 to about 90 nucleotides (e.g., from about 4 to about 80, from about 5 to about 70, from about 10 to about 60, from about 15 to about 50, from about 20 to about 45, from about 25 to about 40, from about 30 to about 35, from about 3 to about 35,Attorney Docket No. 10046-635W01 from about 4 to about 30, from about 5 to about 25, from about 10 to about 20, from about 30 to about 90, from about 35 to about 80, from about 40 to about 70, from about 45 to about 60).

[0079] In some aspects, the first segment of nucleic acid can include about 3 or more nucleotides (e.g., about 4 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more). In some aspects, the first segment of nucleic acid can include about 90 or less nucleotides (e.g., about 80 or less, about 70 or less, about 60 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less, about 5 or less, about 4 or less, about 3 or less).

[0080] It is considered that the first segment of nucleic acid can include a number of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the first segment of nucleic acid can include from about 3 to about 90 nucleotides (e.g., from about 4 to about 80, from about 5 to about 70, from about 10 to about 60, from about 15 to about 50, from about 20 to about 45, from about 25 to about 40, from about 30 to about 35, from about 3 to about 35, from about 4 to about 30, from about 5 to about 25, from about 10 to about 20, from about 30 to about 90, from about 35 to about 80, from about 40 to about 70, from about 45 to about 60).

[0081] In some aspects, the second segment of nucleic acid can include about 10 or more nucleotides (e.g., about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more). In some aspects, the second segment of nucleic acid can include about 90 or less nucleotides (e.g., about 80 or less, about 70 or less, about 60 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less).

[0082] It is considered that the second segment of nucleic acid can include a number of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the second segment of nucleic acid can include from about 10 to about 90 nucleotides (e.g., from about 15 to about 80, from about 20 to about 70, from about 25 to about 60, from about 30 to about 50, from about 35 to about 45, from about 10 to about 40, from about 15 to about 35, from about 20 to about 30, from about 40 to about 90, from about 45 to about 80, from about 50 to about 70).

[0083] In some aspects, the second segment of nucleic acid can be longer than the first segment of nucleic acid by at least one nucleotide (e.g., about 2 or more, about 3 or more, aboutAttorney Docket No. 10046-635W014 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 35 or more, about 40 or more, about 45 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more). In some aspects, the second segment of nucleic acid can be longer than the first segment of nucleic acid by about 80 or less nucleotides (e.g., about 70 or less, about 60 or less, about 50 or less, about 45 or less, about 40 or less, about 35 or less, about 30 or less, about 25 or less, about 20 or less, about 15 or less, about 10 or less, about 5 or less, about 4 or less, about 3 or less, about 2 or less, about 1 or less).

[0084] It is considered that the second segment of nucleic acid can be a number of nucleotides longer than the first segment of nucleic acid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the second segment of nucleic acid can be longer than the first segment of nucleic acid by from about 1 to about 80 nucleotides (e.g., from about 2 to about 70, from about 3 to about 60, from about 4 to about 50, from about 5 to about 45, from about 10 to about 40, from about 15 to about 35, from about 20 to about 30, from about 1 to about 25, from about 2 to about 20, from about 3 to about 15, from about 4 to about 10, from about 25 to about 80, from about 30 to about 70, from about 35 to about 60, from about 40 to about 50).

[0085] In some aspects, the first segment can have a melting temperature of at least about 10°C (e.g., at least about 11°C, at least about 12°C, at least about 13°C, at least about 14°C, at least about 15°C, at least about 16°C, at least about 17°C, at least about 18°C, at least about 19°C, at least about 20°C, at least about 21 °C, at least about 22°C, at least about 23°C, at least about 24°C, at least about 25 °C, at least about 26°C, at least about 27°C, at least about 28°C, at least about 29°C, at least about 30°C). In some aspects, the first segment can have a melting temperature of up to about 30°C (e.g., up to about 29°C, up to about 28°C, up to about 27°C, up to about 26°C, up to about 25°C, up to about 24°C, up to about 23°C, up to about 22°C, up to about 21°C, up to about 20°C, up to about 19°C, up to about 18°C, up to about 17°C, up to about 16°C, up to about 15°C, up to about 14°C, up to about 13°C, up to about 12°C, up to about 11 °C, up to about KFC).

[0086] It is considered that the first segment can have a melting temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the first segment can have a melting temperature of from about KFC to about 30°C (e.g., from about 11 °C to about 29°C, from about 12°C to about 28°C, from about 13°C to about 27°C, from about 14°C to about 26°C, from about 15°C to about 25°C, from about 16°C to about 24°C, from about 17°C to about 23°C, from about 18°C toAttorney Docket No. 10046-635W01 about 22°C, from about 19°C to about 21 °C, from about 10°C to about 20°C, from about 11 °C to about 19°C, from about 12°C to about 18°C, from about 13°C to about 17°C, from about 14°C to about 16°C, from about 20°C to about 30°C, from about 21°C to about 29°C, from about 22°C to about 28°C, from about 23°C to about 27°C, from about 24°C to about 26°C).

[0087] In some aspects, the second segment can have a melting temperature of at least about 30°C (e.g., at least about 32°C, at least about 34°C, at least about 36°C, at least about 40°C, at least about 42°C, at least about 44°C, at least about 46°C, at least about 48°C, at least about 50°C, at least about 52°C, at least about 54°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 62°C, at least about 64°C, at least about 66°C, at least about 68°C, at least about 70°C). In some aspects, the second segment can have a melting temperature of up to about 70°C (e.g., up to about 68°C, up to about 66°C, up to about 64°C, up to about62°C, up to about 60°C, up to about 58°C, up to about 56°C, up to about 54°C, up to about52°C, up to about 50°C, up to about 48°C, up to about 46°C, up to about 44°C, up to about42°C, up to about 40°C, up to about 38°C, up to about 36°C, up to about 34°C, up to about32°C, up to about 30°C).

[0088] It is considered that the second segment can have a melting temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the second segment can have a melting temperature of from about 30°C to about 70°C (e.g., from about 32°C to about 68°C, from about 34°C to about 66°C, from about 36°C to about 64°C, from about 38°C to about 62°C, from about 40°C to about 60°C, from about 42°C to about 58°C, from about 44°C to about 56°C, from about 46°C to about 54°C, from about 48°C to about 52°C, from about 30°C to about 50°C, from about 32°C to about 48°C, from about 34°C to about 46°C, from about 36°C to about 44°C, from about 38°C to about 42°C, from about 50°C to about 70°C, from about 52°C to about 68°C, from about 54°C to about 66°C, from about 56°C to about 64°C, from about 58°C to about 62°C).

[0089] In some aspects, the substrate strand can include SEQ ID NO: 1. In some aspects, the substrate strand can include a variant of SEQ ID NO: 1. In some aspects, the substrate strand can include SEQ ID NO: 2. In some aspects, the substrate strand can include a variant of SEQ ID NO: 2.

[0090] In some aspects, the enzyme strand can include SEQ ID NO: 3. In some aspects, the enzyme strand can include a variant of SEQ ID NO: 3. In some aspects, the enzyme strand can include SEQ ID NO: 4. In some aspects, the enzyme strand can include a variant of SEQ ID NO: 4.Attorney Docket No. 10046-635W01

[0091] In some aspects, the substrate strand and / or the enzyme strand can include DNA. In some aspects, the substrate strand can further include at least one non-natural nucleic acid. For example, in some such aspects, the at least one non-natural nucleic acid can be a locked nucleic acid (LNA) or a 2’ -fluoro arabino nucleic acid (FANA).

[0092] In some aspects, the cleavage site can include at least one RNA base. For example, in some such aspects, the cleavage site can include 1 RNA base, 2 RNA bases, 3 RNA bases, 4 RNA bases, or 5 RNA bases. In some such aspects, the cleavage site can include from 1 RNA base to 5 RNA bases (e.g., from 2 RNA bases to 5 RNA bases, from 3 RNA bases to 5 RNA bases, from 4 RNA bases to 5 RNA bases, from 1 RNA base to 4 RNA bases, from 2 RNA bases to 4 RNA bases, from 3 RNA bases to 4 RNA bases, from 1 RNA base to 3 RNA bases, from 2 RNA bases to 3 RNA bases, from 1 RNA base to 2 RNA bases). In other such aspects, the cleavage site can include more than 5 RNA bases. In some aspects, when more than one RNA base is present, all RNA bases may be immediately adjacent. In other aspects, when more than one RNA base is present, at least one RNA base may be distanced from the other RNA bases by at least one other nucleotide.

[0093] In some aspects, the enzyme strand can include at least one loop region. In some aspects, the enzyme strand can include a Li+binding region.

[0094] In some aspects, the detectable signal can be a fluorophore or a fluorescent dye. In some such aspects, the fluorophore or fluorescent dye can be, but is not limited to Hydroxycoumarin, Alexa fluor, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, Alexa fluor 430, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, Cy2, TruRed, FluorX, Fluorescein, FAM, BODIPY-FL, TET, Alexa fluor 532, HEX, TRITC, Cy3, TMR, Alexa fluor 546, Alexa fluor 555, Tamara, X-Rhodamine, Lissamine Rhodamine B, ROX, Alexa fluor 568, Cy3.5 581, Texas Red, Alexa fluor 594, Alexa fluor 633, LC red 640, Allophycocyanin (APC), Alexa fluor 633, APC-Cy7 conjugates, Cy5, Cy5.5, LC red 705, Cy7, IRDye 800 CW, IRDye 700, Cy7.5, Dy780, Dy781, DyLight 800, IRDye 800 CW, Alexa Fluor 647, Alexa Fluor 488, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 750, Alexa Fluor 790, JOE, or MAX.

[0095] In some aspects, the detectable signal can be a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal can be activated upon exposure to an acoustic signal. In some such aspects, the detectable signal can be indocyanine green, methylene blue, Evans blue, trypan blue, patent blue, IRDye800CW, DiR, Cy7, Cy7.5, or porphyrin.Attorney Docket No. 10046-635W01

[0096] In some aspects, the detectable signal can be conjugated to a first end of the substrate strand, and a quencher can be conjugated to a complementary end of the enzyme strand. In other aspects, the detectable signal can be conjugated to a first end of the substrate strand, and a quencher can be conjugated to a second end of the substrate strand. In yet other aspects, the detectable signal can be conjugated to a first end of the substrate strand, a first quencher can be conjugated to a complementary end of the enzyme strand, and a second quencher can be conjugated to a second end of the substrate strand.

[0097] In some aspects, the quencher can be, but is not limited to, DQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, BHQ-3, IRDye QC-1, or ZEN. In some aspects, when more than one quencher is present, each quencher can be the same or different.

[0098] In some aspects the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+(e.g., at least about 2 ppm Li+, at least about 3 ppm Li+, at least about 4 ppm Li+, at least about 5 ppm Li+, at least about 10 ppm Li+, at least about 15 ppm Li+, at least about 20 ppm Li+, at least about 30 ppm Li+, at least about 40 ppm Li+, at least about 50 ppm Li+, at least about 60 ppm Li+, at least about 70 ppm Li+, at least about 80 ppm Li+, at least about 90 ppm Li+, at least about 100 ppm Li+, at least about 110 ppm Li+, at least about 120 ppm Li+, at least about 130 ppm Li+, at least about 140 ppm Li+, at least about 150 ppm Li+, at least about 160 ppm Li+, at least about 170 ppm Li+, at least about 180 ppm Li+, at least about 190 ppm Li+, at least about 200 ppm Li+, at least about 225 ppm Li+, at least about 250 ppm Li+, at least about 275 ppm Li+, at least about 300 ppm Li+, at least about 325 ppm Li+, at least about 350 ppm Li+, at least about 375 ppm Li+, at least about 400 ppm Li+, at least about 425 ppm Li+, at least about 450 ppm Li+, at least about 475 ppm Li+, at least about 500 ppm Li+, at least about 525 ppm Li+, at least about 550 ppm Li+, at least about 575 ppm Li+, at least about 600 ppm Li+, at least about 625 ppm Li+, at least about 650 ppm Li+, at least about 675 ppm Li+, at least about 700 ppm Li+, at least about 725 ppm Li+, at least about 750 ppm Li+, at least about 775 ppm Li+, at least about 800 ppm Li+, at least about 825 ppm Li+, at least about 850 ppm Li+, at least about 875 ppm Li+, at least about 900 ppm Li+, at least about 925 ppm Li+, at least about 950 ppm Li+, at least about 975 ppm Li+, at least about 1000 ppm Li+).KITS

[0099] In an aspect, provided is a kit including: a DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving theAttorney Docket No. 10046-635W01 substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; and a leaching agent, a precipitating agent, and / or a chelating agent.

[0100] In some aspects, the DNAzyme sensor can be any of the disclosed DNAzyme sensors. These are discussed in detail above.

[0101] In some aspects, the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+(e.g., at least about 2 ppm Li+, at least about 3 ppm Li+, at least about 4 ppm Li+, at least about 5 ppm Li+, at least about 10 ppm Li+, at least about 15 ppm Li+, at least about 20 ppm Li+, at least about 30 ppm Li+, at least about 40 ppm Li+, at least about 50 ppm Li+, at least about 60 ppm Li+, at least about 70 ppm Li+, at least about 80 ppm Li+, at least about 90 ppm Li+, at least about 100 ppm Li+, at least about 110 ppm Li+, at least about 120 ppm Li+, at least about 130 ppm Li+, at least about 140 ppm Li+, at least about 150 ppm Li+, at least about 160 ppm Li+, at least about 170 ppm Li+, at least about 180 ppm Li+, at least about 190 ppm Li+, at least about 200 ppm Li+, at least about 225 ppm Li+, at least about 250 ppm Li+, at least about 275 ppm Li+, at least about 300 ppm Li+, at least about 325 ppm Li+, at least about 350 ppm Li+, at least about 375 ppm Li+, at least about 400 ppm Li+, at least about 425 ppm Li+, at least about 450 ppm Li+, at least about 475 ppm Li+, at least about 500 ppm Li+, at least about 525 ppm Li+, at least about 550 ppm Li+, at least about 575 ppm Li+, at least about 600 ppm Li+, at least about 625 ppm Li+, at least about 650 ppm Li+, at least about 675 ppm Li+, at least about 700 ppm Li+, at least about 725 ppm Li+, at least about 750 ppm Li+, at least about 775 ppm Li+, at least about 800 ppm Li+, at least about 825 ppm Li+, at least about 850 ppm Li+, at least about 875 ppm Li+, at least about 900 ppm Li+, at least about 925 ppm Li+, at least about 950 ppm Li+, at least about 975 ppm Li+, at least about 1000 ppm Li+).

[0102] In some aspects, the kit can further include an inactive DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand including at least one mutation; wherein the at least one mutation can prevent the inactive enzyme strand from cleaving the substrate strand.

[0103] In some aspects, the substrate strand can include SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and the inactive enzyme strand can include SEQ ID NO: 5 or a variant thereof. In some aspects, the substrate strand can include SEQ ID NO: 1 . In some aspects, the substrate strand can include a variant of SEQ ID NO: 1. In some aspects, the substrate strand can include SEQ ID NO: 2. In some aspects, the substrate strand can include a variant of SEQ ID NO: 2. In some aspects, the inactive enzyme strand can include SEQ ID NO: 5. In some aspects, the inactive enzyme strand can include a variant of SEQ ID NO: 5.Attorney Docket No. 10046-635W01

[0104] In some aspects, the leaching agent can include an acid (i.e., a mineral acid and / or an organic acid). Acids suitable for leaching, particularly for leaching lithium, could be reasonably selected by one of skill in the art. For example, in some aspects, the acid can include, but is not limited to, sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, boric acid, hydrofluoric acid, hydrobromic acid, perchloric acid, hydroiodic acid, or any combination thereof.

[0105] In some aspects, the acid can have a pH of at least about 0 (e.g. at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2). In some aspects, the acid can have a pH of up to about 2 (e.g., up to about 1.9, up to about 1.8, up to about 1.7, up to about1.6, up to about 1.5, up to about 1.4, up to about 1.3, up to about 1.2, up to about 1.1, up to about 1, up to about 0.9, up to about 0.8, up to about 0.7, up to about 0.6, up to about 0.5, up to about 0.4, up to about 0.3, up to about 0.2, up to about 0.1, up to about 0).

[0106] It is considered that the acid can have a pH ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the acid can have a pH of from about 0 to about 2 (e.g. , from about 0.1 to about 1.9, from about 0.2 to about 1.8, from about 0.3 to about 1.7, from about 0.4 to about 1.6, from about 0.5 to about 1.5, from about 0.6 to about 1.4, from about 0.7 to about 1.3, from about 0.8 to about 1.2, from about 0.9 to about 1.1, from about 0 to about 1, from about 0.1 to about 0.9, from about 0.2 to about 0.8, from about 0.3 to about 0.7, from about 0.4 to about 0.6, from about 1 to about 2, from about 1.1 to about 1.9, from about 1.2 to about 1.8, from about 1.3 to about1.7, from about 1.4 to about 1.6).

[0107] In some aspects, the precipitating agent can form a precipitate with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof. In some aspects, the precipitating agent can include OH", COr", SO42", or any combination thereof.

