Highly multiplexed target species determination using thermal barcodes

Abstract

Methods and articles for selectively amplifying nucleic acid barcodes to determine target molecules are generally provided. Some methods relate to selective amplification of barcodes in solution, e.g., using proximity of nucleic acids as a mechanism for selectivity. Some methods relate to selective amplification of barcodes bound to substrates, e.g., using substrate-bound target molecules to selectively amplify barcodes. Of particular note, the use of thermal barcodes for highly multiplexed detection (e.g., in conjunction with droplet-based methods) is generally provided.

Description

[0001] HIGHLY MULTIPLEXED TARGET SPECIES DETERMINATION USING THERMAL BARCODES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 652,645, filed May 28, 2024, entitled “Highly Multiplexed Target Species Determination Using Thermal Barcodes,” by Weitz, et al., incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] Systems and methods for determination of target molecules, e.g., by selective amplification of nucleic acid barcodes, are generally provided.

[0006] BACKGROUND

[0007] Determination of target molecules is an important aspect of proteomics, genomics, and metabolomics. However, conventional methods for determining target molecules (e.g., sequencing methods) may be slow and may require considerable infrastructure. Simpler methods would be desirable.

[0008] SUMMARY

[0009] Methods and articles for selectively amplifying nucleic acid barcodes to determine target molecules are generally provided. Some methods relate to selective amplification of barcodes in solution, e.g., using proximity of nucleic acids as a mechanism for selectivity. Some methods relate to selective amplification of barcodes bound to substrates, e.g., using substrate-bound target molecules to selectively amplify barcodes. Of particular note, the use of thermal barcodes for highly multiplexed detection (e.g., in conjunction with droplet-based methods) is generally provided. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] In one aspect, a method of determining a target species is provided. According to some embodiments, the method comprises: selectively amplifying a first nucleic acid and a second nucleic acid to produce a third nucleic acid when both the first nucleic acid and the second nucleic acid are bound to the target species, wherein the first nucleic acid comprises a first barcode, wherein the second nucleic acid comprises a second barcode, wherein the third nucleic acid comprises: (a) the first barcode or a sequence complementary to the first barcode, and (b) the second barcode or a sequence complementary to the second barcode, wherein the first barcode is a thermal barcode sequence exhibits a first peak melting point and the second thermal barcode sequence exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1 °C.

[0011] In another aspect, an article is provided. In some embodiments, the article comprises: a plurality of compartments comprising a first compartment comprising a first target species, a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first target species is bound to the first nucleic acid and the second nucleic acid, wherein the first nucleic acid comprises a first barcode, wherein the second nucleic acid comprises a second barcode, and wherein the third nucleic acid comprises: (a) the first barcode or a sequence complementary to the first barcode, and (b) the second barcode or a sequence complementary to the second barcode, wherein the first barcode is a thermal barcode sequence exhibits a first peak melting point and the second thermal barcode sequence exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1 °C.

[0012] In still another aspect, a method of determining a target species is provided. According to some embodiments, the method comprises: selectively amplifying a first nucleic acid and a second nucleic acid to produce a third nucleic acid when both the first nucleic acid and the second nucleic acid are bound to the target species, wherein the first nucleic acid comprises a first barcode, wherein the second nucleic acid comprises a second barcode, wherein the third nucleic acid comprises: (a) the first barcode or a sequence complementary to the first barcode, and (b) the second barcode or a sequence complementary to the second barcode.

[0013] In yet another embodiment, a method of determining a target species is provided. According to some embodiments, the method comprises: binding the target species to a solid substrate and a nucleic acid in a solution and selectively amplifying the nucleic acid bound to the target species, wherein the nucleic acid comprises a first barcode, wherein the first barcode exhibits a peak melting point differing by at least 0.1 °C from a second peak melting point of a second barcode present in the solution.

[0014] In one embodiment, an article is provided. According to some embodiments, the article comprises: a plurality of compartments comprising a first compartment comprising a first target species bound to a first nucleic acid and a first solid substrate and a second compartment comprising a second target species, a second nucleic acid, and a second solid substrate, wherein the first nucleic acid comprises a first barcode, and wherein the first thermal barcode exhibits a first peak melting point differing by at least 0.1 °C from a second peak melting point of a second barcode present in the first compartment.

[0015] In another aspect, a method of determining a target species is provided. According to some embodiments, the method comprises: amplifying a first nucleic acid to produce a second nucleic acid, wherein during the amplification: the first nucleic acid is bound to a recognition species; the recognition species is bound to a target species; and the target species is bound to a substrate, wherein the first barcode is a thermal barcode sequence exhibits a first peak melting point and the second thermal barcode sequence exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1 °C.

[0016] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale unless otherwise indicated. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0019] FIGS. 1A-1E provide a non-limiting, schematic illustration of a method of determining a target species by selectively amplifying a nucleic acid, according to some embodiments;

[0020] FIG. 2A provides a non-limiting, schematic illustration of a target species complexed with a first nucleic acid and a second nucleic acid, according to some embodiments;

[0021] FIG. 2B provides a non-limiting, schematic illustration of a target species complexed with a first nucleic acid and a second nucleic acid, according to some embodiments;

[0022] FIG. 3A provides a non-limiting, schematic illustration of a target species and a second species complexed with a first nucleic acid and a second nucleic acid, according to some embodiments;

[0023] FIG. 3B provides a non-limiting, schematic illustration of a target species and a second species complexed with a first nucleic acid and a second nucleic acid, according to some embodiments;

[0024] FIGS. 4A-4D provide a non-limiting, schematic illustration of a method of determining a target species by selectively amplifying a nucleic acid, according to some embodiments;

[0025] FIG. 5A provides a non-limiting, schematic illustration of a target species complexed with a nucleic acid and a solid substrate, according to some embodiments;

[0026] FIG. 5B provides a non-limiting, schematic illustration of a target species complexed with a nucleic acid and a solid substrate, according to some embodiments;

[0027] FIG. 6A presents a non-limiting, schematic illustration of a melting profile and the behavior of melting barcoded nucleic acids, according to some embodiments;

[0028] FIG. 6B presents a non-limiting, schematic illustration of a melting profile, according to some embodiments;

[0029] FIGS. 7A-7B present non-limiting, schematic representations of binding of auxiliary barcodes to barcoded nucleic acids, according to some embodiments; FIG. 8 presents a non-limiting, schematic representation of a method of barcoding and determining target molecules, according to some embodiments;

[0030] FIGS. 9A-9D present exemplary micrographs of droplets comprising selectively amplified barcodes, according to some embodiments;

[0031] FIG 10 presents an exemplary comparison of target protein concentration and signal intensity, according to some embodiments;

[0032] FIG. 11 presents exemplary melting profiles of selectively amplified barcodes, according to some embodiments;

[0033] FIG. 12 presents exemplary target protein concentrations determined using selective amplification of barcodes, according to some embodiments;

[0034] FIG. 13A presents non-limiting measurements of target protein concentrations determined using selective amplification of barcodes, according to some embodiments; and

[0035] FIG. 13B presents non-limiting measurements of target protein concentrations analogous to the concentrations of FIG. 13 A, but instead determined using ELISA, according to some embodiments.