[0108] In some aspects, the chelating agent can chelate with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof. In some aspects, the chelating agent can include ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DEPA), and 1 ,2-cyclohexane diamine tetraacetic acid (CDTA), nitrilotriacetic acid (NTA), thiosulfate, iminodiacetic acid, alkylenepolyamino polycarboxylic acids, soaps, amines, citrates, carboxylic acids, diamines, triamines, ammonium nitrate, gluconates, or any combination thereof.Attorney Docket No. 10046-635W01

[0109] In some aspects, the kit can further include a filter. In some aspects, the filter can have a pore size of at least about 0.1 pm (e.g., at least about 0.2 pm, at least about 0.3 pm, at least about 0.4 pm, at least about 0.5 pm, at least about 0.6 pm, at least about 0.7 pm, at least about 0.8 pm, at least about 0.9 pm, at least about 1 pm). In some aspects, the filter can have a pore size of up to about 1 pm (e.g., up to about 0.9 pm, up to about 0.8 pm, up to about 0.7 pm, up to about 0.6 pm, up to about 0.5 pm, up to about 0.4 pm, up to about 0.3 pm, up to about 0.2 pm, up to about 0.1 pm).

[0110] It is considered that the filter can have a pore size ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the filter can have a pore size of from about 0.1 pm to about 1 pm (e.g., from about 0.2 pm to about 0.9 pm, from about 0.3 pm to about 0.8 pm, from about 0.4 pm to about 0.7 pm, from about 0.5 pm to about 0.6 pm, from about 0.1 pm to about 0.6 pm, from about 0.2 pm to about 0.5 pm, from about 0.3 pm to about 0.4 pm, from about 0.5 pm to about 1 pm, from about 0.6 pm to about 0.9 pm, from about 0.7 pm to about 0.8 pm).

[0111] In some aspects, the kit can further include a fluorometer. In some aspects, the fluorometer can be a portable fluorometer.METHODS

[0112] In an aspect, provided is a method of detecting Li+, the method including: a) exposing a sample to any of the disclosed DNAzyme sensors; and b) identifying the detectable signal, thereby detecting Li+in the sample.

[0113] In another aspect, provided is a method of detecting Li+in a sample, including: a) treating the sample with a leaching agent, a precipitating agent, and / or a chelating agent; and b) exposing the sample to a DNAzyme sensor, the DNAzyme sensor including: a substrate strand including a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage can activate the detectable signal; and c) identifying the detectable signal, thereby detecting Li+in the sample.

[0114] In some aspects, before step a), the sample can include an ionic salt. In some aspects, the ionic salt can be Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

[0115] In some aspects, the sample can include Na+and / or K+each in a concentration greater than 0 mM (e.g., at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least aboutAttorney Docket No. 10046-635W0130 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 275 mM, at least about 300 mM, at least about 325 mM, at least about 350 mM, at least about 375 mM, at least about 400 mM, at least about 425 mM, at least about 450 mM, at least about 475 mM, at least about 500 mM). In some aspects, the sample can include Na+and / or K+each in a concentration of up to about 500 mM (e.g., up to about 475 mM, up to about 450 mM, up to about 425 mM, up to about 400 mM, up to about 375 mM, up to about 350 mM, up to about 325 mM, up to about 300 mM, up to about 275 mM, up to about 250 mM, up to about 225 mM, up to about 200 mM, up to about 175 mM, up to about 150 mM, up to about 125 mM, up to about 100 mM, up to about 75 mM, up to about 50 mM, up to about 40 mM, up to about 30 mM, up to about 20 mM, up to about 10 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM, up to about 1 mM, about 0 mM).

[0116] It is considered that the sample can include Na+and / or K+each in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the sample can include Na+and / or K+each in a concentration of from about 0 mM to about 500 mM (e.g., from about 1 mM to about 475 mM, from about 2 mM to about 450 mM, from about 3 mM to about 425 mM, from about 4 mM to about 400 mM, from about 5 mM to about 375 mM, from about 10 mM to about 350 mM, from about 20 mM to about 325 mM, from about 30 mM to about 300 mM, from about 40 mM to about 275 mM, from about 50 mM to about 250 mM, from about 75 mM to about 225 mM, from about 100 mM to about 200 mM, from about 125 mM to about 175 mM, from about 0 mM to about 150 mM, from about 1 mM to about 125 mM, from about 2 mM to about 100 mM, from about 3 mM to about 75 mM, from about 4 mM to about 50 mM, from about 5 mM to about 40 mM, from about 10 mM to about 30 mM, from about 150 mM to about 500 mM, from about 175 mM to about 475 mM, from about 200 mM to about 450 mM, from about 225 mM to about 425 mM, from about 250 mM to about 400 mM, from about 275 mM to about 375 mM, from about 300 mM to about 350 mM).

[0117] In some aspects, the sample can include Mg2+and / or Ca2+each in a concentration greater than 0 mM (e.g., at least about 0.5 mM, at least about 1 mM, at least about 1.5 mM, at least about 2 mM, at least about 2.5 mM, at least about 3 mM, at least about 3.5 mM, at least about 4 mM, at least about 4.5 mM, at least about 5 mM, at least about 5.5 mM, at least about 6 mM, at least about 6.5 mM, at least about 7 mM, at least about 7.5 mM, at least about 8 mM, at least about 8.5 mM, at least about 9 mM, at least about 9.5 mM, at least about 10 mM). InAttorney Docket No. 10046-635W01 some aspects, the sample can include Mg2+and / or Ca2+each in a concentration of up to about 10 mM (e.g., up to about 9.5 mM, up to about 9 mM, up to about 8.5 mM, up to about 8 mM, up to about 7.5 mM, up to about 7 mM, up to about 6.5 mM, up to about 6 mM, up to about 5.5. mM, up to about 5 mM, up to about 4.5 mM, up to about 4 mM, up to about 3.5 mM, up to about 3 mM, up to about 2.5 mM, up to about 2 mM, up to about 1.5 mM, up to about 1 mM, up to about 0.5 mM, about 0 mM).

[0118] It is considered that the sample can include Mg2+and / or Ca2+each in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the sample can include Mg2+and / or Ca2+each in a concentration of from about 0 mM to about 10 mM (e.g., from about 0.5 mM to about 9.5 mM, from about 1 mM to about 9 mM, from about 1.5 mM to about 8.5 mM, from about 2 mM to about 8 mM, from about 2.5 mM to about 7.5 mM, from about 3 mM to about 7 mM, from about 3.5 mM to about 6.5 mM, from about 4 mM to about 6 mM, from about 4.5 mM to about5.5 mM, from about 0 mM to about 5 mM, from about 0.5 mM to about 4.5 mM, from about 1 mM to about 4 mM, from about 1.5 mM to about 3.5 mM, from about 2 mM to about 3 mM, from about 5 mM to about 10 mM, from about 5.5 mM to about 9.5 mM, from about 6 mM to about 9 mM, from about 6.5 mM to about 8.5 mM, from about 7 mM to about 8 mM).

[0119] In some aspects, the sample can include Mn2+and / or Ni2+each in a concentration greater than 0 mM (e.g., at least about 0.25 mM, at least about 0.5 mM, at least about 0.75 mM, at least about 1 mM, at least about 1.25 mM, at least about 1.5 mM, at least about 1.75 mM, at least about 2 mM, at least about 2.25 mM, at least about 2.5 mM, at least about 2.75 mM, at least about 3 mM, at least about 3.25 mM, at least about 3.5 mM, at least about 3.75 mM, at least about 4 mM). In some aspects, the sample can include Mn2+and / or Ni2+each in a concentration of up to about 4 mM (e.g., up to about 3.75 mM, up to about 3.5 mM, up to about 3.25 mM, up to about 3 mM, up to about 2.75 mM, up to about 2.5 mM, up to about 2.25 mM, up to about 2 mM, up to about 1.75 mM, up to about 1.5 mM, up to about 1.25 mM, up to about 1 mM, up to about 0.75 mM, up to about 0.5 mM, up to about 0.25 mM, about 0 mM).

[0120] It is considered that the sample can include Mn2+and / or Ni2+each in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the sample can include Mn2+and / or Ni2+each in a concentration of from about 0 mM to about 4 mM (e.g., from about 0.25 mM to about 3.75 mM, from about 0.5 mM to about 3.5 mM, from about 0.75 mM to about 3.25 mM, from about 1 mM to about 3 mM, from about 1.25 mM to about 2.75 mM, from about 1.5 mM to about2.5 mM, from about 1.75 mM to about 2.25 mM, from about 0 mM to about 2 mM, from aboutAttorney Docket No. 10046-635W010.25 mM to about 1.75 mM, from about 0.5 mM to about 1.5 mM, from about 0.75 mM to about 1.25 mM, from about 2 mM to about 4 mM, from about 2.25 mM to about 3.75 mM, from about 2.5 mM to about 3.5 mM, from about 2.75 mM to about 3.25 mM).

[0121] In some aspects, the sample can include Fe3+and / or Al3+each in a concentration greater than 0 mM (e.g., at least about 0.05 mM, at least about 0.1 mM, at least about 0.15 mM, at least about 0.2 mM, at least about 0.25 mM, at least about 0.3 mM, at least about 0.35 mM, at least about 0.4 mM). In some aspects, the sample can include Fe3+and / or Al3+each in a concentration of up to about 0.4 mM (e.g., up to about 0.35 mM, up to about 0.3 mM, up to about 0.25 mM, up to about 0.2 mM, up to about 0.15 mM, up to about 0.1 mM, up to about 0.05 mM, about 0 mM).

[0122] It is considered that the sample can include Fe3+and / or Al3+each in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the sample can include Fe3+and / or Al3+each in a concentration of from about 0 mM to about 0.4 mM (e.g., from about 0.05 mM to about 0.35 mM, from about 0.1 mM to about 0.3 mM, from about 0.15 mM to about 0.25 mM, from about 0 mM to about 0.2 mM, from about 0.05 mM to about 0.15 mM, from about 0.2 mM to about 0.4 mM, from about 0.25 mM to about 0.35 mM).

[0123] In some aspects, step a) can reduce concentration of the at least one ionic salt by at least about 10% (e.g., at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%). In some aspects, step a) can reduce concentration of the at least one ionic salt by up to about 100% (e.g., up to about 99%, up to about 98%, up to about 97%, up to about 96%, up to about 95%, up to about 94%, up to about93%, up to about 92%, up to about 91%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about25%, up to about 20%, up to about 15%, up to about 10%).

[0124] It is considered that step a) can reduce concentration of the at least one ionic salt by a percentage ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, step a) can reduce concentration of the at least one ionic salt by from about 10% to about 100% (e.g., from about 15% to about 99%,Attorney Docket No. 10046-635W01 from about 20% to about 98%, from about 25% to about 97%, from about 30% to about 96%, from about 35% to about 95%, from about 40% to about 94%, from about 45% to about 93%, from about 50% to about 92%, from about 55% to about 91%, from about 60% to about 90%, from about 65% to about 85%, from about 70% to about 80%, from about 10% to about 75%, from about 15% to about 70%, from about 20% to about 65%, from about 25% to about 60%, from about 30% to about 55%, from about 35% to about 50%, from about 40% to about 45%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 91 % to about 100%, from about 92% to about 100%, from about 93% to about 100%, from about 94% to about 100%, from about 95% to about 100%, from about 96% to about 100%, from about 97% to about 100%, from about 98% to about 100%, from about 99% to about 100%).

[0125] In some aspects, the sample can be brine water. In other aspects, the sample can be rock. In yet other aspects, the sample can be a portion of a lithium ion battery.

[0126] In some aspects, the DNAzyme sensor can be any of the disclosed DNAzyme sensors. These are discussed in detail above.

[0127] In some aspects, the method can further include, before step a), providing any of the disclosed kits. These are discussed in detail above.

[0128] In some aspects, the leaching agent can be any of the leaching agents discussed in detail above. In some aspects, treating the sample with the leaching agent in step a) can include: i) optionally crushing the sample; ii) incubating the sample with the leaching agent for from about 1 hour to about 2 hours; and iii) collecting a supernatant from the incubated sample.

[0129] In some aspects, step ii) can include incubating the sample with the leaching agent for at least about 1 hour (e.g., at least about 1.1 hours, at least about 1.2 hours, at least about 1.3 hours, at least about 1.4 hours, at least about 1.5 hours, at least about 1.6 hours, at least about 1.7 hours, at least about 1.8 hours, at least about 1.9 hours, at least about 2 hours). In some aspects, step ii) can include incubating the sample with the leaching agent for up to about 2 hours (e.g., up to about 1.9 hours, up to about 1.8 hours, up to about 1.7 hours, up to about 1.6 hours, up to about 1.5 hours, up to about 1.4 hours, up to about 1.3 hours, up to about 1.2 hours, up to about 1.1. hours, up to about 1 hour).

[0130] It is considered that step ii) can include incubating the sample with the leaching agent for a duration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, step ii) can include incubating the sample with the leaching agent for from about 1 hour to about 2 hours (e.g., from about 1.1 hours to about 1.9 hours, from about 1.2 hours to about 1.8 hours, from aboutAttorney Docket No. 10046-635W011.3 hours to about 1.7 hours, from about 1.4 hours to about 1.6 hours, from about 1 hour to about 1.5 hours, from about 1.1 hours to about 1.4 hours, from about 1.2 hours to about 1.3 hours, from about 1.5 hours to about 2 hours, from about 1.6 hours to about 1.9 hours, from about 1.7 hours to about 1.8 hours).

[0131] In some aspects, the precipitating agent be any of the precipitating agents discussed in detail above. In some aspects, treating the sample with the precipitating agent in step a) comprises: i) adding the precipitating agent in a concentration of from about 0 mM to about 50 mM, thereby forming a precipitate; and ii) optionally filtering the sample to remove the precipitate.

[0132] In some aspects, step i) can include adding the precipitating agent in a concentration of greater than 0 mM (e.g., at least about 0.1 mM, at least about 0.2 mM, at least about 0.3 mM, at least about 0.4 mM, at least about 0.5 mM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM). In some aspects, step i) can include adding the precipitating agent in a concentration of up to about 50 mM (e.g., up to about 45 mM, up to about 40 mM, up to about 35 mM, up to about 30 mM, up to about 25 mM, up to about 20 mM, up to about 15 mM, up to about 10 mM, up to about 5 mM, up to about 4 mM, up to about 3 mM, up to about 2 mM, up to about 1 mM, up to about 0.5 mM, up to about 0.4 mM, up to about 0.3 mM, up to about 0.2 mM, up to about 0.2 mM).

[0133] It is considered that step i) can include adding the precipitating agent in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, step i) can include adding the precipitating agent in a concentration of from about 0 mM to about 50 mM (e.g., from about 0.1 mM to about 45 mM, from about 0.2 mM to about 40 mM, from about 0.3 mM to about 35 mM, from about 0.4 mM to about 30 mM, from about 0.5 mM to about 25 mM, from about 1 mM to about 20 mM, from about 2 mM to about 15 mM, from about 3 mM to about 10 mM, from about 4 mM to about 5 mM, from about 0 mM to about 5 mM, from about 0.1 mM to about 4 mM, from about 0.2 mM to about 3 mM, from about 0.3 mM to about 2 mM, from about 0.4 mM to about 1 mM, from about 5 mM to about 50 mM, from about 10 mM to about 45 mM, from about 15 mM to about 40 mM, from about 20 mM to about 35 mM, from about 25 mM to about 30 mM).

[0134] In some aspects, the chelating agent can be any of the chelating agents discussed in detail above. In some aspects, treating the sample with the chelating agent in step a) canAttorney Docket No. 10046-635W01 include: i) adding the chelating agent in a concentration of from about 0 mM to about 10 mM, thereby forming a chelate; and ii) optionally filtering the sample to remove the chelate.

[0135] In some aspects, step i) can include adding the chelating agent in a concentration of greater than 0 mM (e.g., at least about 0.1 mM, at least about 0.2 mM, at least about 0.3 mM, at least about 0.4 mM, at least about 0.5 mM, at least about 1 mM, at least about 1.5 mM, at least about 2 mM, at least about 2.5 mM, at least about 3 mM, at least about 3.5 mM, at least about 4 mM, at least about 4.5 mM, at least about 5 mM, at least about 5.5 mM, at least about 6 mM, at least about 6.5 mM, at least about 7 mM, at least about 7.5 mM, at least about 8 mM, at least about 8.5 mM, at least about 9 mM, at least about 9.5 mM, at least about 10 mM). In some aspects, step i) can include adding the chelating agent in a concentration of up to about 10 mM (e.g., up to about 9.5 mM, up to about 9 mM, up to about 8.5 mM, up to about 8 mM, up to about 7.5 mM, up to about 7 mM, up to about 6.5 mM, up to about 6 mM, up to about 5.5 mM, up to about 5 mM, up to about 4.5 mM, up to about 4 mM, up to about 3.5 mM, up to about 3 mM, up to about 2.5 mM, up to about 2 mM, up to about 1.5 mM, up to about 1 mM, up to about 0.5 mM, up to about 0.4 mM, up to about 0.3 mM, up to about 0.2 mM, up to about 0.1 mM).

[0136] It is considered that step i) can include adding the chelating agent in a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, step i) can include adding the chelating agent in a concentration of from about 0 mM to about 10 mM (e.g., from about 0. 1 mM to about 9.5 mM, from about 0.2 mM to about 9 mM, from about 0.3 mM to about 8.5 mM, from about 0.4 mM to about 8 mM, from about 0.5 mM to about 7.5 mM, from about 1 mM to about 7 mM, from about 1.5 mM to about 6.5 mM, from about 2 mM to about 6 mM, from about 2.5 mM to about 5.5 mM, from about 3 mM to about 5 mM, from about 3.5 mM to about 4.5 mM, from about 0 mM to about 4 mM, from about 0.1 mM to about 3.5 mM, from about 0.2 mM to about 3 mM, from about 0.3 mM to about 2.5 mM, from about 0.4 mM to about 2 mM, from about 0.5 mM to about 1 mM, from about 4 mM to about 10 mM, from about 4.5 mM to about 9.5 mM, from about 5 mM to about 9 mM, from about 5.5 mM to about 8.5 mM, from about 6 mM to about 8 mM, from about 6.5 mM to about 7.5 mM).

[0137] In some aspects, step a) can further include neutralizing excess leaching agent, precipitating agent, and / or chelating agent before step c). In some aspects, neutralization can include adding a buffer (e.g., Tris-HCl, MOPS, MES, HEPES, PIPES, PBS, Bis-Tris, or any combination thereof) to a final concentration of at least about 50 mM (e.g., at least about 60 mM, at least about 70 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM,Attorney Docket No. 10046-635W01 at least about 110 mM, at least about 120 mM, at least about 130 mM, at least about 140 mM, at least about 150 mM, at least about 160 mM, at least about 170 mM, at least about 180 mM, at least about 190 mM, at least about 200 mM). In some aspects, neutralization can include adding a buffer to a final concentration of up to about 200 mM (e.g., up to about 190 mM, up to about 180 mM, up to about 170 mM, up to about 160 mM, up to about 150 mM, up to about 140 mM, up to about 130 mM, up to about 120 mM, up to about 110 mM, up to about 100 mM, up to about 90 mM, up to about 80 mM, up to about 70 mM, up to about 60 mM, up to about 50 mM).

[0138] It is considered that neutralization can include adding a buffer to a concentration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, neutralization can include adding a buffer to a concentration of from about 50 mM to about 200 mM (e.g., from about 60 mM to about 190 mM, from about 70 mM to about 180 mM, from about 80 mM to about 170 mM, from about 90 mM to about 160 mM, from about 100 mM to about 150 mM, from about 110 mM to about 140 mM, from about 120 mM to about 130 mM, from about 50 mM to about 130 mM, from about 60 mM to about 120 mM, from about 70 mM to about 1 10 mM, from about 80 mM to about 100 mM, from about 120 mM to about 200 mM, from about 130 mM to about 190 mM, from about 140 mM to about 180 mM, from about 150 mM to about 170 mM).

[0139] In some aspects, step b) can further include exposing the sample to an acoustic signal; wherein the detectable signal can be a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal can be activated upon exposure to the acoustic signal. In some aspects, the acoustic signal can be high frequency ultrasound (HIFU).

[0140] In some aspects, the method can further include, before step a), annealing the substrate strand and the enzyme strand together. For example, in some specific aspects, the substrate strand and the enzyme strand can be mixed together in 100 mM Tris-HCl, pH 7.4 and incubated at 37 °C.

[0141] In some aspects, the substrate strand and the enzyme strand can be incubated together for at least about 5 minutes (e.g., at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 1.25 hours, at least about 1.5 hours, at least about 1.75 hours, at least about 2 hours, at least about 2.25 hours, at least about 2.5 hours, at least about 2.75 hours, at least about 3 hours, at least about 3.25 hours, at least about 3.5 hours, at least about 3.75 hours, at least about 4 hours). In some aspects, the substrate strand and the enzyme strand can be incubated together for up to about 4 hours (e.g.,Attorney Docket No. 10046-635W01 up to about 3.75 hours, up to about 3.5 hours, up to about 3.25 hours, up to about 3 hours, up to about 2.75 hours, up to about 2.5 hours, up to about 2.25 hours, up to about 2 hours, up to about 1.75 hours, up to 1.5 hours, up to 1.25 hours, up to 1 hour, up to 50 minutes, up to 40 minutes, up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, up to 10 minutes, up to 5 minutes).

[0142] It is considered that the substrate strand and the enzyme strand can be incubated together for a duration ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the substrate strand and the enzyme strand can be incubated together for from about 5 minutes to about 4 hours (e.g., from about 10 minutes to about 3.75 hours, from about 15 minutes to about 3.5 hours, from about 20 minutes to about 3.25 hours, from about 25 minutes to about 3 hours, from about 30 minutes to about 2.75 hours, from 40 minutes to about 2.5 hours, from about 50 minutes to about 2.25 hours, from about 1 hour to about 2 hours, from about 1.25 hours to about 1.75 hours, from about 5 minutes to about 1.5 hours, from about 10 minutes to about 1.25 hours, from about 15 minutes to about 1 hour, from about 20 minutes to about 50 minutes, from about 25 minutes to about 40 minutes, from about 1.5 hours to about 4 hours, from about 1.75 hours to about 3.75 hours, from about 2 hours to about 3.5 hours, from about 2.25 hours to about 3.25 hours, from about 2.5 hours to about 3 hours).

[0143] In some aspects, the substrate strand and the enzyme strand can be incubated together in a ratio of at least about 1:0.5 (e.g., at least about 1:0.6, at least about 1:0.7, at least about 1:0.8, at least about 1:0.9, at least about 1 :1, at least about 1 : 1.1, at least about 1 :1.2, at least about 1 :1.3, at least about 1:1.4, at least about 1: 1.5, at least about 1: 1.6, at least about 1:1.7, at least about 1 : 1.8, at least about 1 :1.9, at least about 1:2). In some aspects, the substrate strand and the enzyme strand can be incubated together in a ratio of up to about 1 :2 (e.g., up to about 1:1.9, up to about 1 :1.8, up to about 1: 1.7, up to about 1: 1.6, up to about 1 :1.5, up to about 1 :1.4, up to about 1:1.3, up to about 1 :1.2, up to about 1:1.1, up to about 1 :1, up to about 1:0.9, up to about 1:0.8, up to about 1 :0.7, up to about 1:0.6, up to about 1 :0.5).

[0144] It is considered that the substrate strand and the enzyme strand can be incubated together in a ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the substrate strand and the enzyme strand can be incubated together in a ratio of from about 1:0.5 to about 1:2 (e.g., from about 1 :0.6 to about 1:1.9, from about 1 :0.7 to about 1 :1.8, from about 1 :0.8 to about 1 : 1.7, from about 1:0.9 to about 1 : 1.6, from about 1 : 1 to about 1 : 1.5, from about 1: 1.1 to about 1: 1.4, from about 1 :1.2 to about 1 :1.3, from about 1:0.5 to about 1:1.3, from about 1 :0.6 to aboutAttorney Docket No. 10046-635W011:1.2, from about 1:0.7 to about 1 : 1.1, from about 1:0.8 to about 1:1, from about 1 :1.2 to about 1 :2, from about 1 : 1.3 to about 1: 1.9, from about 1 : 1.4 to about 1:1.8, from about 1 : 1.5 to about 1 :1.7).

[0145] In some aspects, step c) can include measuring a level of the detectable signal using a fluorometer (e.g., a portable fluorometer).

[0146] In some aspects, the method can be used to detect a concentration of Li+in the sample of at least about 1 ppm (e.g., at least about 2 ppm, at least about 3 ppm, at least about 4 ppm, at least about 5 ppm, at least about 10 ppm, at least about 15 ppm, at least about 20 ppm, at least about 30 ppm, at least about 40 ppm, at least about 50 ppm, at least about 60 ppm, at least about 70 ppm, at least about 80 ppm, at least about 90 ppm, at least about 100 ppm, at least about 110 ppm, at least about 120 ppm, at least about 130 ppm, at least about 140 ppm, at least about 150 ppm, at least about 160 ppm, at least about 170 ppm, at least about 180 ppm, at least about 190 ppm, at least about 200 ppm, at least about 225 ppm, at least about 250 ppm, at least about 275 ppm, at least about 300 ppm, at least about 325 ppm, at least about 350 ppm, at least about 375 ppm, at least about 400 ppm, at least about 425 ppm, at least about 450 ppm, at least about 475 ppm, at least about 500 ppm, at least about 525 ppm, at least about 550 ppm, at least about 575 ppm, at least about 600 ppm, at least about 625 ppm, at least about 650 ppm, at least about 675 ppm, at least about 700 ppm, at least about 725 ppm, at least about 750 ppm, at least about 775 ppm, at least about 800 ppm, at least about 825 ppm, at least about 850 ppm, at least about 875 ppm, at least about 900 ppm, at least about 925 ppm, at least about 950 ppm, at least about 975 ppm, at least about 1000 ppm).

[0147] In some aspects, the method can further include, before step c), providing a reference level of the detectable signal by: i) providing to the sample an inactive DNAzyme sensor, the inactive DNAzyme sensor including: a substrate strand comprising a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand including at least one mutation; wherein the at least one mutation can prevent the inactive enzyme strand from cleaving the substrate strand; and ii) measuring a level of the detectable signal using the fluorometer, thereby providing a reference level of the detectable signal; wherein the reference level of the detectable signal can be used to eliminate background noise the level of the detectable signal determined in step d).

[0148] In some aspects, the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and the inactive enzyme strand can include SEQ ID NO: 5 or a variant thereof. In some aspects, the substrate strand can include SEQ ID NO: 1. In some aspects, the substrate strand can include a variant of SEQ ID NO: 1. In some aspects, the substrate strand can includeAttorney Docket No. 10046-635W01SEQ ID NO: 2. In some aspects, the substrate strand can include a variant of SEQ ID NO: 2. In some aspects, the inactive enzyme strand can include SEQ ID NO: 5. In some aspects, the inactive enzyme strand can include a variant of SEQ ID NO: 5.

[0149] In some aspects, the method can be used to quantify the concentration of Li+in the sample.

[0150] In some aspects, the method can be used to determine the absence of Li+in the sample.

[0151] In some aspects, the method can he carried out at a temperature of at least about 10°C (e.g., at least about 12°C, at least about 14°C, at least about 16°C, at least about 18°C, at least about 20°C, at least about 22°C, at least about 24°C, at least about 26°C, at least about 28°C, at least about 30°C, at least about 32°C, at least about 34°C, at least about 36°C, at least about 38°C, at least about 40°C). In some aspects, the method can be carried out at a temperature of up to about 40°C (e.g., up to about 38°C, up to about 36°C, up to about 34°C, up to about 32°C, up to about 30°C, up to about 28°C, up to about 26°C, up to about 24°C, up to about 22°C, up to about 20°C, up to about 18°C, up to about 16°C, up to about 14°C, up to about 12°C, up to about 10°C).

[0152] It is considered that the method can be carried out at a temperature ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the method can be carried out at a temperature of from about 10°C to about 40°C (e.g., from about 12°C to about 38°C, from about 14°C to about 36°C, from about 16°C to about 34°C, from about 18°C to about 32°C, from about 20°C to about 30°C, from about 22°C to about 28°C, from about 24°C to about 26°C, from about 10°C to about 26°C, from about 12°C to about 24°C, from about 14°C to about 22°C, from about 16°C to about 20°C, from about 24°C to about 40°C, from about 26°C to about 38°C, from about 28°C to about 36°C, from about 30°C to about 34°C).

[0153] In some aspects, the sample can be a cell or tissue sample. In some such aspects, the method can further include spatially identifying Li+in the cell or tissue sample (i.e., intracellularly or extracellularly). In other aspects, the sample can be cell lysate or a biological fluid (e.g., sweat, urine, blood, etc.).

[0154] In some aspects, the sample can be derived from a subject having a neurological disorder or a psychiatric disorder. In some aspects, the sample can be derived from a subject having bipolar disorder. In some aspects, the sample can be derived from a subject having a traumatic brain injury.Attorney Docket No. 10046-635W01

[0155] In some aspects, step b) can include imaging the sample and / or analyzing the sample with a fluorometer.

[0156] In some aspects, the method can be carried out in vivo, ex vivo, or in vitro.

[0157] In another aspect, provided is a method of determining usefulness of a test compound in modulating Li+, the method including: a) exposing the test compound to a sample; b) exposing the sample to any of the disclosed DNAzyme sensors; c) identifying the detectable signal, thereby detecting Li+in the sample; and d) using said detectable signal to determine an effect of the test compound on Li+.

[0158] In some aspects, the effect of the test compound on Li+can include an increase in Li+amount or activity, and the detectable signal can be greater than a reference signal produced by the sample not exposed to the test compound. In other aspects, the effect of the test compound on Li+can include a decrease in Li+amount or activity, and the detectable signal can be lesser than a reference signal produced by the sample not exposed to the test compound. In yet other aspects, the effect of the test compound on Li+can include substantially no change in Li+amount or activity, and the detectable signal can be substantially the same as a reference signal produced by the sample not exposed to the test compound.

[0159] In some aspects, the sample can be a cell or tissue sample. In some such aspects, the method can further include spatially identifying Li+in the cell or tissue sample (i.e., intracellularly or extracellularly). In other aspects, the sample can be cell lysate or a biological fluid (e.g., sweat, urine, blood, etc.).

[0160] In some aspects, the therapeutic agent can be medicinal lithium. In other aspects, the therapeutic agent can influence lithium functioning in signaling pathways, either as a primary therapeutic mechanism or a side-effect.

[0161] In some aspects, the therapeutic agent can be used to treat a neurological disorder or a psychiatric disorder. In some aspects, the therapeutic agent can be used to treat bipolar disorder. In some aspects, the therapeutic agent can be used to treat traumatic brain injury.

[0162] In some aspects, the sample can be derived from a subject having a neurological disorder or a psychiatric disorder. In some aspects, the sample can be derived from a subject having bipolar disorder. In some aspects, the sample can be derived from a subject having a traumatic brain injury.

[0163] In some aspects, the method can be carried out in vivo, ex vivo, or in vitro.

[0164] In yet another aspect, provided is a method of treating and / or preventing dysregulation of Li+in a subject in need thereof, the method including: a) administering to theAttorney Docket No. 10046-635W01 subject any of the disclosed DNAzyme sensors; b) identifying the detectable signal, thereby detecting Li+in the subject; and c) using said detectable signal to trigger a therapeutic event.

[0165] In some aspects, the subject can have a neurological disorder or a psychiatric disorder. In some aspects, the subject can have bipolar disorder. In some aspects, the subject can have a traumatic brain injury.

[0166] In some aspects, the detectable signal can be used to determine timing, dosage, or administration route of a therapeutic agent.

[0167] In some aspects, the detectable signal can be identified by imaging the patient.

[0168] In some aspects, the detectable signal can be identified in a sample of a biological fluid (e.g., sweat, urine, blood, etc.) collected from the patient.

[0169] In some aspects, the therapeutic agent can include a small molecule, a biologic agent, a peptide, a nucleic acid, radiation, chemotherapy, and / or surgery.EXAMPLESExample 1: DNAzymes for on-site portable detection of lithium and other metal ions

[0170] Disclosed herein is a DNAzyme-based fluorescent sensing system for the sensitive and robust detection of Li+in complex environments to overcome the current technological limitations of Li+detection in the LIB industry. Importantly, this system can be used for specific on-site sample preparation methods for the extraction of Li+from the relevant Li+- containing materials, i.e., brine, rock, and batteries. Using a simple portable fluorometer, the sensors are capable of detecting Li+down to 10 ppm, a dynamic range of 0 - 200 ppm, and minimal interference from non-target metal ions, in brine mimic samples, spodumene rocks, and electrodes of LIBs. Furthermore, sample preparation and Li+detection can be achieved with basic laboratory equipment and minimal training, making the DNAzyme-based system suitable for on-site and in-the-field applications.

[0171] A study carried out the in vitro selection and development of two DNAzyme-based fluorescence sensors. They are made of two different DNA strands, one called the substrate strand, which contains an RNA base in the middle of the DNAs (rS), and the other called the enzyme strand (E) which can catalyze the cleavage of the RNA base in the presence of lithium.

[0172] The sequences of the sensors are listed in TABLE 2:TABLE 2. Sequences of sensors.Attorney Docket No. 10046-635W01

[0173] When using the sensors, the enzyme strand and substrate strand were annealed with rS:E = 1 : 1.6 ratio. The sensors can be used for selective detection of Li+.

[0174] These core sequence of sensors are based on previously published Li+DNAzyme sequence. However, the binding arms of the original Li+DNAzyme sensor were systematically redesigned to achieve a sensor with improved thermal stability that works under broad range room temperatures (20-40 degrees). This re-engineering of binding arm sequences leads to effective Li+detection under field settings with portable devices, which could not be achieved with original sensors.