[0036] DETAILED DESCRIPTION

[0037] An important frontier in cell biology is the specific, multiplex labeling and identification of target species selected from the proteome, genome, or metabolome of an organism. The present disclosure is directed, according to some embodiments, towards methods and articles for labeling and identifying target species with a relatively high throughput, without reliance on limited throughput analytic techniques like protein sequencing, thereby providing a number of analytic advantages for proteomics, genomics, and / or metabolomics. In particular, the present application is directed, in some aspects, towards methods and articles for specifically binding nucleic acid barcodes to target species and selectively amplifying the barcodes specifically bound to the target species. The approaches provided herein may provide significant advantages for high-throughput omics, particularly when implemented using microfluidic articles and methods suitable for performing the methods iteratively and at large scale. In a non-limiting embodiment, provided solely for the sake of illustration, a sample is collected from a subject suspected of having a disease. The sample may be a raw fluid sample (e.g., a saliva sample) that includes a target species, and that may include one or more off-target species. The method may comprise forming a plurality of droplets (or other compartments) from the sample and introducing a plurality of nucleic acids to the droplets, wherein at least some of the nucleic acids are configured to bind to the target species. For example, at least one of the nucleic acids may be bound to a recognition species (e.g., a recognition antibody) configured to specifically bind to the target species. The nucleic acid may comprise a thermal barcode selected to be identifiable via its melting profile. According to some embodiments, nucleic acids specifically bound to the target species are selectively amplified, while unbound nucleic acids are not. Selective amplification of specifically bound nucleic acids may be accomplished by binding multiple nucleic acids to the same target species and using an overlap between the first nucleic acid and the second nucleic acid or ligation of the first nucleic acid to the second nucleic acid, followed by amplification, to form a third nucleic acid comprising nucleic acids complementary to the first nucleic acid and the second nucleic acid, according to some embodiments. Finally, the amplified nucleic acid may be determined in order to determine the target species.

[0038] Of course it should be recognized that a method described above is included solely for the purpose of illustration, and that the present disclosure is not in any way restricted to such a method. A more general description is provided below.

[0039] In one aspect, a method of determining a target species by selectively amplifying a nucleic acid is provided. FIGS. 1A-1E provide a non-limiting, schematic illustration of such a method, according to some embodiments. In FIG. 1A, a target species 103 is provided (e.g., as a component of a sample obtained from a subject). A first nucleic acid 111 comprising a first barcode (e.g., thermal barcode) 121 and a second nucleic acid 113 comprising a second barcode (e.g., thermal barcode) 123 are also provided. The first nucleic acid and / or the second nucleic acid may be configured to bind to the target species, according to some embodiments. One or more of the nucleic acids may be complexed with (e.g., bound to) a recognition species configured to recognize the target species. For example, in the non-limiting embodiment presented in FIG. 1A, first nucleic acid 111 is bound (e.g., covalently bound) to recognition species 131 that is configured to recognize site 141 of target species 103, while second nucleic acid 113 is bound (e.g., covalently bound) to recognition species 133 that is configured to recognize site 143 of target species 103. Any of a variety of recognition species may be used, as discussed in greater detail below. For example, in some embodiments, the recognition species are antibodies configured to recognize the target species. However, it should of course be understood that other recognition species are also possible.

[0040] In some embodiments, all provided nucleic acids are configured to bind to the same target species. However, according to some embodiments, at least some nucleic acids are provided that are not configured to bind to the target species. For example, FIG. 1A shows a non-limiting nucleic acid 115 that is configured to bind to a different target species (not present) using recognition species 135. Such embodiments may be advantageous for methods of determining multiple target species, or for determining binding of a target species, as discussed in greater detail elsewhere herein. For example, barcodes 121, 123, and 125 may be distinguishable from one another, e.g., based on their melting profiles, such that they may be used to uniquely determine their binding targets. Nucleic acids not configured to bind to the target species may also be provided. For example, the solution may include one or more primers suitable for amplifying a bound nucleic acid, depending on the embodiment.

[0041] In FIG. 1A, recognition species 131, 133, and 135 are bound to the 5’ termini of nucleic acids 111, 113, and 115 respectively. The free 3’ ends of nucleic acids 111, 113 and 115 are indicated in FIG. 1A by text in the figure and by the terminal arrow-tip of nucleic acids 111, 113 and 115. However, it should be understood that other embodiments, e.g., where the 5’ termini of the nucleic acids are free while the 3’ termini are bound to recognition species are also possible, or where mixtures of free 5’ termini and 3’ termini are used, are also possible.

[0042] According to some embodiments, a method comprises binding a first nucleic acid and a second nucleic acid to the target species. For example, FIG. IB shows nucleic acids 111 and 113 bound to target species 103 via recognition species 131 and 135. Binding the first nucleic acid and the second nucleic acid to the same molecule may, according to some embodiments, facilitate the selective amplification of bound nucleic acids, e.g., without amplifying other nucleic acids such as nucleic acid 115 shown in FIG. 1A. As illustrated in FIG. IB, the first nucleic acid (e.g., using the first recognition species) and the second nucleic acid (e.g., using the second recognition species) are, according to some embodiments, configured to bind to the same target species simultaneously (e.g., because they recognize different portions of the target species). It should, of course be understood that other embodiments, where only a single nucleic acid binds to a target species are also possible. To provide a specific example, in some embodiments, the first nucleic acid is bound to a target species and the second nucleic acid is bound to a second species in order to determine a binding interaction of the target species and the second species, as discussed in greater detail below.

[0043] FIGS. 1C-1E schematically illustrate one embodiment of selective amplification of nucleic acids 111 and 113. The first nucleic acid and the second nucleic acid may be bound to the target species to form a complex. For example in FIG. 1C, both nucleic acids 111 and 113 are bound to target species 103 to form complex 101. Complexed nucleic acids 111 and 113 may be amplified by polymerase chain reaction (PCR) using overlap between the nucleic acids. For example, nucleic acid 111 comprises overlap portion 161 that complements overlap portion 171 of nucleic acid 113 as shown in FIG. 1C. It should, of course, be recognized that other amplification schemes may be used, including other types of PCR, as the disclosure is not so limited.

[0044] FIG. ID, illustrates the result of extension of nucleic acids 111 and 113 as shown in FIG. 1C. As shown, both nucleic acids comprise two thermal barcode sequences. Nucleic acid 111 comprises original thermal barcode sequence 121 and thermal barcode sequence 193 complementary to original thermal barcode sequence 123, while nucleic acid 113 comprises original thermal barcode sequence 123 and thermal barcode sequence 191 complementary to original thermal barcode sequence 121. In some embodiments the method may comprise forming a third nucleic acid comprising both the first barcode and a sequence complementary thereto and the second barcode or a sequence complementary thereto. FIG. ID illustrates this process, according to some embodiments, schematically illustrating the use of primers 148 and 149 to further amplify nucleic acids 111 and 113 as part of an amplification reaction. Each of primers 148 and 149, when amplified against nucleic acids 111 and 113, will produce a nucleic acid comprising thermal barcode sequences 121 and 193 or comprising thermal barcode sequences 123 and 191.

[0045] FIG. IE shows the result of the non-limiting PCR reaction illustrated in FIG. 1C, the synthesis of a third nucleic acid 181 and a fourth nucleic acid 183. Third nucleic acid 181 comprises barcode 121 and further comprises sequence 193 complementary to barcode 123. Likewise, fourth nucleic acid 183 comprises barcode 123 and further comprises sequence 191 complementary to barcode 121.

[0046] Finally, the target species may be determined using the amplified thermal barcodes (e.g., barcodes 121 or 123), as discussed in greater detail below. In some embodiments, the target species may be determined based at least in part on the identity of the amplified barcodes. For example, in the context of FIGS. 1A-1E, barcodes 121 and 123 are thermal barcodes that are amplified such that they may be detected during a melting profile assay, while thermal barcode 125, keyed to a different target species not present in solution, was not amplified and would not be detected during a melting profile assay. Accordingly the detected barcodes may be used to determine the presence of target species 103. Of course, it should be understood that signaling entities other than thermal barcodes may also be used, as discussed in greater detail below. And it should further be understood that in some embodiments, related methods may be used to detect other phenomena (e.g., binding interactions of target species) rather than the mere presence or absence of a target species, as discussed in greater detail below. The method of FIGS. 1A-1E should generally be understood to be non-limiting, and may be modified or supplemented as discussed in greater detail below.