[0175] Detecting Li+using these sensors has advantages of high specificity, portable detection, on-site sample preparation, and quick extraction of Li+from relevant Li+-containing materials, i.e., brine, rock, and batteries. Using a simple portable fluorometer, these sensors can sensitively detect Li+down to 10 ppm, a dynamic range of 0 - 200 ppm, and minimal interference from non-target metal ions, in brine mimic samples, spodumene rocks, and electrodes of LIBs. Furthermore, sample preparation and Li+detection can be achieved with basic laboratory equipment and minimal training, making this DNAzyme-based system suitable for on-site and in-the-field applications.

[0176] The sample preparation strategy has the potential to be generalized to portable detection of other types of minerals. The detection method can be adapted to various mining and recycling industries of materials.Example 2: DNAzymes for on-site portable detection of lithium and other metal ions

[0177] The amount of lithium that constitutes an economically valuable resource is determined by different threshold values in each industry. In brine mining, lithium is considered enriched in the field when detected at 50 ppm or higher, and commercially valuable when detected at > 200 ppm [7], In rock mining, lithium oxide (LFO)-containing spodumeneAttorney Docket No. 10046-635W01 is the most important lithium ore mineral, and while pure spodumene contains 8% LEO, ~6% IJ2C) spodumene-rich concentrate (SC6) is the commercial standard. In contrast, economic viability in the recycling industry is in part evaluated by determining whether battery aging is due to lithium loss or mechanical damage. Each sector of the lithium mining industry currently utilizes a specific state-of-the-art on-site lithium detection technique, due to differences in the sample types. The brine mining sector utilizes a hand-held laser-induced breakdown spectrometer (LIBS), whereas the rock mining sector utilizes a portable x-ray diffraction (XRD) device. However, each of these state-of-the-art technologies has limitations. The LIBS device is prone to fluctuations in measurement precision as a direct result of interfering metal ions present in brine solutions [8], specifically K+and Na+, while the result from portable XRD device is not quantitative and highly depends on the crystal size [4,9]. As such, potentially valuable brine reserves may be overlooked from a measurement skewed by interference ions, whereas the value of a rock source may be hit-or-miss due to the lack of quantification.

[0178] Additionally, both methods require additional safety precautions and regulations given the inherent instrumental reliance on high-energy lasers and x-ray equipment

[0010] . Thus, a convenient approach to lithium detection in the field for its identification in environmental samples is not yet available. In the recycling industry, there is an additional area in which portable lithium detection methods are lacking. A method for the simple on-site measurement of residual lithium in spent batteries may allow better choice of LIB recycling options such as direct recycling (relithiation), hydrometallurgy, or pyrometallurgy, for economic viability decisions. Inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectroscopy (ICP-OES), and their derivative techniques are the current state-of-the-art method for lithium detection for this purpose. However, the process is complex, time-consuming and requires trained specialists and labs [6]. As a finite resource, lithium detection throughout the LIB industrial process holds great value, yet the ability to do so currently presents a significant challenge to the field, demonstrating the need for a portable, simple, on-site lithium detection technology.

[0179] This study utilizes DNAzymes, deoxyribozymes or catalytic DNA, as a versatile platform for developing metal ion sensors [11,12], which the study proposes can be used to overcome these challenges in lithium detection. Discovered in 1994, DNAzymes are metalloenzymes that are obtained through the process of in vitro selection and employ metal ions as cofactors for catalysis [13,14]. RNA-cleaving DNAzymes, especially, stand out for their fast reaction rates, high metal ion cofactor selectivity, and ease of signal detection through the “catalytic beacon” strategy [15-18]. In contrast to traditional screening methods, theAttorney Docket No. 10046-635W01 method of in vitro selection allows identification of DNAzymes with a desired sensitivity and specificity for a metal ion of interest from a vast library of DNA molecules [19,20]. Notably, this approach does not require prior knowledge of metal ion binding sites. The in vitro selection process also enables optimization of metal ion binding affinity and selectivity by tuning selection pressure stringency through varying reaction times, manipulating metal ion concentrations, and introducing counter selection to remove DNAzymes dependent on competing metal ions

[0021] , With cost-effective DNA synthesis and biocompatible properties, DNAzyme-based sensors are excellent tools for environment detection and cell imaging of metal ions, including Pb2+[22,23], Cu+

[0024] , Cu2+[24-27], UO22+

[0028] , Zn2+[29-31], Mg2+[32-34], Hg2+

[0035] , Ag+

[0036] , Ca2+

[0037] , Tl3+

[0038] , Cd2+

[0039] , C+

[0040] , Co2+

[0041] , Ni2+

[0042] , Na+

[0043] , Li+

[0044] , Fe2+, Fe3+

[0045] , and Mn2+

[0046] . Even though the Li+DNAzyme has been applied for Li+imaging in living cells under mild and physiological conditions

[0044] ,

[0047] ,

[0054] , it has not yet been applied in more complex conditions, such as those found in throughout the lithium industry, which typically involve harsh pH’s, strong ionic strengths, and high concentrations of non-target metal ions.

[0180] This work developed a DNAzyme-based fluorescent sensing system for the sensitive and robust detection of Li+in complex environments to overcome the current technological limitations of Li+detection in the LIB industry. Importantly, this study has also developed specific on-site sample preparation methods for the extraction of Li+from the relevant Li+-containing materials, i.e., brine, rock, and batteries. Using a simple portable fluorometer, this study has achieved the selective in-field detection of Li+down to 10 ppm, differentiation of several industrial relevant thresholds including 0 - 200 ppm in brine mimic samples, 0 - 8% in spodumene rocks, and new from cycle-aged electrodes in LIBS, all with minimal interference from non-target metal ions. The simplicity of the DNAzyme-based fluorescent Li+sensor design can overcome several limitations of current technologies such as complex operation, interference from competing metal ions, and crystal size filtering, in addition to the advantage of being robust enough to be applied in a variety of environments. Furthermore, sample preparation and Li+detection can be achieved with basic laboratory equipment and minimal training, making the DNAzyme-based system suitable for on-site and in-the-field applications.Materials and Methods

[0181] DNA: All DNA was obtained from International DNA Technologies (IDT) with HPLC purification. Sequences (5’ - 3’) include those in TABLE 2.Attorney Docket No. 10046-635W01

[0182] Chemicals and Reagents: All chemicals were obtained from Sigma Aldrich, Thermo Fisher, and Fisher Scientific. The chemicals were sodium chloride (99.998%), calcium chloride hydrate (99.9965%), potassium chloride (technical grade), lithium chloride (> = 98.7%), magnesium chloride (99.9%), sulfuric acid (95-98%), calcium oxide 99.95% (metals basis) and were used to prepare all stock solutions (IM LiCl, IM NaCl, IM MgC12, IM CaCl2, IM Tris, pH 7.4, 5M H2SO4, 5M KOH, and IM CaO).

[0183] Instruments: Instruments used for the collection of fluorescence data include BioTek Synergy Hl Microplate Reader (Agilent Technologies, Winooski, VT), ISS Chronos Digital Frequency Domain Spectrofluorometer (ISS Inc., Champaign, IL), DeNovix QFX Fluorometer (DeNovix Inc., Wilmington, DE).

[0184] Software: All the fluorometer data were exported and analyzed with Microsoft Excel. Figures were created with GraphPad Prism 8 and Microsoft PowerPoint.

[0185] Sensor Preparation: Melting temperature simulations were performed using IDT’s sequence tool (OligoAnalyzer) and the UNAFold Web Server (DNA Folding Form) to determine the best binding arm combination for the design of the sensor. Melting temperatures were assessed for designs under 150 mM monovalent and 2 mM divalent metal ions with the same tool. DNAzyme sensors were synthesized and HPLC purified directly by IDT with fluorophore and quencher modifications. Quantifications were performed by Nanodrop to verify sensor component concentrations.

[0186] Sensor Annealing: The enzyme-substrate complex was formed by annealing a mixture of 20-4RT enzyme and 20-4RT substrate strands for sensor in 150 mM Tris pH 7.4 with the ratio of 1.6:1 with volumes no larger than 50 pL. The final concentration of substrate strand in the annealing system is 5 p M for the annealing system. The enzyme-substrate mixes were enclosed in 0.2 mL PCR tubes and annealed in a thermocycler to gradually decrease the temperature from 95 °C to 25°C with the time range of 2 - 3 hours to ensure the highest annealing efficiency. Sensors were diluted to lOx working solutions in 150 mM Tris pH 7.4 prior to the addition to the samples.

[0187] Sample preparation: As described in the main text, samples are prepared with spiked lithium addition or with certain expected lithium content percentage. For brine mining tests, 1 M LiCl solution was spiked into brine samples containing 465 mM NaCl, 10 mM KC1, 53 mM MgCL, and 10 mM CaCl2with different final concentrations (10, 50, 200 ppm). Samples were precipitated as introduced in the main text and were filtered with 0.2 pm syringe filter with a 5 mL syringe. Rock samples were broken into small pieces by hammer and grinded into powder by mortar and pestle. Different percentages of Li2O content rock samples wereAttorney Docket No. 10046-635W01 prepared by mixing spodumene (8% LizO content) with limestone (0% LizO content) powder at different ratios. Acid leaching was performed for 1 hour at room temperature. Extracts were filtered by 0.2 pm syringe filter with a 5 mL syringe. Filtered extracts were neutralized and precipitated with different methods as introduced in the main text and were filtered again for portable detections. For safely disassemble batteries, new and aged batteries were fully discharged by a short circuit connecting the cathode and anode. Batteries were then disassembled, and black matters were scratched off and collected from the cathode and the anode. Leaching and precipitation were carried out as described in the main text. Extracts were filtered by 0.2 pm syringe filter with a 5 mL syringe before portable detections.

[0188] Fluorescence Measurements: Fluorescence assays were carried out using multiple spectrometers, including a plate reader and a portable fluorometer.

[0189] The kinetic curve assays were performed in a BioTek Synergy Hl Multimode Reader (Agilent Technologies, California). Fluorescence data were collected with 96-well format in triplicate using the plate reader to simultaneously assess multiple conditions in parallel. Samples were preloaded to 96-well black flat bottom plates. 10 pL lOx sensor in Tris buffer was added into each well containing 90 pL samples. Measurements were collected with fluorescence intensity mode with single-excitation single-wavelength multiple timepoints setup. Intervals were ranging from 3 - 30 minutes over the course of 12 hours with continuous shaking. Shaking mode was set to be double-orbital slow mode. Excitation wavelengths were set to be 480 nm, and emission were collected at 525 nm. Reading distance was 7 nm, and gaining was 100%.

[0190] Portable fluorescence detections were performed with the DeNovix QFX Fluorometer (DeNovix Inc., Delaware) with the basic fluorometer mode. Samples were prepared as introduced and mixed with annealed and diluted sensor with 190 pL to 10 pL combination. Sensor concentrations were 25 nM for condition optimization and 125 nM for on-site detection experiments. The samples were briefly mixed and detected with blue LED excitation filter 442 - 497 nm and green emission filter 514 - 567 nm. Fluorescence intensity values were collected across four hours for condition optimization, and two hours for on-site detection experiments.

[0191] Calculation of Sensor Activities: (F-Fo) / Fo was used to calculate the relative change in fluorescence intensity compared to the baseline. FQ was measured at 0-minute timepoint after mixing the sensor with samples as initial or baseline fluorescence intensity. F was measured after a certain time point to calculate the sensor activity at that time point.Attorney Docket No. 10046-635W01Results and Discussion

[0192] Re-engineering Li+-DNAzyme Sensor for Detection at Room Temperature: The fundamental principle of DNAzyme-based sensing by the catalytic beacon method relies upon differences in the melting temperatures (Tm) of the uncleaved and cleaved DNAzymes, relative to the target application temperature. Since the application of the previously reported 20-4 DNAzyme sensor was intended for cellular imaging, the Tmof the hybridized DNAzyme / substrate was designed to be 56.7°C before cleavage and 36.1 °C and 33.7°C after the cleavage (FIG. 1A). This design allowed for sufficient hybridization and fluorescence quenching in the absence of Li+, and fluorescence signal turn-on in the presence of Li+under the cell imaging temperature of 37°C. However, when tested in the presence of 10 mM Li+at room temperature, the fluorescence increase was 12% less than its previously reported activity at 37 °C

[0044] , potentially because the T,,, of the cleaved binding arms were higher than the testing temperature. Consequently, the development of a DNAzyme-based detection method for Li+in mining and recycling applications required optimization of the Li+DNAzyme sensor under ambient conditions. To this end, the study systematically redesigned the binding arms of the original 20-4 DNAzyme sensor for optimal fluorophore release following Li+-induced cleavage at room temperature. The study subsequently developed an alternate version of the Li+sensor, named 20-4RT (FIG. IB), to improve signal output at room temperature by elongating one of the binding arms, shortening the other binding arm labeled with fluorophore, FAM and altering the nucleotide contents in both binding arms to achieve specific melting temperature thresholds, while maintaining the ability to form Watson-Crick base pairs. Specifically, 20-4RT DNAzyme was designed to have Tmof 62.6°C for the hybridized DNAzyme / substrate strands to enhance the overall stability of the uncleaved sensor. After the Li+-dependent cleavage, the FAM-labeled binding arm has a Tmof only 20.8°C and thus will dehybridize under room temperature. To assess whether the 20-4RT DNAzyme sensor outperformed the original 20-4 DNAzyme sensor at the LIB target application temperature, the study first monitored the fluorescent signal output of each sensor at room temperature under different concentrations of Li+(FIG. 1C).

[0193] The 20-4RT DNAzyme sensor showed a 120% fluorescence increase in the presence of 10 mM at room temperature relative to the original 20-4 DNAzyme sensor (FIG. 1C), outperforming the 20-4 DNAzyme sensor. The study also tested the 20-4RT DNAzyme sensor at 40°C, as some field applications necessitate higher temperature conditions. As shown in FIG. 2, the 20-4RT DNAzyme sensor was able to differentiate different lithium levels up to 200 ppm. Next, to determine whether the re-engineering of the DNAzyme sensor affected itsAttorney Docket No. 10046-635W01 selectivity toward other metal ions, the study compared the performance of the original and sensor in the presence of non-target metal ions that would be present in the final application environment, including Na+, K+, Mg2+, and Ca2+. As shown in FIG. ID, the 20-4RT DNAzyme sensor displayed a substantially greater fluorescence increase in the presence of 200 mM Li+than in the presence of 200 mM Na+or K+, or 4 mM Mg2+or Ca2+. These results demonstrate that the selectivity of the 20-4RT DNAzyme sensor for Li+over other metal ions is comparable to the original 20-4 DNAzyme sensor (FIG. ID). Taken together, the enhanced signal response and high selectivity of the re-engineered Li+DNAzyme design make the 20-4RT DNAzyme sensor a promising candidate for DNAzyme-based Li+detection in LIB industry applications.

[0194] Quantifying Li+in the Presence of High Concentrations of Other Metal Ions: Unlike previous success in imaging Li+in living cells in the presence of - 12 mM Na+, - 150 mM K+, - 2.1 mM Mg2+and - 100 nM Ca2+, or portable detection of Li+in serum in the presence of 1.1 mM Mg2+and 2.5 mM Ca2+using a spectrometry method,

[0057] the detection of Li+in industrial samples necessitates the consideration of these metal ions at much higher concentrations than biological samples, such as -500 mM Na+and K+, -50 mM of Mg2+and Ca2+. Additionally, it has been shown that high concentrations of divalent metal ions may influence DNAzyme activity.[55,56] Therefore, such high levels of competing metal ions raised concerns on whether the selectivity and performance of the 20-4RT DNAzyme sensor would still be sufficient to reliably differentiate Li+from other metal ions. To address this concern, the study evaluated the response of the 20-4RT DNAzyme sensor towards different levels of Li+in the presence of high levels of competing metal ions. Li+was detected above 10 ppm in the presence of 500 mM Na+, K+or 10 mM Mg2+, Ca2+(FIGS. 3A-3D and FIGS. 4A- 4D), further supporting the use of the 20-4RT DNAzyme sensor in complex environmental samples.

[0195] Developing a Method for On-site Detection of Lithium in Brine Water: While the study was able to achieve Li+detection down to 10 ppm in the presence of high concentrations of individual non-target metal ions, it remained challenging to overcome the effects of exceptionally high concentrations of Mg2+and Ca2+together (> 10 mM each), even with 200 ppm of Li+spiked into the sample (FIG. 5).

[0196] Heating water to remove Ca2+and Mg2+as carbonate compounds is a common method used in industry

[0049] , but it is unsuitable for on-site detection methods due to power and time requirements, as well as difficulty controlling sample size and metal ion concentration. Therefore, this study designed and evaluated alternative methods of reducing Mg2+and Ca2+concentrations (FIG. 6A). Lime softening is also used in industry to reduceAttorney Docket No. 10046-635W01Mg2+concentrations through the addition of limewater, Ca(0H)2 or simply CaO, to raise the pH and precipitate Mg2+as Mg(0H)2

[0050] ,

[0051] . Based on the Kspof Mg(OH)2 (1.5 x 10n), it was reasoned that increasing the sample pH from 7.4 to 11 ([OH ] = 10‘3M) would promote Mg(OH)2 precipitation to yield a final Mg2+concentration in solution of ~15 pM. As demonstrated in FIG. 14, there is little decrease in the fluorescence signal in the presence of 5 mM level of Mg2+in comparison with that of non-target metal ions. Therefore, Mg2+at such a low concentration of 15 pM should have little effect on the sensor’s performance. To test this method, KOH was added to a simulated brine water sample containing 465 mM Na+, 10 mM K+, 50 mM Mg2+and 10 mM Ca2+, with or without 200 ppm Li+, to raise the pH to 11 and precipitates (ppt) were removed using a syringe filter. The filtered water samples were then neutralized by adding Tris-HCl (pH 7.4) to a final concentration of 150 mM. Indeed, the study observed fluorescence turn-on from the brine-mimic water samples containing 200ppm Li+, and could differentiate the samples from the controls (0 ppm Li+) using a simple portable fluorometer.

[0197] The study tested two additional approaches to further reduce the influence of high Ca2+concentrations on sensor activity (FIG. 6A). In the first approach, 5 mM of EDTA was added to the filtered samples and an improved signal turn-on of the sensor was observed (FIGS. 7A-7B). In the second approach, 10 mM of CO32" was introduced into the filtered sample prior to Tris neutralization to precipitate Ca2+ions to a final solution concentration of - 62 pM, which would not interfere with sensor activity ( Kspof CaCCh = 3.8 x 10‘9). The supernatant was filtered a second time to remove CaCCh precipitate and neutralized by Tris buffer addition. The CaCCh approach achieved comparable results to the EDTA-based approach, suggesting that both methods can effectively reduce Ca2+interference from brine-mimic samples and for on-site Li+detection in field samples. The EDTA-based chelation approach is the simpler of the two methods, requiring only one round of precipitation and filtration, whereas the CaCCb- based precipitation method, while it can further reduce Ca2+, does not necessarily improve the signal output. Therefore, the study chose to utilize the EDTA-based approach (FIG. 6B) as the primary method in subsequent brine-mimic Li+detection experiments with a portable fluorometer to simulate field conditions. The study also tested if the amount of sensor contributes to the performance of the sensor. Upon titrating the sensor amount (FIG. 8), it was found that 125 nM sensor concentration performs the best and decided to use the amount for further experiments. Using this method and the 20-4RT DNAzyme sensor, the study achieved the detection of Li+in brine water samples with the ability to differentiate Li+at theAttorney Docket No. 10046-635W01 commercially valuable level of 200 ppm and an enriched level of 50 ppm from either the lower level of 10 ppm or no Li+using a simple handheld portable fluorometer (FIG. 6C).

[0198] Developing a Method for On-site Detection of Lithium in Hard Rocks: Rock mining is the second major source of Li+for industrial use. Assessing Li+content typically requires the pretreatment of rock samples with high temperatures prior to its leaching and extraction, which is not realistic to perform in on-site settings [3]. Thus, the study sought out a method for Li+extraction that did not require extreme temperatures and was appropriate for in-the-field use and subsequent Li+detection using the 20-4RT DNAzyme sensor. For on-site field tests of Li+from hard rocks, the study used spodumene as the target Li+-containing rock samples with limestone and glass as negative controls. In this approach, Li+was extracted by pulverizing spodumene rocks with a mortar and pestle, then added different concentrations of sulfuric acid (0.05 M H2SO4, 4 M H2SO4, 50% H2SO4 or 98% H2SO4) to leach the Li+from the rock powders, following a previously reported protocol

[0052] . After the extraction process, the supernatant was collected and samples were prepared for ICP-MS analysis to evaluate the leaching methods (FIG. 9A). It was found that 0.05 M H2SO4 incubation provided the highest leaching efficiency, and that there was a negative correlation between the leaching efficiency and the concentration of H2SO4. No detectable Li+was extracted from limestone and glass sample controls, supporting the purity of extraction materials and the reliability of the leaching method. However, while the leaching method proved to be successful, the study still needed to address other ionic components in the sample that could affect the sensor’ s activity.

[0199] The lithium rock mining industry often uses limewater to neutralize the leached acidic sample to remove most frequently observed crust elements such as iron and aluminum

[0052] . As such, the study adopted this acid neutralization method by treating H2SO4-leached samples with CaO, generating soluble Ca(OH)2 and CaSO4precipitate in the process. Based on the Kspof CaSCb (2.4 x 1 O'5), the study calculated the final concentration of Ca2+to be ~5 mM following the neutralization and filtration of the leaching solution. A concentration of Ca2+at this level will not significantly affect the sensor (FIG. 15). The study also evaluated the possibility of utilizing KOH for the neutralization step to avoid introducing additional Ca2+ions that may interfere with detection, as well as a combination of the CaO and KOH neutralization methods (50% of each reagent used). The study evaluated these neutralization approaches using a solution containing 0.05 M H2SO4 spiked with 200 ppm Li+as the leaching product mimic. After neutralization, the samples were filtered and mixed with Tris to a final concentration of 150 mM, and the 20-4RT DNAzyme sensor was added to detect the Li+in the final neutralized, leach-mimic samples. The resulting fluorescent readouts demonstrated thatAttorney Docket No. 10046-635W01 while all three methods produced an increased fluorescent signal, the combination CaO-KOH neutralization method showed the greatest signal turn-on (FIG. 9B). The study further applied this method for Li+detection in leaching product mimics and tested the effect of EDTA on signal output to remove residual Ca2+interference introduced during the neutralization step (FIG. 10). The study observed - 50% signal improvement with the addition of 2 mM EDTA, validating the incorporation of a supplemental EDTA clean-up step into the method (FIG. 9C). Applying the on-site detection method for Li+from hard rocks, including the optimized steps of 0.05M H2SO4 extraction, CaO-KOH combination neutralization, and EDTA clean-up (FIG. 9D), the study observed a significant signal increase from rock samples with higher lithium content than those without. More specifically, the study was able to differentiate samples of pure spodumene (8% Li2O) and SC6 commercial standard (6% Li2O) from samples containing lower or no Li+(4%, 2%, and 0% Li2O), as shown in FIG. 9E. As such, these results support the efficiency and validity of the method in distinguishing economically valuable sources of Li+in spodumene rock using a simple handheld fluorometer (FIG. 9E).

[0200] Developing a Method for On-site Detection of Lithium from Recycled Batteries: While all Li+-ion batteries have materials that are worthy of recycling, the loss of Li+through formation of solid-electrolyte interphase, dendrite growth, and side reactions may reduce the amount of recoverable Li+and impact effectiveness of recycling

[0053] . Therefore, an on-site detection method for recoverable Li+in spent LIBs utilized before initiating the recycling process will be vital for assessing its economic viability. To provide an on-site evaluation of Li+amount in aged battery electrodes, the study looked to adopt the Li+leaching and detection methods developed in this work for the comparison of Li+loss from electrodes in batteries at different stages of cycle-induced aging. After disassembling a new Li+-ion battery, the study utilized a leaching method based on the hard rock leaching method using 0.05 M H2SO4, and a neutralization method similar to Mg2+reduction method (FIG. 11 A), both demonstrated in the previous sections. Although Mg2+and Ca2+are not commonly used in battery electrodes, electrode materials such as Mn2+, Co2+, and Ni2+all share the property of a low Kspof their hydroxide products (4.6 x 1014for Mn(0H)2, 2.5 x IO16for Co(OH)2, and 2.8 x 1016for Ni(0H)2) that may precipitate at pH >10, allowing only nM to pM levels of cation impurities in the supernatant. The LIBs utilized in this study were composed of a lithium cobalt oxide (LiCoCL) cathode and graphite anode

[0053] . Following hydroxide product precipitation, the supernatant was filtered and neutralized with Tris to a final concentration of 150 mM for subsequent sensor addition and portable detection (FIG. 11A). To assess the Li+loss of aged batteries, the study completed multiple cycles of charging and discharging. As shown in FIG.Attorney Docket No. 10046-635W0111B, the on-site leaching and detection method could detect Li+enrichment in the cathode compared with the anode in new LIBs. Moreover, the method was able to differentiate decreasing Li+levels in battery cathodes that had been cycle-aged with ~70 and -150 cycles, relative to the new battery cathode (FIG. 11B). In addition, the opposite trend was observed when detecting Li+in the anode, in which the method could differentiate increasing Li+levels in cycle-aged battery anodes, relative to the new battery anode (FIG. 11B). These results suggest that the method reliably reflects information regarding the amount of recoverable Li+in battery electrodes, which would help on-site quality control and evaluation of Li+content in battery manufacturing and recycling.Conclusion

[0201] This work developed a Li+DNAzyme-based fluorescent sensor for portable on-site detection and quantification of Li+in brine water, hard rocks and recycled lithium batteries, addressing limitations faced by current methods in the LIB industry. To adapt the system for diverse applications in brine mining, rock mining, and battery recycling, the study introduced tailored on-site sample preparation methods for efficient Li+extraction from various natural and battery samples. By integrating the DN Azyme sensor and sample preparation methods with portable fluorescence detection devices, the study achieved sensitive detection of lithium down to 10 ppm, with minimal interference from non- target metal ions and the ability to differentiate a series of lithium thresholds for different industries. Specifically, the study was able to differentiate between 0, 10, 50, and 200 ppm Li+in brine mimic samples, 0%, 2%, 4%, 6% (the SC6 standard) and 8% Li2O in spodumene rock, and between new, -70 cycle-aged, and -150 cycle-aged LIB cathodes and anodes.

[0202] Significantly, the on-site detection method demonstrated in this work simplified sample preparation steps and equipment requirements for better in-field applications. All the portable detection is achieved under field conditions, with ambient temperature, conventional sample collection equipment (harmer, mortar and pestle), portable fluorometer, syringe filters, pipettors and a few consumables (tubes, tips, gloves). The streamlined equipment requirements minimize both space and weight in a travel package, crucial for in-field applications where portability is paramount. Furthermore, the method showcased here avoids the need for excessive heat, lasers, or X-ray sources, reducing safety concerns and precautions.

[0203] Moreover, the detection method does not necessitate complex sample pretreatment techniques, unlike the combined acid digestion and total dissolved solids adjustment in ICP- MS, argon purging and matrix matching in LIBS, and crystal size filtering and internal calibration in XRD. The simplicity of the method also reduces the expertise required forAttorney Docket No. 10046-635W01 operation. With straightforward steps such as sample weighing, liquid transfer and mixing, pH adjustment, syringe filtering, and portable fluorometer reading, individuals with basic lab training will be proficient enough to conduct lithium detection, resulting in significant cost savings on trained personnel.

[0204] In summary, the methods demonstrated in this work achieved highly selective lithium detection down to 10 ppm with the ability to distinguish key threshold lithium levels that identify economic value across several industries, including 200 ppm Li+for brine mining, 6% Li2O or SC6 for rock mining, and Li+-specific aging of LIBs. This workflow can be conducted with minimal laboratory equipment or training, rendering them suitable for on-site and in-field applications.Example 3: Cellular applications

[0205] FIG. 12 and FIG. 13 show the use of the disclosed DNAzyme sensors for use in detecting Li+in cells. These DNAzyme sensors can detect Li+in cells at a concentration of about 0.5 mM, which is lower than other lithium DNAzyme sensors. These DNAzyme sensors can be used in neuron cells as biomarkers to screen for better drugs for bipolar disorder and traumatic brain injury.Example 4: On-Site Portable Lithium Detection in Mining and Recycling Industries Based on a DNAzyme Fluorescent Sensor

[0206] The past decade has witnessed an enormous expansion of applications for lithium- ion batteries (LIB), leading to a high demand for additional lithium resources and the development of methods for lithium recycling [1, 2], There are two major mining resources from which this highly valuable metal can be extracted: high-lithium brines (natural brines or enriched industrial brines), which account for approximately 60% of global lithium, and pegmatite ores (spodumene and other high-lithium rocks and clays), which account for approximately 20% of global lithium [3]. Additionally, these resources are rare and geographically constrained, making the accurate and timely identification of commercially valuable lithium reserves a top priority in the LIB industry [4]. The third and final lithium resource comes from the opposing end of the LIB industrial process, battery recycling. However, not all recycled LIBs are cost-effective for lithium extraction, as the amount and quality of remaining and extractable lithium can vary between aged electrodes [5, 6], To meet the demands of improving both the search for additional lithium resources and the efficiency of lithium recycling, an on-site portable method of detection for lithium will play a major role.

[0207] The amount of lithium that constitutes an economically valuable resource varies among different sectors of the industry. In brine mining, lithium is considered enriched in theAttorney Docket No. 10046-635W01 field when detected at 50 ppm or higher, and commercially valuable when detected at over 200 ppm [7]. In rock mining, lithium oxide (Li2O) -containing spodumene is the most important lithium ore mineral, and while pure spodumene contains 8% LEO. approximately 6% Li2O spodumene-rich concentrate (SC6) is the commercial standard. In contrast, economic viability in the recycling industry is partly evaluated by determining whether battery aging is due to lithium loss or mechanical damage. Each sector of the lithium mining industry utilizes a specific state-of-the-art on-site lithium detection technique due to differences in sample types. The brine mining sector utilizes a hand-held laser-induced breakdown spectrometer (LIBS), whereas the rock mining sector utilizes a portable x-ray diffraction (XRD) device. However, each of these state-of-the-art technologies has limitations. The LIBS device is prone to fluctuations in measurement precision due to interfering metal ions in brine solutions [8], specifically K+and Na+, while the results from the portable XRD device are not quantitative and highly depend on crystal size [4, 9]. As a result, potentially valuable brine reserves may be overlooked due to measurement skewed by interfering ions, while the value of a rock source may be uncertain due to the lack of quantification. Moreover, both methods require additional safety precautions and regulations given the inherent instrumental reliance on high-energy lasers and x-ray equipment

[0010] . Thus, a convenient field approach for lithium detection in environmental samples is needed. In the recycling industry, there is an additional area in which portable lithium detection methods would be beneficial. A simple on-site method for measuring residual lithium in spent batteries may improve the selection of LIB recycling options such as direct recycling (relithiation), hydrometallurgy, or pyrometallurgy for economic viability decisions. Inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectroscopy (ICP-OES), and their derivative techniques are the current state-of-the-art method for lithium detection for this purpose. However, the process is complex, time-consuming, and requires specialized training and laboratory facilities [6]. As a finite resource, lithium requires careful detection throughout the LIB industrial process. Given the requirement, current challenges in the field underscore the need for a portable, simple, onsite lithium detection technology.

[0208] DNAzymes, also known as deoxyribozymes or catalytic DNA, are utilized as a versatile platform for developing metal ion sensors [11, 12], which are proposed here to effectively address the challenges in lithium ion (Li+) detection. Discovered in 1994, DNAzymes are metalloenzymes obtained through in vitro selection that employ metal ions as cofactors for catalysis [13, 14]. RNA-cleaving DNAzymes are particularly notable for their fast reaction rates, high metal ion cofactor selectivity, and ease of signal detection through theAttorney Docket No. 10046-635W01“catalytic beacon” strategy [15-18]. In contrast to traditional screening methods, the method of in vitro selection allows identification of DNAzymes with a desired sensitivity and specificity for a metal ion of interest from a vast library of DNA molecules [19, 20]. Notably, this approach does not require prior knowledge of metal ion binding sites. The in vitro selection process also enables optimization of metal ion binding affinity and selectivity by tuning selection pressure stringency through varying reaction times, manipulating metal ion concentrations, and introducing counter selection to remove DNAzymes dependent on competing metal ions

[0021] . With cost-effective DNA synthesis and biocompatible properties, DNAzyme-based sensors are excellent tools for environmental detection and cell imaging of metal ions, including Pb2+[22, 23], Cu+

[0024] , Cu2+[24-27], UO22+

[0028] , Zn2+[29-31], Mg2+[32-34], Hg2+

[0035] , Ag+

[0036] , Ca2+

[0037] , Tl3+

[0038] , Cd2+

[0039] , Cr3+

[0040] , Co2+

[0041] , Ni2+

[0042] , Na+

[0043] , Li+

[0044] , Fe2+, Fe3+

[0045] , and Mn2+

[0046] . Although the Li+DNAzyme has been applied for Li+imaging in living cells under mild and physiological conditions [44, 47, 54], it has not yet been applied in more complex conditions found throughout the lithium industry, which typically involve harsh pH conditions, strong ionic strengths, and high concentrations of non-target metal ions.

[0209] This study developed a DNAzyme-based fluorescent sensing system for the sensitive and robust detection of Li+in complex environments, overcoming the current technological limitations in the LIB industry. Importantly, the study also developed specific on-site sample preparation methods for the extraction of Li+from relevant materials, including brine, rock, and batteries. Using a simple portable fluorometer, this study has achieved selective in-field detection of Li+down to 10 ppm. The study successfully differentiated several industrial relevant thresholds including 0-200 ppm in brine mimic samples, 0-8% in spodumene rocks, and new from cycle-aged electrodes in LIBS, all with minimal interference from non-target metal ions. The simplicity of the DNAzyme-based fluorescent Li+sensor design overcomes several limitations of current technologies, including complex operation, interference from competing metal ions, and crystal size filtering. Additionally, it is robust enough for application in a variety of environments. Furthermore, sample preparation and Li+detection can be accomplished with basic laboratory equipment and minimal training, making the DNAzyme-based system suitable for on-site and in-field applications.Materials and Methods

[0210] DNA: All DNA was obtained from International DNA Technologies (IDT) with HPLC purification. Sequences (5’ - 3’) are shown in TABLE 2.