[0047] Methods such as that of FIGS. 1A-1E may be used to determine any of a variety of suitable target species, including nucleic acids, as discussed in greater detail below. However, it is noteworthy that the methods provided herein do not, according to some embodiments, include amplification of the target species — even if the target species is a nucleic acid (e.g., a single- stranded DNA or an RNA). For example, although target species 103 shown in FIG. 1A-1E may be a nucleic acid, according to some embodiments, target species 103 is not amplified because it is not connected to nucleic acid 111 or nucleic acid 113 as part of a single nucleic acid chain, and because it is not configured to be amplified by a provided primer. Non-amplification of a target nucleic acid species may be advantageous for detection of the target nucleic acid species, e.g., when detection is performed by measuring melting profiles of thermal barcodes 121 and 123 as discussed in greater detail below. For example, without wishing to be bound by any particular theory, non-amplification of a target nucleic acid species during amplification of the thermal barcodes may reduce or eliminate the contribution of the target nucleic acid species to the overall melting profile, improving contrast associated with the melting profiles of the amplified thermal barcodes. Another advantage is that, when the target species is not a nucleic acid (e.g., when it is a protein or a metabolite), detection of amplified thermal barcodes is possible even without concentrating or amplifying the target species in solution.

[0048] It should, of course, be understood that while FIGS. 1A-1E schematically illustrate one method of forming a third nucleic acid, other methods may also be suitable. For example, according to some embodiments, the first nucleic acid and the second nucleic acid may be ligated to form a single nucleic acid that may subsequently be amplified, as the disclosure is not so limited.

[0049] FIGS. 2A-2B illustrate specific and non-limiting embodiments of nucleic acids bound to a target species using species recognition groups. Differences between FIGS. 2A-2B illustrate different ways in which recognition species may be used to facilitate detection of target species as part of the methods provided herein.

[0050] In FIG. 2A, first nucleic acid 211 is covalently bound to recognition species 231 and recognition species 231 is configured to bind to target species 203 to form a complex 200. Likewise, second nucleic acid 213 is covalently bound to recognition species 233 and recognition species 233 is configured to bind to target species 203. In the embodiment of FIG. 2A, target species 203 is a protein and recognition species 231 and 231 are both antibodies that specifically bind to distinct portions of target species 203.

[0051] Unlike the embodiment of FIGS. 1A-1E, nucleic acids 211 and 213 are not complementary. Rather, nucleic acids 211 and 213 are complementary and bound to ligation splint 249. Nucleic acids 211 and 213 may thus be ligated as discussed in greater detail below, thereby joining nucleic acids 211 and 213. Joined nucleic acids 211 and 213 may subsequently be amplified using complementary primers (not shown) in order to produce a third nucleic acid comprising the thermal barcode sequences (or sequences complementing the thermal barcode sequences) of nucleic acids 211 and 213. Ligation may be performed using any of a variety of suitable ligation reagents. In particular, in some embodiments, ligation is performed using a T4 enzyme.

[0052] In contrast, FIG. 2B illustrates nucleic acids 261 and 263 which are not covalently bound to recognition species 281 and 283. Like nucleic acids 211 and 213 of FIG. 2A, nucleic acids 261 and 263 are bound to ligation splint 299 and are thus configured to be ligated to form a single nucleic acid. And like recognition species 231 and 233 of FIG. 2A, recognition species 281 and 283 are antibodies configured to specifically bind to distinct portions of target species 203. However, in the example of FIG. 2B, nucleic acid 261 is covalently bound to intermediate recognition (e.g., intermediate recognition) species 291 and nucleic acid 263 is covalently bound to intermediate recognition species 293. In turn, intermediate recognition species 291 is configured to specifically bind to recognition species 281 and intermediate recognition species 293 is configured to specifically bind to recognition species 283. In some embodiments, intermediate recognition species 291 and 293 are specifically configured to recognize and bind to recognition species 281 and 283, respectively; however, according to some embodiments, one or both of intermediate recognition species 291 and 293 is capable of specifically binding to both recognition species 281 and recognition species 283 (e.g., such that the relative position of nucleic acids 261 and 263 may be reversed), as amplification of nucleic acids 261 and 263 is still possible in such an embodiment. For example, intermediate recognition species 291 and / or intermediate recognition species 293 could simply be configured to recognize any antibody, such that if both recognition species 281 and recognition species 283 are target species recognizing antibodies, one or both of the intermediate recognition species recognize both antibody 281 and antibody 283. Accordingly, in some embodiments, intermediate recognition species 291 is the same as intermediate recognition species 293 while, in some embodiments, intermediate recognition species 291 is different from intermediate recognition species 293.

[0053] Noncovalent binding of nucleic acids 261 and 263 to recognition species 281 and 283 as shown in FIG. 2B may be disadvantageous, in some embodiments (e.g., may reduce the total proportion of target species-nucleic acid complexes that result in barcode amplification). However, noncovalent binding of nucleic acids 261 and 263 to recognition species 281 and 283 may be advantageous, in some embodiments, e.g., because nucleic acids 261 and 263 may be used in a wider range of assays without synthetic modification. For example, to modify the method schematized in FIG. 2A for use with a different target species, the nucleic acids would have to be covalently bonded to entirely new recognition species. However, to modify the method schematized in FIG. 2B for use with a different target species, the recognition antibodies may simply be replaced with other recognition antibodies, since the other recognition antibodies may be selected to be recognized by intermediate recognition species 291 and 293. Accordingly, the embodiment of FIG. 2B is, in some embodiments, more modular and easier to use than the assay of FIG. 2A. Since both methods present relative advantages and disadvantages, the person of ordinary skill would recognize that either method (or a combination of the methods) may be used, depending on the embodiment.

[0054] FIGS 3A-3B illustrate complexes 300 and 301 suitable for determining binding of a target species 303a to a second species 303b. FIG. 3A is similar to FIG. 2A, depicting first nucleic acid 311 covalently bound to first recognition species 331 and second nucleic acid 313 covalently bound to second recognition species 333. However, unlike in FIG. 2A, first recognition species 331 is bound to target species 303a, while second recognition species 333 is bound to second species 303b, which is not the same as target species 303a. Consequently, in complex 300, amplification of nucleic acids 311 and 313 occurs only when target species 303a is bound to second species 303b, and amplification does not occur if binding does not occur. Ligation splint 349 illustrates how nucleic acids 311 and 313 may be joined for subsequent amplification, according to some embodiments. Of note, as shown in FIG. 3A, both nucleic acid 311 and 313 are bound to target species 303a, but second nucleic acid 313 is linked by two intermediary species (second recognition species 333 and second species 303b) whereas first nucleic acid 311 is linked by a single intermediary species (first recognition species 331) prior to amplification. The term “second species” is used generically and generally a second species may be a second target species if it is also chosen to be determined as part of a method. However, in some embodiments the second species is not a target species, as the disclosure is not so limited. Likewise, FIG. 3B is similar to FIG. 2B, except that complex 301 is suitable for determining binding of target species 303a rather than to simply determine the concentration of target species 303a. In complex 301, first nucleic acid 361 is bound to target species 303a via covalent attachment to intermediate recognition species 391, which in turn binds recognition species 381, which is bound to target species 303a. Similarly, second nucleic acid 363 is bound to second species 303b via covalent attachment to intermediate recognition species 393, which in turn binds recognition species 383, which is bound to second species 303b. As in FIG. 3A, second species 303b binds second nucleic acid 363 to target species 303a to form complex 301, where amplification may occur after ligation using ligation splint 399. Complex 301, like complex 300, may be used to determine target species 303a by determining binding of target species 303a to target species 303b.

[0055] In another aspect, a method of selective nucleic acid amplification to determine a target species comprises use of a solid substrate. According to some embodiments, the solid substrate may be used to selectively bind the target species as well as a nucleic acid configured to selectively bind to the target species. The method may then comprise washing the solid substrate to remove unbound nucleic acids, such that only nucleic acids that specifically recognize the target species may be amplified to determine the target species, according to some embodiments.

[0056] FIGS. 4A-4D present a non-limiting method of determining a target species 403, according to some embodiments. In FIG. 4A, target species 403 is present in a solution with nucleic acid 413, which is configured to bind to target species 403 via optional recognition species 433. As shown, nucleic acid 413 comprises barcode 423. Other nucleic acids, not configured to bind to target species 403 may also be present. For example, nucleic acid 415 comprising barcode 425 is covalently bound to recognition species 435, which is not capable of specific binding to target species 403, but which may be capable of binding another target species (not shown). Barcodes 423 and 425 may be distinguishable from one another, e.g., based on their melting profiles, such that they may be used to uniquely determine their binding targets.