[0211] Chemicals and Reagents: All chemicals were obtained from Sigma Aldrich, Thermo Fisher, and Fisher Scientific. The chemicals were sodium chloride (99.998%), calciumAttorney Docket No. 10046-635W01 chloride hydrate (99.9965%), potassium chloride (technical grade), lithium chloride (> - 98.7%), magnesium chloride (99.9%), sulfuric acid (95-98%), calcium oxide 99.95% (metals basis) and were used to prepare all stock solutions (IM LiCl, IM NaCl, IM MgCh, IM CaCh, IM Tris, pH 7.4, 5M H2SO4, 5M KOH, and IM CaO).

[0212] Instruments: Instruments used for the collection of fluorescence data include BioTek Synergy Hl Microplate Reader (Agilent Technologies, Winooski, VT), ISS Chronos Digital Frequency Domain Spectrofluorometer (ISS Inc., Champaign, IL), DeNovix QFX Fluorometer (DeNovix Inc., Wilmington, DE).

[0213] Sensor Preparation : Melting temperature simulations were performed using IDT ’ s sequence tool (OligoAnalyzer) and the UNAFold Web Server (DNA Folding Form) to determine the best binding arm combination for the design of the new sensor. Melting temperatures were assessed for designs under 150 mM monovalent and 2 mM divalent metal ions with the same tool. DNAzyme sensors were synthesized and HPLC purified directly by IDT with fluorophore and quencher modifications. Quantifications were performed by Nanodrop to verify sensor component concentrations.

[0214] Sensor Annealing: The enzyme-substrate complex was formed by annealing a mixture of 20-4RT enzyme and 20-4RT substrate strands for sensor in 150 mM Tris pH 7.4 with the ratio of 1.6:1 with volumes no larger than 50 L. The final concentration of substrate strand in the annealing system is 5 p M for the annealing system. The enzyme-substrate mixes were enclosed in 0.2 mL PCR tubes and annealed in a thermocycler to gradually decrease the temperature from 95 °C to 25°C with the time range of 2 - 3 hours to ensure the highest annealing efficiency. Sensors were diluted to lOx working solutions in 150 mM Tris pH 7.4 prior to the addition to the samples.

[0215] Sample Preparation: Samples are prepared with spiked lithium addition or with certain expected lithium content percentage. For brine mining tests, 1 M LiCl solution was spiked into brine samples containing 465 mM NaCl, 10 mM KC1, 53 mM MgCh, and 10 mM CaCL with different final concentrations (10, 50, 200 ppm). Samples were precipitated as introduced in the main text and were filtered with 0.2 pm syringe filter with a 5 mL syringe. Rock samples were broken into small pieces by hammer and grinded into powder by mortar and pestle. Different percentages of Li2O content rock samples were prepared by mixing spodumene (8% Li2O content) with limestone (0% Li2O content) powder at different ratios. Acid leaching was performed for 1 hour at room temperature. Extracts were filtered by 0.2 pm syringe filter with a 5 mL syringe. Filtered extracts were neutralized and precipitated with different methods as introduced in the main text and were filtered again for portable detections.Attorney Docket No. 10046-635W01For safely disassemble batteries, new and aged batteries were fully discharged by a short circuit connecting the cathode and anode. Batteries were then disassembled, and black matters were scratched off and collected from the cathode and the anode. Leaching and precipitation were carried out as described in the main text. Extracts were filtered by 0.2 pm syringe filter with a 5 mL syringe before portable detections.

[0216] Fluorescence Measurements: Fluorescence assays were carried out using multiple spectrometers, including a fluorescence spectrophotometer, a plate reader and a portable fluorometer.

[0217] The sensor response at certain time points were tested in a ChronosBH TCSPC lifetime spectrometer (ISS Inc, Illinois). Fluorescence data were collected using 1.5mL SemiMicro Spectrophotometry Desposable UV Cuvettes (BRAND, Germany). 400 pL of diluted sensor in Tris buffer was preloaded into the cuvette to 25 nM concentration. Reaction was initiated by mixing 400 pL 2x metal mix into each cuvette. Fluorescence intensity at initial and selected timepoints were collected under single wavelength intensity mode. Excitation wavelengths were set to be 488 nm, and emission were collected at 525 nm. 1.0 excitation slit and 1.0 emission slit.

[0218] The kinetic curve assays were performed in a BioTek Synergy Hl Multimode Reader (Agilent Technologies, California). Fluorescence data were collected with 96-well format in triplicate using the plate reader to simultaneously assess multiple conditions in parallel. Samples were preloaded to 96-well black flat bottom plates. 10 pL lOx sensor in Tris buffer was added into each well containing 90 pF samples. Measurements were collected with fluorescence intensity mode with single-excitation single-wavelength multiple timepoints setup. Intervals were ranging from 3 - 30 minutes over the course of 12 hours with continuous shaking. Shaking mode was set to be double-orbital slow mode. Excitation wavelengths were set to be 480 nm, and emission were collected at 525 nm. Reading distance was 7 nm, and gaining was 100%.

[0219] Portable fluorescence detections were performed with the DeNovix QFX Fluorometer (DeNovix Inc., Delaware) with the basic fluorometer mode. Samples were prepared as introduced and mixed with annealed and diluted sensor with 190 pL to 10 pL combination. Sensor concentrations were 25 nM for condition optimization and 125 nM for on-site detection experiments. The samples were briefly mixed and detected with blue LED excitation filter 442 - 497 nm and green emission filter 514 - 567 nm. Fluorescence intensity values were collected across four hours for condition optimization, and two hours for on-site detection experiments.Attorney Docket No. 10046-635W01

[0220] Calculation of Sensor Activities: (F-Fo) / Fo was used to calculate the relative change in fluorescence intensity compared to the baseline. Fo was measured at 0-minute timepoint after mixing the sensor with samples as initial or baseline fluorescence intensity. F was measured after a certain time point to calculate the sensor activity at that time point.Results and Discussion

[0221] Re-Engineering Li+-DNAzyme Sensor for Detection at Room Temperature: The fundamental principle of DNAzyme-based sensing by the catalytic beacon method relies upon differences in the melting temperatures (Tm) between the uncleaved and cleaved substrate strands to the enzyme strand in DNAzymes, relative to the target application temperature. Since the application of the previously reported 20-4 DNAzyme sensor was intended for cellular imaging, the Tmof the hybridized DNAzyme / substrate was designed to be 56.7 °C before cleavage and 36.1 °C and 33.7 °C after the cleavage (FIG. 1A). This design allowed for sufficient hybridization and fluorescence quenching in the absence of Li+, and fluorescence signal tum-on in the presence of Li+under the cell imaging temperature of 37 °C. However, when tested in the presence of 10 mM Li+at room temperature, the fluorescence increase was 12% less than its previously reported activity at 37 °C 1441. This result was likely because the Tmof the cleaved binding arms were higher than the testing temperature. Consequently, the development of a DNAzyme-based detection method for Li+in mining and recycling applications required optimization of the Li+DNAzyme sensor under ambient conditions. To this end, the study developed a new version of the Li+sensor, named 20-4RT (FIG. IB), to improve signal output at room temperature by elongating one of the binding arms, shortening the other binding arm labeled with FAM fluorophore, and altering the nucleotide contents in both binding arms to achieve specific melting temperature thresholds, while maintaining the ability to form Watson-Crick base pairs. Specifically, 20-4RT DNAzyme was designed to have an increased overall Tmof 62.6 °C for the hybridized DNAzyme / substrate strands before Li+- dependent cleavage to lower the detection background. After the Li+-dependent cleavage, the shortened FAM-labeled binding arm has a lower Tmof only 20.8 °C and thus will more easily dehybridize under room temperature. To assess whether the newly designed 20-4RT DNAzyme sensor outperformed the original 20-4 DNAzyme sensor at the LIB target application temperature, the study first monitored the fluorescent signal output of each sensor at room temperature under different concentrations of Li+(FIG. 1C).

[0222] The 20-4RT DNAzyme sensor showed a greater relative increase in fluorescence intensity at room temperature compared to the original 20-4 DNAzyme sensor as measured by the (F-Fo) / Fo, where Fo represents the baseline fluorescence intensity measured at the zeroAttorney Docket No. 10046-635W01 timepoint upon mixing the sensor with the samples. (FIG. 1C). Thus, enhancing the overall stability of the sensor before the cleavage while shortening the FAM-labeled binding arm after the cleavage improved the performance of the sensor for applications. The study also tested the new 20-4RT DNAzyme sensor at 40 °C, as some field applications necessitate higher temperature conditions. As shown in FIG. 2, the new 20-4RT DNAzyme sensor was able to differentiate different lithium levels up to 200 ppm. Next, to determine whether the reengineering of the DNAzyme sensor affected its selectivity toward other metal ions, the study compared the performance of the original and new sensor in the presence of non-target metal ions that would be present in the final application environment, including Na+, K+, Mg2+, and Ca2+. As shown in FIG. ID, the new 20-4RT DNAzyme sensor displayed a substantially greater fluorescence increase in the presence of 200 mM Li+than in the presence of 200 mM Na+or K+, or 4 mM Mg2+or Ca2+. These results demonstrate that the selectivity of the 20-4RT DNAzyme sensor for Li+over other metal ions was comparable to the original 20-4 DNAzyme sensor (FIG. ID). Taken together, the enhanced signal response and high selectivity of the reengineered Li+DNAzyme design make the 20-4RT DNAzyme sensor a promising candidate for DNAzyme-based Li+detection in LIB industry applications.

[0223] Quantifying Li+in the Presence of High Concentrations of Other Metal Ions: Unlike previous success in imaging Li+in living cells in the presence of ~12 mM Na+, -150 mM K+, -2.1 mM Mg2+and -100 nM Ca2+, or portable detection of Li+in serum in the presence of 1.1 mM Mg2+and 2.5 mM Ca2+using a spectrometry method

[0057] , the detection of Li+in industrial samples necessitates the consideration of these metal ions at much higher concentrations than biological samples, such as -500 mM Na+and K+, -50 mM of Mg2+and Ca2+. Additionally, it has been shown that high concentrations of divalent metal ions may influence DNAzyme activity [55, 56]. Therefore, such high levels of competing metal ions raised concerns on whether the selectivity and performance of the 20-4RT DNAzyme sensor would still be sufficient to reliably differentiate Li+from other metal ions. To address this concern, the study evaluated the response of the 20-4RT DNAzyme sensor towards different levels of Li+in the presence of high levels of competing metal ions. The study was able to detect Li+above 10 ppm in the presence of 500 mM Na+, K+or 10 mM Mg2+, Ca2+(FIGS. 3A- 3D and FIGS. 4A-4D), further supporting the use of the 20-4RT DNAzyme sensor in complex environmental samples.

[0224] Developing a Method for On-site Detection of Lithium in Brine Water: While the study was able to achieve Li+detection down to 10 ppm in the presence of high concentrations of individual non-target metal ions, it remained challenging to overcome theAttorney Docket No. 10046-635W01 effects of exceptionally high concentrations of Mg“+and Ca~+together (>10 mM each), even with 200 ppm of Li+spiked into the sample (FIG. 5).

[0225] Heating water to remove Ca2+and Mg2+as carbonate compounds is a common method used in industry

[0049] , but it is unsuitable for on-site detection methods due to power and time requirements, as well as difficulty controlling sample size and metal ion concentration. Therefore, the study designed and evaluated alternative methods of reducing Mg2+and Ca2+concentrations (FIG. 6A). Lime softening is also used in industry to reduce Mg2+concentrations through the addition of limewater, Ca(OH)2 or simply CaO, to raise the pH and precipitate Mg2+as Mg(OH)2 [50, 51]. Based on the Kspof Mg(OH)2 (1.5xl0-11), it was reasoned that increasing the sample pH from 7.4 to 11 ([OH-] = 10-3M) would promote Mg(OH)2 precipitation to yield a final Mg2+concentration in solution of ~15 pM. As demonstrated in FIG. 14, there is little decrease in the fluorescence signal in the presence of 5 mM level of Mg2+in comparison with that of non-target metal ions. Therefore, Mg2+at such a low concentration of 15 pM should have little effect on the sensor's performance. To test this method, the study added KOH to a simulated brine water sample containing 465 mM Na+, 10 mM K+, 50 mM Mg2+and 10 mM Ca2+, with or without 200 ppm Li+, to raise the pH to 11 and precipitates (ppt) were removed using a syringe filter. The filtered water samples were then neutralized by adding Tris-HCl (pH 7.4) to a final concentration of 150 mM. Indeed, the study observed fluorescence tum-on from the brine-mimic water samples containing 200 ppm Li+, and could differentiate the samples from the controls (0 ppm Li+) using a simple portable fluorometer.

[0226] The study tested two additional approaches to further reduce the influence of high Ca2+concentrations on sensor activity (FIG. 6A). In the first approach, the study added 5 mM of EDTA to the filtered samples and observed an improved signal turn-on of the sensor (FIGS. 7A-7B). In the second approach, the study introduced 10 mM of CCh2-into the filtered sample prior to Tris neutralization to precipitate Ca2+ions to a final solution concentration of ~62 pM, which would not interfere with sensor activity ( Kspof CaCO = 3.8xl0-9). The supernatant was filtered a second time to remove CaCCL precipitate and neutralized by Tris buffer addition. The CaCCh approach achieved comparable results to the EDTA-based approach, suggesting that both methods can effectively reduce Ca2+interference from brine-mimic samples and for on-site Li+detection in field samples. In both Ca2+removal approaches, the signals in the 0 ppm Li+samples were higher than that from the OH- precipitation group without Ca2+removal, suggesting that the background signal elevation became more apparent as the Ca2+level decreases. The EDTA-based chelation approach is the simpler of the two methods, requiringAttorney Docket No. 10046-635W01 only one round of precipitation and filtration, whereas the CaCCU-based precipitation method, while it can further reduce Ca2+, does not necessarily improve the signal output. Therefore, the study chose to utilize the EDTA-based approach (FIG. 6B) as the primary method in subsequent brine-mimic Li+detection experiments with a portable fluorometer to simulate field conditions. The study also tested whether the amount of sensor contributes to the performance of the sensor. Upon titrating the sensor amount (FIG. 8), the study found 125 nM sensor concentration performs the best and decided to use this amount for further experiments. Using this method and the 20-4RT DNAzyme sensor, the study achieved the detection of Li+in brine water samples with the ability to differentiate Li+at the commercially valuable level of 200 ppm and an enriched level of 50 ppm from either the lower level of 10 ppm or no Li+using a simple handheld portable fluorometer (FIG. 6C). The OH precipitation method without Ca2+removal, which has fewer number of steps and lower background signal, or the CaCO3-based precipitation method, which result in lower final Ca2+interferences, can also be viable options for sample preparation in different industrial applications.

[0227] Developing a Method for On-site Detection of Lithium in Hard Rocks: Rock mining is the second major source of Li+for industrial use. Assessing Li+content typically requires the pretreatment of rock samples with high temperatures prior to its leaching and extraction, which is not realistic to perform in on-site settings [3]. Thus, the study sought out a method for Li+extraction that did not require extreme temperatures and was appropriate for in- the-field use and subsequent Li+detection using the 20-4RT DNAzyme sensor. For on-site field tests of Li+from hard rocks, the study used spodumene as the target Li+-containing rock samples with limestone and glass as negative controls. In this approach, the study extracted Li+by pulverizing spodumene rocks with a mortar and pestle, then added different concentrations of sulfuric acid (0.05 M H2SO4, 4 M H2SO4, 50% H2SO4 or 98% H2SO4) to leach the Li+from the rock powders, following a previously reported protocol

[0052] . After the extraction process, the supernatant was collected and samples were prepared for ICP-MS analysis to evaluate the leaching methods (FIG. 9A). It was found that 0.05 M H2SO4 incubation provided the highest leaching efficiency, and that there was a negative correlation between the leaching efficiency and the concentration of H2SO4. No detectable Li+was extracted from limestone and glass sample controls, supporting the purity of extraction materials and the reliability of the leaching method. However, while the leaching method proved to be successful, the method still needed to address other ionic components in the sample that could affect the sensor's activity.

[0228] The lithium rock mining industry often uses limewater to neutralize the leached acidic sample to remove most frequently observed crust elements such as iron and aluminumAttorney Docket No. 10046-635W01

[0052] . As such, the study adopted this acid neutralization method by treating [ hSCh-leached samples with CaO, generating soluble Ca(0H)2 and CaSO4 precipitate in the process. Based on the Kspof CaSOr (2.4xl0-5), the study calculated the final concentration of Ca2+to be ~5 mM following the neutralization and filtration of the leaching solution. A concentration of Ca2+at this level will not significantly affect the sensor (FIG. 15). The study also evaluated the possibility of utilizing KOH for the neutralization step to avoid introducing additional Ca2+ions that may interfere with detection, as well as a combination of the CaO and KOH neutralization methods (50% of each reagent used). The study evaluated these neutralization approaches using a solution containing 0.05 M H2SO4 spiked with 200 ppm Li+as the leaching product mimic. After neutralization, the samples were filtered and mixed with Tris to a final concentration of 150 mM, and the 20-4RT DNAzyme sensor was added to detect the Li+in the final neutralized, leach-mimic samples. The resulting fluorescent readouts demonstrated that while all three methods produced an increased fluorescent signal, the combination CaO-KOH neutralization method showed the greatest signal tum-on (FIG. 9B). The study further applied this method for Li+detection in leaching product mimics and tested the effect of EDTA on signal output to remove residual Ca2+interference introduced during the neutralization step (FIG. 16). The study observed -50% signal improvement with the addition of 2 mM EDTA, validating the incorporation of a supplemental EDTA clean-up step into the method (FIG. 9C). Applying the on-site detection method for Li+from hard rocks, including the optimized steps of 0.05 M H2SO4 extraction, CaO-KOH combination neutralization, and EDTA clean-up (FIG. 9D), the study observed a significant signal increase from rock samples with higher lithium content than those without. More specifically, the study was able to differentiate samples of pure spodumene (8% Li2O) and SC6 commercial standard (6% Li2O) from samples containing lower or no Li+(4%, 2%, and 0% Li2O), as shown in FIG. 9E. As such, these results support the efficiency and validity of the method in distinguishing economically valuable sources of Li+in spodumene rock using a simple handheld fluorometer (FIG. 9E).

[0229] Developing a Method for On-site Detection of Lithium from Recycled Batteries: While all Li+-ion batteries have materials that are worthy of recycling, the loss of Li+through formation of solid-electrolyte interphase, dendrite growth, and side reactions may reduce the amount of recoverable Li+and impact effectiveness of recycling

[0053] , Therefore, an on-site detection method for recoverable Li+in spent LIBs utilized before initiating the recycling process will be vital for assessing its economic viability. To provide an on-site evaluation of Li+amount in aged battery electrodes, the study sought to adopt the Li+leaching and detection methods developed in this work for the comparison of Li+loss from electrodesAttorney Docket No. 10046-635W01 in batteries at different stages of cycle-induced aging. After disassembling a new Li+-ion battery, the study utilized a leaching method based on the hard rock leaching method using 0.05 M H2SO4, and a neutralization method similar to Mg2+reduction method (FIG. 11A), both demonstrated in the previous sections. Although Mg2+and Ca2+are not commonly used in battery electrodes, electrode materials such as Mn2+, Co2+, and Ni2+all share the property of a low KSp of their hydroxide products (4.6xl0“14for Mn(0H)2, 2.5xl0"16for Co(OH)2, and 2.8xl0“16for Ni(0H)2) that may precipitate at pH >10, allowing only nM to pM levels of cation impurities in the supernatant. The LIBs utilized in this study were composed of a lithium cobalt oxide (LiCoO2) cathode and graphite anode

[0053] . Following hydroxide product precipitation, the supernatant was filtered and neutralized with Tris to a final concentration of 150 mM for subsequent sensor addition and portable detection (FIG. 11A). To assess the Li+loss of aged batteries, the study completed multiple cycles of charging and discharging. As shown in FIG. 11B, the on-site leaching and detection method could detect Li+enrichment in the cathode compared with the anode as expected in new LIBs. Moreover, the method was able to differentiate decreasing Li+levels in battery cathodes that had been cycle-aged with ~70 and -150 cycles, relative to the new battery cathode (FIG. 11B). In addition, the opposite trend was observed when detecting Li+in the anode, in which the method could differentiate increasing Li+levels in cycle-aged battery anodes, relative to the new battery anode (FIG. 11B) These results suggest that the method reliably reflects information regarding the amount of recoverable Li+in battery electrodes, which would help on-site quality control and evaluation of Li+content in battery manufacturing and recycling.Conclusion

[0230] This study developed a Li+DNAzyme-based fluorescent sensor for portable on-site detection and quantification of Li+in brine water, hard rocks, and recycled lithium batteries, addressing the limitations of current methods in the LIB industry. To adapt this system for diverse applications in brine and rock mining, as well as battery recycling, the study developed on-site sample preparation methods for efficient Li+extraction tailored for a variety of natural and battery-derived samples. By integrating the DNAzyme sensor with sample preparation methods and portable fluorescence detection devices, the study achieved sensitive detection of lithium down to 10 ppm. The detection was accomplished with minimal interference from nontarget metal ions and enabled differentiation of a series of lithium thresholds relevant to different industries. Specifically, the study was able to differentiate between 0, 10, 50, and 200 ppm Li+in brine mimic samples, 0%, 2%, 4%, 6% (the SC6 standard) and 8% Li2O inAttorney Docket No. 10046-635W01 spodumene rock, and between new, ~70 cycle-aged, and -150 cycle-aged LIB cathodes and anodes.

[0231] Significantly, the on-site detection method demonstrated in this work simplified sample preparation steps and equipment requirements, enhancing in-field applications. All portable detection was performed under field conditions using conventional sample collection equipment (harmer, mortar and pestle), a portable fluorometer, syringe filters, pipettors, and a few consumables (tubes, tips, gloves) at ambient temperature. The streamlined equipment requirements reduce space and weight in a travel package, crucial for in-field applications where portability is paramount. Furthermore, this method eliminates the need for excessive heat, lasers, or X-ray sources, reducing safety concerns and precautions.

[0232] Moreover, this detection method avoids complex sample pretreatment techniques. It does not require the combined acid digestion and total dissolved solids adjustment needed for ICP-MS, argon purging and matrix matching in LIBS, or crystal size filtering and internal calibration in XRD. The simplicity of this method significantly reduces the expertise required for operation. With straightforward steps such as sample weighing, liquid transfer and mixing, pH adjustment, syringe filtering, and portable fluorometer reading, individuals with basic lab training can efficiently conduct lithium detection. This approach results in significant cost savings on trained personnel.

[0233] While the catalytic beacon method can be used under multi- turnover conditions to take advantage of the signal amplification capability of the RNA-cleaving DNAzyme, the study chose the single turnover condition to avoid dehybridization of the excess amount of the substrate strand that may result in a high background signal. Since this study has achieved the goal of differentiating a set of commercially valuable lithium levels in the lithium-enriched sources, signal amplification is not absolutely required for this application.

[0234] In summary, the methods demonstrated in this work achieved highly selective lithium detection down to 10 ppm. These methods can distinguish key threshold lithium levels that indicate economic value across several industries, including 200 ppm Li+for brine mining, 6% Li2O or SC6 for rock mining, and Li+-specific aging in LIBs. This workflow can be conducted with minimal laboratory equipment or training, making it suitable for on-site and in-field applications.EXAMPLE ASPECTS

[0235] Example 1 : A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrateAttorney Docket No. 10046-635W01 strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; wherein the substrate strand comprises, from 5’ to 3’, a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid comprises from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid comprises from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid hy at least one nucleotide.

[0236] Example 2: The DNAzyme sensor of any examples herein, particularly Example 1, wherein the substrate strand comprises SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the enzyme strand comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[0237] Example 3: The DNAzyme sensor of any examples herein, particularly Examples 1-2, wherein the substrate strand comprises a variant of SEQ ID NO: 1 or a variant of SEQ ID NO: 2; and wherein the enzyme strand comprises a variant of SEQ ID NO: 3 or a variant of SEQ ID NO: 4.

[0238] Example 4: The DNAzyme sensor of any examples herein, particularly Examples 1-3, wherein the first segment has a melting temperature of up to about 30°C; and wherein the second segment has a melting temperature of at least about 30°C.

[0239] Example 5: The DNAzyme sensor of any examples herein, particularly Example 4, wherein the first segment has a melting temperature of up to about 25 °C; and wherein the second segment has a melting temperature of at least about 50°C.

[0240] Example 6 : A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the enzyme strand comprises SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.

[0241] Example 7: The DNAzyme sensor of any examples herein, particularly Example 6, wherein the substrate strand comprises SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the enzyme strand comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[0242] Example 8: The DNAzyme sensor of any examples herein, particularly Examples 6-7, wherein the substrate strand comprises a variant of SEQ ID NO: 1 or a variant of SEQ IDAttorney Docket No. 10046-635W01NO: 2; and wherein the enzyme strand comprises a variant of SEQ ID NO: 3 or a variant of SEQ ID NO: 4.

[0243] Example 9: The DNAzyme sensor of any examples herein, particularly Examples 6-8, wherein the cleavage site is interspersed between two segments of nucleic acid.

[0244] Example 10: The DNAzyme sensor of any examples herein, particularly Example9, wherein the two segments of nucleic acid have a same length.

[0245] Example 11: The DNAzyme sensor of any examples herein, particularly Example10, wherein the two segments of nucleic acid have different lengths.

[0246] Example 12: The DNAzyme sensor of any examples herein, particularly Examples 9-11, wherein each of the two segments of nucleic acid comprises about 3 to about 90 nucleotides.

[0247] Example 13: The DNAzyme sensor of any examples herein, particularly Examples 1-12, wherein the substrate strand and / or the enzyme strand comprise DNA.

[0248] Example 14: The DNAzyme sensor of any examples herein, particularly Examples 1-13, wherein the cleavage site comprises at least one RNA base.

[0249] Example 15: The DNAzyme sensor of any examples herein, particularly Example 14, wherein the cleavage site comprises 1 to 5 RNA bases.

[0250] Example 16: The DNAzyme sensor of any examples herein, particularly Examples 1-15, wherein the substrate strand further comprises at least one non-natural nucleic acid.

[0251] Example 17: The DNAzyme sensor of any examples herein, particularly Example 16, wherein the at least one non-natural nucleic acid is a locked nucleic acid (LNA) or a 2’- fluoro arabino nucleic acid (FAN A).

[0252] Example 18: The DNAzyme sensor of any examples herein, particularly Examples 1-17, wherein the enzyme strand comprises at least one loop region.

[0253] Example 19: The DNAzyme sensor of any examples herein, particularly Examples 1-18, wherein the enzyme strand comprises a Li+binding region.

[0254] Example 20: The DNAzyme sensor of any examples herein, particularly Examples 1-19, wherein the detectable signal is a fluorophore or a fluorescent dye.

[0255] Example 21: The DNAzyme sensor of any examples herein, particularly Example 20, wherein the detectable signal is Alexa Fluor 647 or Alexa Fluor 488.

[0256] Example 22: The DNAzyme sensor of any examples herein, particularly Examples 1-19, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.Attorney Docket No. 10046-635W01

[0257] Example 23: The DNAzyme sensor of any examples herein, particularly Example 22, wherein the detectable signal is indocyanine green, methylene blue, or Evans blue.

[0258] Example 24: The DNAzyme sensor of any examples herein, particularly Examples 1-23, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand.

[0259] Example 25: The DNAzyme sensor of any examples herein, particularly Examples 1-24, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

[0260] Example 26: The DNAzyme sensor of any examples herein, particularly Examples 24-25, wherein the quencher is Iowa Black RQ or Iowa Black FQ.

[0261] Example 27: The DNAzyme sensor of any examples herein, particularly Examples 1 -26, wherein the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+.

[0262] Example 28: A kit comprising: a DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; and a leaching agent, a precipitating agent, and / or a chelating agent.

[0263] Example 29: The kit of any examples herein, particularly Example 28, wherein the cleavage site comprises at least one RNA base.

[0264] Example 30: The kit of any examples herein, particularly Example 29, wherein the cleavage site comprises 1 to 5 RNA bases.

[0265] Example 31: The kit of any examples herein, particularly Examples 28-30, wherein the cleavage site is interspersed between two segments of nucleic acid.

[0266] Example 32: The kit of any examples herein, particularly Example 31, wherein the two segments of nucleic acid have a same length.

[0267] Example 33: The kit of any examples herein, particularly Example 32, wherein the two segments of nucleic acid have different lengths.

[0268] Example 34: The kit of any examples herein, particularly Examples 31-33, wherein each of the two segments of nucleic acid comprises about 3 to about 90 nucleotides.

[0269] Example 35: The kit of any examples herein, particularly Examples 28-34, wherein the substrate strand and / or the enzyme strand comprise DNA.Attorney Docket No. 10046-635W01

[0270] Example 36: The kit of any examples herein, particularly Examples 28-35, wherein the substrate strand further comprises at least one non-natural nucleic acid.

[0271] Example 37: The kit of any examples herein, particularly Example 36, wherein the at least one non-natural nucleic acid is a locked nucleic acid (LN A) or a 2’ -fluoro arabino nucleic acid (FANA).

[0272] Example 38: The kit of any examples herein, particularly Examples 28-37, wherein the enzyme strand comprises at least one loop region.

[0273] Example 39: The kit of any examples herein, particularly Examples 28-38, wherein the enzyme strand comprises a Li+binding region.

[0274] Example 40: The kit of any examples herein, particularly Examples 28-39, wherein the substrate strand comprises SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the enzyme strand comprises SEQ ID NO: 3 or SEQ ID NO: 4.

[0275] Example 41: The kit of any examples herein, particularly Examples 28-40, wherein the substrate strand comprises a variant of SEQ ID NO: 1 or a variant of SEQ ID NO: 2; and wherein the enzyme strand comprises a variant of SEQ ID NO: 3 or a variant of SEQ ID NO: 4.

[0276] Example 42: The kit of any examples herein, particularly Examples 28-41 , wherein the detectable signal is a fluorophore or a fluorescent dye.

[0277] Example 43: The kit of any examples herein, particularly Example 42, wherein the detectable signal is Alexa Fluor 647 or Alexa Fluor 488.

[0278] Example 44: The kit of any examples herein, particularly Examples 28-43, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

[0279] Example 45: The kit of any examples herein, particularly Example 44, wherein the detectable signal is indocyanine green, methylene blue, or Evans blue.

[0280] Example 46: The kit of any examples herein, particularly Examples 28-45, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand.

[0281] Example 47: The kit of any examples herein, particularly Examples 28-46, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

[0282] Example 48: The kit of any examples herein, particularly Examples 46-47, wherein the quencher is Iowa Black RQ or Iowa Black FQ.Attorney Docket No. 10046-635W01

[0283] Example 49: The kit of any examples herein, particularly Examples 28-48, wherein the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+.

[0284] Example 50: The kit of any examples herein, particularly Examples 28-49, further comprising an inactive DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand comprising at least one mutation; wherein the at least one mutation prevents the inactive enzyme strand from cleaving the substrate strand.

[0285] Example 51: The kit of any examples herein, particularly Example 50, wherein the substrate strand comprises SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the inactive enzyme strand comprises SEQ ID NO: 5.

[0286] Example 52: The kit of any examples herein, particularly Examples 50-51 , wherein the substrate strand comprises a variant of SEQ ID NO: 1 or a variant of SEQ ID NO: 2; and wherein the inactive enzyme strand comprises a variant of SEQ ID NO: 5.

[0287] Example 53: The kit of any examples herein, particularly Examples 28-52, wherein the leaching agent comprises an acid.

[0288] Example 54: The kit of any examples herein, particularly Example 53, wherein the acid has a pH of from about 0 to about 2.

[0289] Example 55: The kit of any examples herein, particularly Examples 53-54, wherein the acid comprises sulfuric acid.

[0290] Example 56: The kit of any examples herein, particularly Examples 28-55, wherein the precipitating agent forms a precipitate with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

[0291] Example 57: The kit of any examples herein, particularly Example 56, wherein the precipitating agent comprises OH', COr', SOr', or any combination thereof.

[0292] Example 58: The kit of any examples herein, particularly Examples 28-57, wherein the chelating agent chelates with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

[0293] Example 59: The kit of any examples herein, particularly Example 58, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

[0294] Example 60: The kit of any examples herein, particularly Examples 28-59, further comprising a filter.

[0295] Example 61: The kit of any examples herein, particularly Example 60, wherein the filter has a pore size of from about 0.1 pm to about 1 pm.Attorney Docket No. 10046-635W01

[0296] Example 62: The kit of any examples herein, particularly Examples 28-61, further comprising a fluorometer.

[0297] Example 62: The kit of any examples herein, particularly Example 62, wherein the fluorometer is a portable fluorometer.

[0298] Example 64: A method of detecting Li+in a sample, comprising: a) treating the sample with a leaching agent, a precipitating agent, and / or a chelating agent; and b) exposing the sample to a DNAzyme sensor, the DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; and c) identifying the detectable signal, thereby detecting Li+in the sample.

[0299] Example 65: The method of any examples herein, particularly Example 64, wherein, before step a), the sample comprises an ionic salt.

[0300] Example 66: The method of any examples herein, particularly Example 65, wherein the ionic salt is: Na+in a concentration of from about 0 to about 500 mM; K+in a concentration of from about 0 to about 500 mM; Mg2+in a concentration of from about 0 to about 10 mM; Ca2+in a concentration of from about 0 to about 10 mM; Mn2+in a concentration of from about 0 to about 4 mM; Co2+in a concentration of from about 0 to about 4 mM; Ni2+in a concentration of from about 0 to about 4 mM; Fe3+in a concentration of from about 0 to about 0.4 mM; Al3+in a concentration of from about 0 to about 0.4 mM; or any combination thereof.

[0301] Example 67: The method of any examples herein, particularly Examples 65-66, wherein step a) reduces concentration of the at least one ionic salt by from about 100% to about 0.00001%.