[0057] The solution further comprises solid substrate 450. Solid substrate 450 is presented as a bead in the non-limiting method of FIG. 4A, but any solid substrate may generally be used. For example, in some embodiments the solid substrate is a solid substrate of a well, as the disclosure is not so limited. Solid substrate 450 is bound to recognition species 431, which is configured to bind target species 403, according to some embodiments. It should, of course, be understood that in some embodiments the target species may be immobilized directly on the substrate (e.g., via covalent bonding) without use of a recognition species. It should further be understood that a plurality of target species may be attached to the same substrate (e.g., via a plurality of recognition species bound to the substrate), depending on the embodiment.

[0058] FIG. 4B illustrates specific binding of target species 403 to substrate 450 and nucleic acid 413 via recognition groups 431 and 433 respectively. It should, of course, be understood that while only a single target species is shown, a plurality of target species may be bound to the same solid substrate (e.g., via a plurality of target species recognition groups 431 located thereon). After binding of target species 403 and nucleic acid 413 to substrate 450, substrate 450 may be washed to remove unbound material (e.g., unbound nucleic acids, off-target species).

[0059] After washing, a primer or plurality of primers (e.g., a plurality of primers) may be provided to amplify remaining nucleic acids. For example, referring to FIG. 4B, primers 461, 463, 465, and 467 have been provided after washing to remove nucleic acid 415 (shown in FIG. 4A but not shown in FIG. 4B). According to some embodiments the primer(s) are barcoded. For example, primers 461, 463, 465 and 467 are barcoded with thermal barcodes 471, 473, 475, and 477. If a plurality of primers is used, at least some primers of the plurality may be configured to amplify a nucleic acid bound to a target. For example, referring to FIG. 4B, primer 463 is configured to bind to nucleic acid 413 to prime amplification of nucleic acid 413, while primers 461, 465, and 467 are not configured to bind to nucleic acid 413. The use of a plurality of primers may thus be used to screen for a plurality of substrate-bound molecules, depending on the embodiment.

[0060] Finally, the method may comprise selectively amplifying the one or more nucleic acids bound to the target species. Amplification of the nucleic acid may amplify the concentration of the barcode of the nucleic acid, resulting in enhanced detection of barcodes associated with the target species. In this way, the barcodes may be used to determine the target species. FIG. 4C illustrates an exemplary first amplification step, where primer 463 has been extended to form nucleic acid 483 complementary to nucleic acid 413, and where nucleic acid 413 has itself been extended by the sequence 493 complementary to barcode 473. As FIG. 4D illustrates, subsequent amplification steps may be used to further amplify barcodes 423 and 473 and their complementary sequences, raising their concentration relative to the concentration of other nucleic acids (e.g., other barcoded primers such as primers 461, 465 and 467 showing in FIG. 4B).

[0061] Like the methods discussed above, substrate-based methods provided herein do not, according to some embodiments, include amplification of the target species — even if the target species is a nucleic acid (e.g., a single- stranded DNA or an RNA). For example, in FIGS. 4A-4D, target species 403 is not amplified. Non-amplification of a target nucleic acid species in methods using solid substrates has the same advantages discussed above.

[0062] FIGS. 5A-5B show additional, non-limiting embodiments suitable for methods of amplifying substrate-bound nucleic acids to identify a substrate-bound target species.

[0063] In FIG. 5A, nucleic acid 511 is covalently bound to recognition species 531 (in the form of an antibody). The method is performed in well 500 comprising solid substrate 502 (in the form of the interior surface of well 500). A plurality of species including target species 503 and off-target species 505 are covalently bound to substrate 502 so that, as shown, recognition species 531 can bind to target species 503.

[0064] As in the solution-based methods described above, according to some embodiments a nucleic acid binds to the target species indirectly. For example, FIG. 5B schematically illustrates an embodiment where nucleic acid 561 is covalently bound to intermediate recognition species 591, which in turn is bound to recognition species 581 that binds target species 503. As in the embodiment of FIG. 5 A, off-target species 505 are also bound to substrate 502 of well 500, but the off target species are not recognized by a recognition species such as recognition species 581 and therefore do not result in nucleic acid amplification. As in the solution-based methods described above, the use of indirect, intermediate recognition species has both advantages and disadvantages for substrate-based methods of determining a target species, and the person of ordinary skill would recognize that either design could be used, depending on the embodiment. While FIGS. 5A-5B do not present an example of determining binding of a target species (as described above with reference to FIGS. 3A-3B). However, it should be understood that determining binding of a target species may be accomplished just as easily using a substrate-based method like the methods of FIGS. 4A-4D and 5A-5B. For example, the target species or its prospective binding partner may be immobilized to a substrate such that only binding between the target species and its binding partner binds a barcoded nucleic acid to the substrate. The substrate may then be washed to remove unbound nucleic acids, and any remaining nucleic acids may subsequently be amplified.

[0065] The methods, articles, and fluidic devices provided herein may generally be used to determine any of a variety of target species. According to some embodiments, for example, the target species is a protein, a nucleic acid, or a metabolite. Accordingly, in some embodiments, the methods, articles and fluidic devices may used to at least partially determine the proteome, genome, or metabolism of an organism.

[0066] Determining the target species may comprise, depending on the embodiment, determining the presence or absence of the target species within a sample. The presence or absence of the target species may have clinical significance. For example, in some embodiments, the presence of the target species is indicative of a disease or condition. As another example, in some embodiments, the absence of the target species is indicative of a disease or condition.

[0067] In some embodiments, determining the target species comprises determining a subject from which the target species originated. For example, in some embodiments, a method comprises analyzing a plurality of samples and determining a target species within each sample. Accordingly, in some embodiments one or more barcodes amplified during performance of a method provided herein comprises amplifying a barcode identifying a sample from which the target species originated.

[0068] According to some embodiments, determining a target species comprises determining its binding to a second species. Such embodiments may be advantageous, for example, during screening assays designed to identify molecules capable of binding to a target species (e.g., a viral antigen). Such assays may also be useful for the quantitative or qualitative comparison of binding interactions between a target species and a plurality of second species (e.g., as part of a binding competition assay). Other embodiments are also possible, as the disclosure is not so limited, and as inter-species binding assays are widely important for applications such as medicine and drug design.

[0069] Although FIGS 1A-5B only specifically illustrate a single target species, according to some embodiments the methods provided herein may be used to determine a plurality of target species. For example, a first nucleic acid or plurality of nucleic acids may be used to determine a first target species and a second nucleic acid or second plurality of nucleic acids may be used to determine a second target species, according to some embodiments. The first nucleic acid and / or first plurality of nucleic acids may be barcoded such that they are distinguishable from the second nucleic acid or plurality of nucleic acids, depending on the embodiment, even if both target species are detected within the same solution. This type of multiplex detection of target species may be extended to facilitate determination (e.g., simultaneous determination) of any of a variety of suitable numbers of target species present in the same solution, compartment, plurality of compartments, article, and / or fluidic system.

[0070] The methods provided herein above may be used to identify any of a variety of appropriate numbers of target molecules. In some embodiments, a method may be used to identify greater than or equal to 1 target molecule, greater than or equal to 10 target molecules, greater than or equal to 20 target molecules, greater than or equal to 50 target molecules, greater than or equal to 100 target molecules, greater than or equal to 200 target molecules, greater than or equal to 300 target molecules, greater than or equal to 400 target molecules, greater than or equal to 500 target molecules, greater than or equal to 600 target molecules, greater than or equal to 700 target molecules, greater than or equal to 800 target molecules, or greater than or equal to 900 target molecules. In some embodiments, a method may be used to identify less than or equal to 1000 target molecules, less than or equal to 900 target molecules, less than or equal to 800 target molecules, less than or equal to 700 target molecules, less than or equal to 600 target molecules, less than or equal to 500 target molecules, less than or equal to 400 target molecules, less than or equal to 300 target molecules, less than or equal to 200 target molecules, less than or equal to 100, less than or equal to 50 target molecules, less than or equal to 20 target molecules, or less than or equal to 10 target molecules. Combinations of these ranges are also possible (e.g., greater than or equal to 1 target molecule and less than or equal to 1000 target molecules, or greater than or equal to 50 target molecules and less than or equal to 100 target molecules). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0071] It should be understood that the above-mentioned ranges may be applicable to both substrate-based and non-substrate-based methods of selective nucleic acid amplification to determine the target substrate(s).