[0302] Example 68: The method of any examples herein, particularly Examples 64-67, wherein the sample is brine water.

[0303] Example 69: The method of any examples herein, particularly Examples 64-67, wherein the sample is rock.

[0304] Example 70: The method of any examples herein, particularly Examples 64-67, wherein the sample is a portion of a lithium ion battery.

[0305] Example 71: The method of any examples herein, particularly Examples 64-70, further comprising, before step a), providing the kit of any examples herein, particularly Examples 28-63.Attorney Docket No. 10046-635W01

[0306] Example 72: The method of any examples herein, particularly Examples 64-71, wherein the leaching agent comprises an acid.

[0307] Example 73: The method of any examples herein, particularly Example 72, wherein the acid has a pH of from about 2 to about 7 .

[0308] Example 74: The method of any examples herein, particularly Examples 72-73, wherein the acid comprises sulfuric acid.

[0309] Example 75: The method of any examples herein, particularly Examples 64-74, wherein treating the sample with the leaching agent in step a) comprises: i) optionally crushing the sample; ii) incubating the sample with the leaching agent for from about 1 hour to about 2 hours; and iii) collecting a supernatant from the incubated sample.

[0310] Example 76: The method of any examples herein, particularly Examples 64-75, wherein the precipitating agent forms a precipitate with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

[0311] Example 77: The method of any examples herein, particularly Example 76, wherein the precipitating agent comprises OH', CO?2', SO42', or any combination thereof.

[0312] Example 78: The method of any examples herein, particularly Examples 64-77, wherein treating the sample with the precipitating agent in step a) comprises: i) adding the precipitating agent in a concentration of from about 0 to about 50 mM, thereby forming a precipitate; and ii) optionally filtering the sample to remove the precipitate.

[0313] Example 79: The method of any examples herein, particularly Examples 64-78, wherein the chelating agent chelates with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Mg2+, Ca2+, Fe3+, Al3+, or any combination thereof.

[0314] Example 80: The method of any examples herein, particularly Example 79, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

[0315] Example 81: The method of any examples herein, particularly Examples 64-80, wherein treating the sample with the chelating agent in step a) comprises: i) adding the chelating agent in a concentration of from about 0 to about 10 mM, thereby forming a chelate; and ii) optionally filtering the sample to remove the chelate.

[0316] Example 82: The method of any examples herein, particularly Examples 64-81, wherein step a) further comprises neutralizing excess leaching agent, precipitating agent, and / or chelating agent before step c).

[0317] Example 83: The method of any examples herein, particularly Examples 64-82, wherein the substrate strand comprises SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the enzyme strand comprises SEQ ID NO: 3 or SEQ ID NO: 4.Attorney Docket No. 10046-635W01

[0318] Example 84: The method of any examples herein, particularly Examples 64-83, wherein the substrate strand comprises a variant of SEQ ID NO: 1 or a variant of SEQ ID NO: 2; and wherein the enzyme strand comprises a variant of SEQ ID NO: 3 or a variant of SEQ ID NO: 4.

[0319] Example 85: The method of any examples herein, particularly Examples 64-84, wherein the detectable signal is a lluorophore or a fluorescent dye.

[0320] Example 86: The method of any examples herein, particularly Example 85, wherein the detectable signal is Alexa Fluor 647 or Alexa Fluor 488.

[0321] Example 87: The method of any examples herein, particularly Examples 64-84, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

[0322] Example 88: The method of any examples herein, particularly Example 87, wherein the detectable signal is indocyanine green, methylene blue, or Evans blue.

[0323] Example 89: The method of any examples herein, particularly Examples 87-88, wherein step b) further comprises exposing the cell to an acoustic signal.

[0324] Example 90: The method of any examples herein, particularly Example 89, wherein the acoustic signal is high frequency ultrasound (HIFU).

[0325] Example 91: The method of any examples herein, particularly Examples 64-90, further comprising, before step b), annealing the substrate strand and the enzyme strand together.

[0326] Example 92: The method of any examples herein, particularly Examples 64-91, wherein step c) comprises measuring a level of the detectable signal using a fluorometer.

[0327] Example 93: The method of any examples herein, particularly Example 92, wherein the method is used to detect a concentration of Li+in the sample of at least about 1 ppm.

[0328] Example 94: The method of any examples herein, particularly Examples 92-93, further comprising, before step c), providing a reference level of the detectable signal by: i) providing to the sample an inactive DNAzyme sensor, the inactive DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand comprising at least one mutation; wherein the at least one mutation prevents the inactive enzyme strand from cleaving the substrate strand; and ii) measuring a level of the detectable signal using the fluorometer, thereby providing a reference level of the detectable signal; wherein the referenceAttorney Docket No. 10046-635W01 level of the detectable signal is used to eliminate background noise the level of the detectable signal determined in step d).

[0329] Example 95: The method of any examples herein, particularly Example 94, wherein the substrate strand comprises SEQ ID NO: 1 or SEQ ID NO: 2; and wherein the inactive enzyme strand comprises SEQ ID NO: 5.

[0330] Example 96: The method of any examples herein, particularly Examples 94-95, wherein the substrate strand comprises a variant of SEQ ID NO: 1 or a variant of SEQ ID NO: 2; and wherein the inactive enzyme strand comprises a variant of SEQ ID NO: 5.

[0331] Example 97: The method of any examples herein, particularly Examples 92-96, wherein the method is used to quantify the concentration of Li+in the sample.

[0332] Example 98: The method of any examples herein, particularly Example 92, wherein the method is used to determine the absence of Li+in the sample.

[0333] Example 99: The method of any examples herein, particularly Examples 64-98, wherein the method is carried out at a temperature of from about 10°C to about 40°C.

[0334] Example 100: A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; wherein the substrate strand comprises, from 5’ to 3’, a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid comprises from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid comprises from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid by at least one nucleotide.

[0335] Example 101: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the enzyme strand comprises SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.

[0336] Example 102: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the first segment has a melting temperature of up to about 30°C; and wherein the second segment has a melting temperature of at least about 30°C.

[0337] Example 103: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the cleavage site comprises at least one RNA base.Attorney Docket No. 10046-635W01

[0338] Example 104: The DNAzyme sensor of any examples herein, particularly Example 103, wherein the cleavage site comprises 1 to 5 RNA bases.

[0339] Example 105: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the substrate strand further comprises at least one non-natural nucleic acid.

[0340] Example 106: The DNAzyme sensor of any examples herein, particularly Example 105, wherein the at least one non-natural nucleic acid is a locked nucleic acid (LNA) or a 2’- fluoro arabino nucleic acid (FANA).

[0341] Example 107: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the enzyme strand comprises at least one loop region.

[0342] Example 108: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the enzyme strand comprises a Li+binding region.

[0343] Example 109: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the detectable signal is a fluorophore or a fluorescent dye.

[0344] Example 110: The DNAzyme sensor of any examples herein, particularly Example 109, wherein the detectable signal is Alexa Fluor 647 or Alexa Fluor 488.

[0345] Example 111: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

[0346] Example 112: The DNAzyme sensor of any examples herein, particularly Example 111, wherein the detectable signal is indocyanine green, methylene blue, or Evans blue.

[0347] Example 113: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand; or wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

[0348] Example 114: The DNAzyme sensor of any examples herein, particularly Example 113, wherein the quencher is Iowa Black RQ or Iowa Black FQ.

[0349] Example 115: The DNAzyme sensor of any examples herein, particularly Example 100, wherein the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+.

[0350] Example 116: A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavageAttorney Docket No. 10046-635W01 site in presence of Li+, wherein said cleavage activates the detectable signal; wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the enzyme strand comprises SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.

[0351] Example 117: A kit comprising: the DNAzyme sensor of any examples herein, particularly Example 100; and a leaching agent, a precipitating agent, and / or a chelating agent.

[0352] Example 118: A method of detecting Li+in a sample, comprising: a) treating the sample with a leaching agent, a precipitating agent, and / or a chelating agent; and b) exposing the sample to the DNAzyme sensor of any examples herein, particularly Example 100; and c) identifying the detectable signal, thereby detecting Li+in the sample.

[0353] Example 119: The method of any examples herein, particularly Example 118, wherein the sample is brine water, rock, or a portion of a lithium ion battery.

[0354] Example 120: A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the enzyme strand comprises SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.

[0355] Example 121: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the cleavage site is interspersed between two segments of nucleic acid; and wherein each of the two segments of nucleic acid comprises about 3 to about 90 nucleotides.

[0356] Example 122: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the cleavage site comprises at least one RNA base.

[0357] Example 123: The DNAzyme sensor of any examples herein, particularly Example 122, wherein the cleavage site comprises 1 to 5 RNA bases.

[0358] Example 124: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the substrate strand further comprises at least one non-natural nucleic acid.

[0359] Example 125: The DNAzyme sensor of any examples herein, particularly Example 124, wherein the at least one non-natural nucleic acid is a locked nucleic acid (LNA) or a 2’- fluoro arabino nucleic acid (FAN A).

[0360] Example 126: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the enzyme strand comprises at least one loop region.

[0361] Example 127: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the enzyme strand comprises a Li+binding region.Attorney Docket No. 10046-635W01

[0362] Example 128: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the detectable signal is a fluorophore or a fluorescent dye.

[0363] Example 129: The DNAzyme sensor of any examples herein, particularly Example 128, wherein the detectable signal is Alexa Fluor 647 or Alexa Fluor 488.

[0364] Example 130: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

[0365] Example 131: The DNAzyme sensor of any examples herein, particularly Example 130, wherein the detectable signal is indocyanine green, methylene blue, or Evans blue.

[0366] Example 132: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand; or wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

[0367] Example 133: The DNAzyme sensor of any examples herein, particularly Example 132, wherein the quencher is Iowa Black RQ or Iowa Black FQ.

[0368] Example 134: The DNAzyme sensor of any examples herein, particularly Example 120, wherein the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+.

[0369] Example 135: A DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; wherein the substrate strand comprises, from 5’ to 3’, a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid comprises from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid comprises from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid by at least one nucleotide.

[0370] Example 136: The DNAzyme sensor of any examples herein, particularly Example 135, wherein the first segment has a melting temperature of up to about 30°C; and wherein the second segment has a melting temperature of at least about 30°C.Attorney Docket No. 10046-635W01

[0371] Example 137: A kit comprising: the DNAzyme sensor of any examples herein, particularly Example 120; and a leaching agent, a precipitating agent, and / or a chelating agent.

[0372] Example 138: A method of detecting Li+in a sample, comprising: a) treating the sample with a leaching agent, a precipitating agent, and / or a chelating agent; and b) exposing the sample to the DNAzyme sensor of any examples herein, particularly Example 120; and c) identifying the detectable signal, thereby detecting Li+in the sample.

[0373] Example 139: The method of any examples herein, particularly Example 138, wherein the sample is brine water, rock, or a portion of a lithium ion battery.

[0374] Example 140: A kit comprising: a DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; and a leaching agent, a precipitating agent, and / or a chelating agent.

[0375] Example 141: The kit of any examples herein, particularly Example 140, wherein the cleavage site comprises at least one RNA base.

[0376] Example 142: The kit of any examples herein, particularly Example 140, wherein the cleavage site is interspersed between two segments of nucleic acid; and wherein each of the two segments of nucleic acid comprises about 3 to about 90 nucleotides.

[0377] Example 143: The kit of any examples herein, particularly Example 140, wherein the substrate strand further comprises at least one non-natural nucleic acid.

[0378] Example 144: The kit of any examples herein, particularly Example 140, wherein the substrate strand comprises, from 5’ to 3’, a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid comprises from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid comprises from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid by at least one nucleotide.

[0379] Example 145: The kit of any examples herein, particularly Example 140, wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the enzyme strand comprises SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.

[0380] Example 146: The kit of any examples herein, particularly Example 140, wherein the detectable signal is a fluorophore or a fluorescent dye.Attorney Docket No. 10046-635W01

[0381] Example 147: The kit of any examples herein, particularly Example 140, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

[0382] Example 148: The kit of any examples herein, particularly Example 140, wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand; or wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

[0383] Example 149: The kit of any examples herein, particularly Example 140, wherein the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+.

[0384] Example 150: The kit of any examples herein, particularly Example 140, further comprising an inactive DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand comprising at least one mutation; wherein the at least one mutation prevents the inactive enzyme strand from cleaving the substrate strand.

[0385] Example 151: The kit of any examples herein, particularly Example 150, wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the inactive enzyme strand comprises SEQ ID NO: 5 or a variant thereof.

[0386] Example 152: The kit of any examples herein, particularly Example 140, wherein the leaching agent comprises an acid.

[0387] Example 153: The kit of any examples herein, particularly Example 140, wherein the precipitating agent forms a precipitate with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

[0388] Example 154: The kit of any examples herein, particularly Example 153, wherein the precipitating agent comprises OH’, CO32’, SO ’, or any combination thereof.

[0389] Example 155: The kit of any examples herein, particularly Example 140, wherein the chelating agent chelates with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

[0390] Example 156: The kit of any examples herein, particularly Example 155, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

[0391] Example 157: The kit of any examples herein, particularly Example 140, further comprising a filter and / or a fluorometer.

[0392] Example 158: A method of detecting Li+in a sample, comprising: a) providing the kit of any examples herein, particularly Example 140; b) treating the sample with the leachingAttorney Docket No. 10046-635W01 agent, precipitating agent, and / or chelating agent; and c) exposing the sample to the DNAzyme sensor; and d) identifying the detectable signal, thereby detecting Li+in the sample.

[0393] Example 159: The method of any examples herein, particularly Example 158, wherein the sample is brine water, rock, or a portion of a lithium battery.

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Claims

Attorney Docket No. 10046-635W01What is claimed is:

1. A kit comprising: a DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; wherein the detectable signal is deactivated when lithium (Li+) is not present; and an enzyme strand at least partially complementary to the substrate strand; wherein the enzyme strand is capable of cleaving the substrate strand at the cleavage site in presence of Li+, wherein said cleavage activates the detectable signal; and a leaching agent, a precipitating agent, and / or a chelating agent.

2. The kit of claim 1, wherein the cleavage site comprises at least one RNA base.

3. The kit of claim 1, wherein the cleavage site is interspersed between two segments of nucleic acid; and wherein each of the two segments of nucleic acid comprises about 3 to about 90 nucleotides.

4. The kit of claim 1, wherein the substrate strand further comprises at least one nonnatural nucleic acid.

5. The kit of claim 1 , wherein the substrate strand comprises, from 5 ’ to 3 ’ , a first segment of nucleic acid, the cleavage site, and then a second segment of nucleic acid, so that the cleavage site is interspersed between the first segment of nucleic acid and the second segment of nucleic acid; wherein the first segment of nucleic acid comprises from about 3 to about 90 nucleotides and the detectable signal; and wherein the second segment of nucleic acid comprises from about 10 to about 90 nucleotides and is longer than the first segment of nucleic acid by at least one nucleotide.

6. The kit of claim 1 , wherein the substrate strand comprises SEQ ID NO: 1 , SEQ ID NO: 2, or a variant thereof; and wherein the enzyme strand comprises SEQ ID NO: 3, SEQ ID NO: 4, or a variant thereof.Attorney Docket No. 10046-635W017. The kit of claim 1, wherein the detectable signal is a fluorophore or a fluorescent dye.

8. The kit of claim 1, wherein the detectable signal is a photoacoustic dye; and wherein, when the substrate strand is cleaved, the detectable signal is activated upon exposure to an acoustic signal.

9. The kit of claim 1 , wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a complementary end of the enzyme strand; or wherein the detectable signal is conjugated to a first end of the substrate strand, and wherein a quencher is conjugated to a second end of the substrate strand.

10. The kit of claim 1, wherein the enzyme strand can activate the detectable signal in the presence of at least about 1 ppm Li+.

11. The kit of claim 1, further comprising an inactive DNAzyme sensor comprising: a substrate strand comprising a cleavage site and a detectable signal; and an inactive enzyme strand at least partially complementary to the substrate strand comprising at least one mutation; wherein the at least one mutation prevents the inactive enzyme strand from cleaving the substrate strand.

12. The kit of claim 11, wherein the substrate strand comprises SEQ ID NO: 1, SEQ ID NO: 2, or a variant thereof; and wherein the inactive enzyme strand comprises SEQ ID NO: 5 or a variant thereof.

13. The kit of claim 1, wherein the leaching agent comprises an acid.

14. The kit of claim 1, wherein the precipitating agent forms a precipitate with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

15. The kit of claim 14, wherein the precipitating agent comprises OH", CO32’, SO42", or any combination thereof.Attorney Docket No. 10046-635W0116. The kit of claim 1 , wherein the chelating agent chelates with Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Fe3+, Al3+, or any combination thereof.

17. The kit of claim 16, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA).

18. The kit of claim 1, further comprising a filter and / or a fluorometer.

19. A method of detecting Li+in a sample, comprising: a) providing the kit of claim 1 ; b) treating the sample with the leaching agent, precipitating agent, and / or chelating agent; and c) exposing the sample to the DNAzyme sensor; and d) identifying the detectable signal, thereby detecting Li+in the sample.

20. The method of claim 19, wherein the sample is brine water, rock, or a portion of a lithium battery.

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