[0072] The target species may be determined by any of a variety of suitable methods. According to some embodiments, the target species may be determined at least in part by determining a barcode associated with the target species. Any of a variety of barcodes may be used, including but not limited to thermal barcodes and barcodes configured to interact with signaling entities (e.g., optical signaling entities), as discussed in greater detail below. The use of thermal barcodes may be particularly advantageous, depending on the embodiment, since thermal barcodes may significantly increase the number of available contrast channels available for distinguishing discrete target species, facilitating highly-multiplexed assays.

[0073] Thermal barcodes may be used to determine target species based on their melting profile. A nucleic acid’s melting profile generally refers to a distribution of an average rate of melting of the nucleic acid as a function of temperature. The melting profile of a nucleic acid or a plurality of nucleic acids may be obtained by using a binding detection agent (e.g., SYBR Green Dye or Eva Green Dye, discussed in greater detail below) as a proxy for a proportion of the nucleic acid present in a solution that is single-stranded or double-stranded. Without wishing to be bound by any particular theory, the rate of melting of the nucleic acid (e.g., the rate of de-binding of the double- stranded nucleic acid to form the single stranded nucleic acid) may be related to the rate of change in the amount of double stranded DNA and the amount of single stranded DNA present in solution, and thus may be directly related to the intensity of the signal produced by the binding detection agent.

[0074] To measure the melting profile of a nucleic acid, according to some embodiments, the nucleic acid may be heated while measuring a signal from the binding detection agent to produce an integrated melting profile. The integrated melting profile may then be differentiated (e.g., using a numerical differentiation algorithm, such as a finite difference algorithm) to determine the melting profile of the nucleic acid. The melting profile may then be rescaled (e.g., normalized to a highest measured value) to eliminate the effects of concentration and / or type of binding detection agent used in the measurement.

[0075] Generally, a nucleic acid in the presence of a complementary strand can melt from a double-stranded state to a single- stranded state over a range of temperatures during quasistatic heating. Quasistatic heating is given its ordinary meaning in the art, and may refer, in some instances, to rates of temperature change less than or equal to 10 °C / s, less than or equal to 5 °C / s, less than or equal to 1 °C / s, less than or equal to 0.5 °C / s, less than or equal to 0.2 °C / s, or less than or equal to 0.1 °C / s. Various aspects of the melting profile, such as the extent to which the nucleic acid is dissociated, the temperature distribution of melting rate, and a peak melting temperature (a temperature at which the rate of melting is greatest), may be estimated by methods known to those of ordinary skill in the art, some of which are discussed in the examples below.

[0076] A thermal barcode is a nucleic acid or a portion of a nucleic acid that can be used to determine a target species based on a melting profile of the thermal barcode. Ordinarily, naturally occurring nucleic acids melt continuously over a single temperature range, since disjointed melting of individual portions of the nucleic acid over separate temperature ranges is improbable in naturally occurring sequences. However, a thermal barcode of a barcoded nucleic acid may be selected such that the thermal barcode melts over a temperature range that is at least partially separated from the temperature range at which another portion of the barcoded nucleic acid melts. For example, a first thermal barcode may be selected to melt over a separate temperature range from a second thermal barcode, from a separate temperature range from an intermediate portion of a nucleic acid, and / or over a separate temperature range from other species (e.g., other nucleic acids, proteins, etc.) which may be present in solution.

[0077] As described in greater detail below, the melting profile of a nucleic acid depends in large part on the sequence of the nucleic acid because, without wishing to be bound by any particular theory, nucleic acid melting is primarily related to local binding interactions between the nucleic acid and its complement. Thus, a thermal barcode may be designed to have an atypically high melting point by the disproportionate inclusion of high-binding-energy nucleotides, or to have an atypically low melting point by the disproportionate inclusion of low-binding-energy nucleotides within the sequence of the thermal barcode, relative to another portion of the nucleic acid.

[0078] FIGS. 6 A and 6B present non-limiting, schematic illustrations of an exemplary melting profile of a nucleic acid comprising a thermal barcode and an intermediate sequence during quasi-static heating, according to some embodiments. FIG. 6A presents a non-limiting melting profile showing signal intensity (corresponding to the rate of melting, along the y-axis) versus temperature (T, along the x-axis). The signal intensity of FIG. 6A is proportional to a derivative of a signal measured during a melting profile measurement, according to some embodiments. Schematic illustrations of amplified nucleic acids 610 and 620 comprising thermal barcodes 623 (curved, white rectangles) and intermediate sequences 621 (curved, black rectangles) are shown for points 601, 603, 605, 607, and 609 along the melting profile, schematically representing the condition of nucleic acids 610 and 620 at points 601, 603, 605, 607, and 609. It should, of course, be understood that the nucleic acids discussed herein need not comprise an intermediate sequence, and that the intermediate sequence 621 merely serves to illustrate how a melting temperature barcode may have a different and identifiable melting profile than a separate nucleic acid portion or than a different nucleic acid present in solution.

[0079] At point 601, nucleic acids 610 and 620 are completely double-stranded and are not melting — they remain double-stranded and in quasi-steady-state (with a signal intensity of near- zero, corresponding to a melting rate of approximately zero). The thermal separation of barcode melting from intermediate sequence melting explains why the melting profile is two-peaked, rather than one-peaked. At point 605, thermal barcodes 623 have totally melted, but intermediate sequences 621 remain doublestranded, resulting in a near- zero rate of melting, and a near- zero signal intensity. Melting of intermediate sequences 621 remains near- zero at point 601, since thermal barcodes 623 were designed to melt at a lower temperature than most naturally-occurring nucleic acids, according to some embodiments. Of course, in some embodiments a melting peak of a thermal barcode at least partially overlaps with a melting peak of another nucleic acid, so it should be understood that a point such as 605, where the signal intensity is near zero between two melting peaks, is not a necessary feature for all thermal barcodes. As the solution is heated from point 605 to point 607, the intermediate sequence begins to melt, ultimately reaching point 607, corresponding to the maximum rate of melting of the intermediate sequence. Finally, at point 609, nucleic acids 610 and 620 are fully melted, so that no further melting can occur with additional heating, and the signal intensity is near zero.

[0080] It should, of course, be understood that while FIG. 6A depicts the melting profile of a single set of nucleic acids, the melting profile of a plurality of nucleic acids present in the same solution may be determined by a similar method, as the disclosure is not so limited. For example, point 603 and point 607 could correspond to the peak melting temperature of distinct thermal barcodes, according to some embodiments. It should further be recognized that the relative intensity of a nucleic acid’s melting profile may depend on the concentration of that nucleic acid in solution, such that amplifying the nucleic acid may make the nucleic acid’ s melting profile easier to detect.

[0081] The temperature-dependent melting profile of a thermal barcode sequence may differ from a temperature-dependent melting profile of another nucleic acid (e.g., the melting profiles of the thermal barcode and the other acid may differ by design). For example, in some embodiments, a thermal barcode sequence melts within a first temperature range and another nucleic acid (e.g., a second thermal barcode sequence, a target species, a primer, another portion of the nucleic acid comprising the thermal barcode, or another nucleic acid present in solution) melts within a second temperature range different from the first temperature range. For example, referring to FIG. 6B (showing the same melting profile presented in FIG. 6A), the thermal barcode melts within first temperature range 651 and the intermediate sequence melts within second temperature range 653. Range 655 represents a range associated with the entire melting profile, beginning at a temperature where the nucleic acids are substantially doublestranded and ending at a temperature where they have substantially melted.

[0082] A first temperature range, wherein a thermal barcode melts, and a second temperature range, wherein another nucleic acid (e.g., a second thermal barcode sequence, a target species, a primer, another portion of the nucleic acid comprising the thermal barcode, or another nucleic acid present in solution) melts, may at least partially overlap. Alternatively, the first temperature range, and the second temperature range may be separated such that the thermal barcode and the other nucleic acid would not melt simultaneously at any temperature during quasistatic heating. For example, in some embodiments the first temperature range is totally below the second temperature range, such that the thermal barcodes totally melt prior to any melting of the other nucleic acid during quasistatic heating. Referring again to FIG. 6B, first temperature range 651 does not overlap with second temperature range 653, meaning that the proportion of the thermal barcode that has melted and the proportion of the intermediate sequence (the other nucleic acid) that has melted does not substantially depend on temperature for at least one temperature between the first temperature range and the second temperature range (e.g., at point 605 between first temperature range 651 and second temperature range 653, the thermal barcode has already substantially melted but the intermediate sequence does not become more melted more in response to a marginal increase in temperature). In some embodiments the first temperature range is totally above the second temperature range, such that the other nucleic totally melts prior to any melting of the thermal barcode during quasistatic heating. The first temperature range and the second temperature range may be separated by a temperature gap. For example, referring again to FIG. 6B, first temperature range 651 and second temperature range 653 are separated by temperature gap 650.

[0083] In some embodiments, the first temperature range and the second temperature range are separated by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 2 °C, greater than or equal to 3 °C, greater than or equal to 5 °C, greater than or equal to 8 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 25 °C, or greater. In some embodiments, the first temperature range and the second temperature range are separated by less than or equal to 50 °C, less than or equal to 25 °C, less than or equal to 15 °C, less than or equal to 10 °C, less than or equal to 8 °C, less than or equal to 5 °C, less than or equal to 3 °C, or less. Combinations of these ranges are possible. For example, in some embodiments, the first temperature range and the second temperature range are separated by greater than or equal to 0.1 °C and less than or equal to 50 °C. Other ranges are also possible. For example, in some embodiments, the first temperature range and the second temperature range overlap, such that there is no gap between them.

[0084] The melting profile of a nucleic acid may be characterized in any of a variety of suitable ways. For example, a nucleic acid may have one or more peak melting temperatures. For example, referring again to FIGS. 6A-6B, points 603 and 607 represent a peak melting temperatures of thermal barcodes 623 and intermediate sequences 621, respectively. Generally, a peak melting temperature is a temperature at which a rate of melting is maximized as a function of temperature during quasistatic heating. Commonly, naturally occurring nucleic acids have one melting temperature, sometimes referred to as their melting point. However, since different portions of nucleic acids can melt over different temperature ranges, in some embodiments, a nucleic acid comprises multiple peak melting temperatures corresponding to the melting points of various portions of the nucleic acid. For example, a nucleic acid comprising thermal barcode connected to another nucleic acid may have a first peak melting temperature corresponding to melting of the thermal barcode and a second peak melting temperature corresponding to melting of the other nucleic acid.

[0085] A first peak melting temperature associated with a first thermal barcode and a second peak melting temperature associated with another nucleic acid (e.g., a second thermal barcode sequence, a target species, a primer, another portion of the nucleic acid comprising the thermal barcode, or another nucleic acid present in solution) may be separated by any of a variety of appropriate temperature gaps (e.g., the temperature gap between points 603 and 607 in FIG. 6A). The first thermal barcode may or may not be connected with the other nucleic acid, depending on the embodiment. In some embodiments, the first peak melting temperature and the second peak melting temperature are separated by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 30 °C, or greater. In some embodiments, the first peak melting temperature and the second peak melting temperature are separated by less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 30 °C, less than or equal to 20 °C, less than or equal to 10 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1 °C, or less. Combinations of these ranges are possible. For example, in some embodiments, the first peak melting temperature and the second peak melting temperature are separated by greater than or equal to 0.1 °C and less than or equal to 60 °C. Other ranges are also possible.

[0086] As another example, a nucleic acid may have one or more median melting temperatures (e.g., associated with different barcodes). For example, referring again to FIGS. 6A-6B, points 603 and 607 represent median melting temperatures of thermal barcodes 623 and intermediate sequences 621, respectively, since the melting profiles are symmetric (with the consequence that the median melting temperature corresponds to the peak melting temperature).

[0087] A first median melting temperature associated with a first thermal barcode and a second median melting temperature associated with another nucleic acid (e.g., a second thermal barcode sequence, a target species, a primer, another portion of the nucleic acid comprising the thermal barcode, or another nucleic acid present in solution) may be separated by any of a variety of appropriate temperature gaps (e.g., the temperature gap between points 603 and 607 in FIG. 6A). The first thermal barcode may or may not be connected with the other nucleic acid, depending on the embodiment. In some embodiments, the first median melting temperature and the second median melting temperature are separated by greater than or equal to 0.1 °C, greater than or equal to 0.5 °C, greater than or equal to 1 °C, greater than or equal to 2 °C, greater than or equal to 5 °C, greater than or equal to 10 °C, greater than or equal to 15 °C, greater than or equal to 30 °C, or greater. In some embodiments, the first median melting temperature and the second median melting temperature are separated by less than or equal to 60 °C, less than or equal to 50 °C, less than or equal to 40 °C, less than or equal to 30 °C, less than or equal to 20 °C, less than or equal to 10 °C, less than or equal to 5 °C, less than or equal to 2 °C, less than or equal to 1 °C, or less. Combinations of these ranges are possible. For example, in some embodiments, the first median melting temperature and the second median melting temperature are separated by greater than or equal to 0.1 °C and less than or equal to 60 °C. Other ranges are also possible.

[0088] The melting point of a nucleic acid sequence has long been understood to be related to the composition and sequence of the nucleic acid itself. For example, without wishing to be bound by any particular theory, the nearest neighbor model may be used to predict the melting point of a particular nucleic acid sequence and its perfect complementary strand based on thermodynamic parameters associated with adjacent pairs of nucleotides within the sequence. For example, Table 1 summarizes the relevant energetic parameters that may be used in calculating the melting point of a DNA sequence.

[0089] Table 1: Nearest-neighbor model energetic parameters for DNA.

[0090] The nearest-neighbor melting temperature of a sequence can generally be determined by totaling the enthalpy of each pair of nearest neighbors to determine AH°totai, totaling the entropy of each pair of nearest neighbors to determine AS°totai, and determining the melting temperature, Tmin temperature (Celsius), by equation (1): 273.15 + 16.6 log[Na+] (1) where C is the total molar DNA concentration, R is the universal gas constant of 0.00199 kcal / (K-mol), A is an initiation constant of -0.0108 kcal / (K-mol), and [Na+] is the molar concentration of sodium ions. For example, the nearest- neighbor melting point of the DNA sequence 5’-CGTTGA-3’ (which binds to complementary strand 3’-GCAACT-5’) can be calculated by finding the total enthalpy (AH°totai= AH°CG / GC+ AH°GT / CA+ AH°TT / AA+ AH°TG / AC+ AH°GA / CT = (-11.9) + (-6.5) + (-9.1) + (-5.8) + (-5.6) = -38.9 kcal / mol) and the total entropy (AS°total= AS°CG / GC+ AS°GT / CA+ AS°TT / AA+ AS°TG / AC + AS°GA / CT= (-0.0278) + (-0.0173) + (-0.0240) + (-0.0129) + (-0.0135) = - 0.0955 kcal / mol-K). Assuming a sodium ion concentration of 5 M and a 500 nanomolar DNA concentration, the nearest neighbor melting temperature may be determined using equation 1 to be:

[0091] Tm

[0092] -0.0108 + (-0.0955) + 0.00199 In

[0093] The melting point, calculated by this method, is Tm= 20.5 °C.

[0094] A nucleic acid or a portion of a nucleic acid may be characterized in terms of parameters of a nearest-neighbor model as described above, in some embodiments. For example, in some embodiments, it may be advantageous to characterize a nucleic acid or nucleic acid portion directly in terms of a ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy (AH°totai / AS°totai). Without wishing to be bound by any particular theory, assuming constant salt conditions, this quantity may approximately scale with melting point of the nucleic acid or nucleic acid portion.

[0095] A first ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy of a first thermal barcode and a second ratio of total nearest-neighbor binding enthalpy to total nearest- neighbor binding entropy of another nucleic acid (e.g., a second thermal barcode sequence, a target species, a primer, another portion of the nucleic acid comprising the thermal barcode, or another nucleic acid present in solution) may differ by any of a variety of appropriate amounts, such that the thermal barcode has a different melting profile than the other nucleic acid. For example, in some embodiments, the first ratio and the second ratio differ by greater than or equal to 1%, greater than or equal to 2%, greater than or equal to 3%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 12%, or greater, versus the value of the second ratio. In some embodiments, in some embodiments, the first ratio and the second ratio differ by less than or equal to 15%, less than or equal to 12%, less than or equal to 10%, less than or equal to 5%, less than or equal to 3%, less than or equal to 2%, or less, versus the value of the second ratio. Combinations of these ranges are possible. For example, in some embodiments, in some embodiments, the first ratio and the second ratio differ by greater than or equal to 1% and less than or equal to 15%, versus the value of the second ratio. Other ranges are also possible.

[0096] Of course, thermal barcodes need not be perfectly complementary to their binding partner to have predictable melting points, and in general, the melting points and melting profiles of thermal barcodes (or of nucleic acids in general) may be predicted from their sequence using software known to those of ordinary skill in the art. For example, melting profiles may be reliably predicted using software such as uMeltSM. Thus, a thermal barcode may be designed such that the melting profile of the thermal barcode differs from the melting profile of another nucleic acid (e.g., a second thermal barcode sequence, a target species, a primer, another portion of the nucleic acid comprising the thermal barcode, or another nucleic acid present in solution) as described above.

[0097] A thermal barcode may have a nucleotide sequence that is atypical of naturally occurring nucleic acids, e.g., because such a sequence could help to distinguish a melting profile of a thermal barcode from a melting profile of other barcodes. For example, the thermal barcode may include a high proportion of A, T, or U, since these nucleobases typically have a weaker bonding energy than G or C.

[0098] Without wishing to be bound by any particular theory, the lower bonding energy of A, T, and U make them easier to melt, and their inclusion in an atypically high proportion in the thermal barcode can help separate the melting profile of the thermal barcode to temperatures below the melting profile of other barcodes. For example, in some embodiments, the thermal barcode comprises A, T, or U (e.g., A or T) in an amount greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 98%, greater than or equal to 99%, or more of the total number of nucleotides in the thermal barcode. For example, it may be advantageous for the barcode to include greater than or equal to 70% A, T, or U, in some embodiments. Of course, A, T, or U may be included in smaller proportions, as the disclosure is not so limited. Conversely, in some embodiments, a thermal barcode comprises G or C in an amount greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 98%, greater than or equal to 99%, or more of the total number of nucleotides in the thermal barcode. For example, it may be advantageous for the thermal barcode to include greater than or equal to 65% G or C, in some embodiments. Without wishing to be operated particular theory, the higher bonding energy of G and C make them harder to melt, and their inclusion in an atypically high proportion in the thermal barcode can help separate the melting profile of the thermal barcode to temperatures above the melting profile of other barcodes. Of course, G or C may be included in smaller proportions, as the disclosure is not so limited.

[0099] A thermal barcode may have any of a variety of appropriate lengths. According to some embodiments, a thermal barcode includes greater than or equal to 5, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20 nucleotides, greater than or equal to 25 nucleotides, greater than or equal to 30 nucleotides, greater than or equal to 35 nucleotides, greater than or equal to 40 nucleotides, greater than or equal to 45 nucleotides, greater than or equal to 50 nucleotides, greater than or equal to 60 nucleotides, greater than or equal to 70 nucleotides, greater than or equal to 80 nucleotides, greater than or equal to 90 nucleotides, greater than or equal to 100 nucleotides, greater than or equal to 125 nucleotides, greater than or equal to 150 nucleotides, or more. According to some embodiments, a thermal barcode includes less than or equal to 500 nucleotides, less than or equal to 400 nucleotides, less than or equal to 300 nucleotides, less than or equal to 200 nucleotides, less than or equal to 175 nucleotides, less than or equal to 150 nucleotides, less than or equal to 125 nucleotides, less than or equal to 100 nucleotides, less than or equal to 90 nucleotides, less than or equal to 80 nucleotides, less than or equal to 70 nucleotides, less than or equal to 60 nucleotides, less than or equal to 50 nucleotides, or fewer. Combinations of these ranges are also possible. For example, in some embodiments, a thermal barcode includes greater than or equal to 5 nucleotides and less than or equal to 500 nucleotides. Other ranges are also possible.

[0100] Multiple thermal barcodes may be used, in some embodiments. For example, in some embodiments, a plurality of primers comprising a plurality of thermal barcode sequences are used to amplify one or more nucleic acids bound to one or more target species, producing barcoded nucleic acids that may be used to determine the target species. As another example, according to some embodiments, one or more nucleic acids comprising thermal barcodes are bound to a target species and are amplified (e.g., using non-barcoded nucleic acids). Thermal barcodes of the plurality may be amplified selectively, based on the presence or absence of the target species, based on the binding of the target species, and / or based on the sample of origin of the thermal barcode, depending on the embodiment.

[0101] Barcodes may, in some embodiments, be used in combination with other signaling entities to facilitate multiplexed detection of target species. For example, according to some embodiments, a barcode may be configured to be detected in using a signaling entity. The signaling entity may be configured to be detected in the presence or absence of the associated barcode, depending on the embodiment. A signaling entity may be detected by any of a variety of suitable methods. For example, a signaling entity may be an optical signaling entity (e.g., a colorimetric signaling entity or a fluorescent signaling entity). Like thermal barcodes, barcodes configured to interact with signaling entities may be used to determine a target species and / or an organism of origin, depending on the embodiment. Barcodes suitable for use with signaling entities may be thermal barcodes, may be used in combination with thermal barcodes, or may be used without thermal barcodes, depending on the embodiments.

[0102] A signaling entity may comprise any of a variety of appropriate moieties that can be detected by a method known to one of ordinary skill. For example, the signaling entity may comprise a fluorophore, a quencher, or a dye.

[0103] In some embodiments, the signaling entity is configured to specifically signal the presence or absence of a nucleic acid. For example, a signaling entity may be designed to interact specifically with a portion of a nucleic acid. The portion of the nucleic acid that is configured to interact with a signaling entity is a type of barcode (though not necessarily a thermal barcode) of the nucleic acid. As another example, a nucleic acid (e.g., a nucleic acid comprising a barcode) may be synthetically modified to include a signaling entity (e.g., an optical signaling entity such as a fluorophore).

[0104] A signaling entity may be a modified nucleic acid, configured to emit a signal (e.g., fluoresce) in the presence of a barcode, but not in its absence. To provide a specific, non-limiting example, the signaling entity could include a fluorophore and a quencher, and could be designed such that, in the absence of a barcode, the fluorophore and the quencher are proximate, but in the presence of the barcode, the signaling entity binds to a portion of the barcode, separating the fluorophore from the quencher to produce a fluorescent signal.

[0105] A signaling entity, like a thermal barcode, can be used to identify a target species. For example, in some embodiments, a plurality of nucleic acids is configured to bind to one or more target species and the plurality includes a first nucleic acid comprising a first thermal barcode and a second nucleic acid comprising a second, identical thermal barcode, wherein the first nucleic acid includes a signaling entity or a barcode configured to interact with a signaling entity, the second nucleic acid does not include a signaling entity or barcode configured to interact with a signaling entity, and the first nucleic acid and the second nucleic acid are bound to different recognition species configured to bind to different target species. In this situation, the melting profile alone might be unable to distinguish the first nucleic acid from the second nucleic acid, since they include the same thermal barcode sequence and would be expected to have similar melting profiles, according to some embodiments. However, the signaling entity could be used to distinguish the first nucleic acid (and, correspondingly, the first target species) from the second nucleic acid (and the second target species) by the presence or the absence of signal from the signaling entity. In this way, the presence or absence of an optical signaling entity can multiply a number of barcoded nucleic acids that can be used simultaneously, by providing an additional mechanism for distinguishing between distinct, barcoded nucleic acids.

[0106] As another example, in some embodiments one or more nucleic acids include a signaling entity without including a thermal barcode. Thus, in some embodiments, a thermally barcoded nucleic acid and a non-thermally -barcoded nucleic acid that both comprise the same signaling entity can be distinguished based on the presence or absence of the melting profile of the thermal barcode.

[0107] More than one type of signaling entity may be used, allowing for even greater multiplexing of the primers described herein. Likewise, more than one type of signaling entity may be used, allowing for even greater multiplexing of nucleic acids configured to identify target species described herein. In some embodiments, greater than or equal to 0, greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, or more signaling entities are used. In some embodiments, less than or equal to 20, less than or equal to 15, less than or equal to 10, less than or equal to 8, less than or equal to 5, less than or equal to 4, less than or equal to 3, less than or equal to 2, or fewer signaling entities are used. Combinations of these ranges are possible. For example, in some embodiments, greater than or equal to 0 and less than or equal to 20 signaling entities are used. Other ranges are also possible.

[0108] Combinations of barcodes and signaling entities (e.g., melting temperature barcodes, optical signaling entities) may be used to provide any of a variety of suitable numbers of uniquely identifiable combinations suitable for distinguishing between target species and / or sample origins. For example, in some embodiments, the number of uniquely identifiable combinations (Nc) is given by Equation (1):

[0109] Nc= ax• a2• b2• N • M + (ax+ a2) • b • N • M + N • M (1) where ai is the number of distinguishable melting temperatures of a first thermal barcode, a2 is the number of distinguishable melting temperatures of a second thermal barcode, b is a number of distinguishable optical signaling entities, N is a number of distinct global melting temperatures (e.g., distinct melting peaks corresponding to the overall melting profile of a first nucleic acid), and M is a number of distinct global melting temperatures (e.g., distinct melting peaks corresponding to the overall melting profile of a second nucleic acid). For example, if ai=l, a2=2, b=4, N=8 and M=8, 2880 unique combinations may be identified.

[0110] In some embodiments, a plurality of target species may be identified using barcodes and / or signaling entities that produce greater than or equal to 1, greater than or equal to 10, greater than or equal to 50, greater than or equal to 100, greater than or equal to 500, greater than or equal to 1,000, greater than or equal to 5,000, greater than or equal to 10,000, or more unique combinations of signaling entities. In some embodiments, a plurality of target species may be identified using signaling entities that produce less than or equal to 100,000, less than or equal to 10,000, less than or equal to 5,000, less than or equal to 1,000, less than or equal to 500, less than or equal to 100, or less unique combinations of signaling entities. Combinations of these ranges are possible. For example, in some embodiments, a plurality of target species may be identified using signaling entities that produce greater than or equal to 1 and less than or equal to 100,000 unique combinations of signaling entities. Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

[0111] According to some embodiments, an auxiliary barcode may be used in combination with a thermal barcode of a barcoded nucleic acid. An auxiliary barcode is, according to some embodiments, a nucleic acid that is configured to bind to an incomplete portion of the thermal barcode of a barcoded nucleic acid. The auxiliary barcode may compete with the ability of the thermal barcode to re- solidify after melting, by binding to the incomplete portion of the thermal barcode.

[0112] In some embodiments, the auxiliary barcode has a different melting profile from the thermal barcode of the thermal barcode of a barcoded nucleic acid. One advantage of using an auxiliary barcode is that, although distinct thermal barcodes may have similar melting profiles, an auxiliary barcode may be configured to bind to a particular thermal barcode sequence (e.g., by including a nucleic acid sequence complementary to the particular thermal barcode sequence). Thus, in some embodiments, the auxiliary barcode, exhibits an observable melting profile only if a thermal barcode to which it is configured to bind is present in a detectable quantity.

[0113] To provide a non-limiting example, in some embodiments, a first thermal barcode and a second thermal barcode are present in a solution (e.g., the first thermal barcode and the second thermal barcode may be comprised by separate nucleic acids of a plurality of nucleic acids). The first thermal barcode and the second thermal barcode may have similar melting profiles to one another, such that it would be difficult to uniquely distinguish between the first thermal barcode and the second thermal barcode based on a melting profile, alone. According to some embodiments, an auxiliary barcode may be added to the solution, wherein the auxiliary barcode is configured to bind to a portion of the first thermal barcode but not the second thermal barcode. A melting profile of the auxiliary barcode would thus only be detectable in the presence of the first thermal barcode. Furthermore, the melting profile of the first thermal barcode would be diminished in the presence of the auxiliary barcode, due to the competitive binding of the auxiliary barcode. Thus, the first thermal barcode may be distinguished from the second thermal barcode using the auxiliary barcode.

[0114] The use of auxiliary barcodes may be particularly advantageous in the context of barcoded nucleic acids that have been barcoded with multiple thermal barcodes. Without wishing to be bound by any particular theory, in some embodiments, a single auxiliary barcode may be auxiliary to both the first thermal barcode and the second thermal barcode, e.g., by being configured to overlap a junction between the first thermal barcode and the second thermal barcode. According to some embodiments, the first thermal barcode and the second thermal barcode of the barcoded nucleic acid have large differences in peak melting temperature, as discussed above. In some embodiments, an auxiliary barcode may have a plurality of peak melting temperatures, including a peak melting temperature associated with the melting of the auxiliary barcode away from the first thermal barcode and a peak melting temperature associated with melting of the auxiliary barcode away from the second thermal barcode. The positions and relative scale of these peak melting temperatures may be controlled, in some embodiments, by changing a length and extent of overlap between the auxiliary barcode and the first thermal barcode, and / or between the auxiliary barcode and the second thermal barcode.

[0115] An auxiliary barcode may be configured to bind to any of a variety of appropriate proportions of a thermal barcode. For example, in some embodiments, an auxiliary barcode is configured to bind to greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, greater than or equal to 25%, greater than or equal to 30%, greater than or equal to 50%, or more of the nucleotides of a thermal barcode. In some embodiments, an auxiliary barcode is configured to bind to less than or equal to 80%, less than or equal to 50%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, or fewer of the nucleotides of a thermal barcode. Combinations of these ranges are possible. For example, in some embodiments, an auxiliary barcode is configured to bind to greater than or equal to 5% and less than or equal to 80% of the nucleotides of a thermal barcode. Other ranges are also possible.

[0116] Any of a variety of appropriate numbers of auxiliary barcodes may be used in combination with the thermal barcodes described herein. In some embodiments, greater than or equal to 1, greater than or equal to 2, greater than or equal to 5, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, or more auxiliary barcodes are used. In some embodiments, less than or equal to 1000, less than or equal to 500, less than or equal to 200, less than or equal to 100, less than or equal to 50, less than or equal to 20, less than or equal to 15, less than or equal to 10, less than or equal to 5, or fewer auxiliary barcodes are used. Combinations of these ranges are possible. For example, in some embodiments, greater than or equal to 1 and less than or equal to 1000 auxiliary barcodes are used. Other ranges are also possible.

[0117] FIGS. 7A-7B present non-limiting, schematic representations of binding of auxiliary barcodes to barcoded nucleic acids, according to some embodiments. FIG. 7A presents binding of auxiliary barcode 798 to thermal barcode 705 of barcoded nucleic acid 701. As shown, the auxiliary barcode is not configured to bind to all nucleotides of thermal barcode 705, but is configured to bind to a subset of the nucleotides of thermal barcode 705. FIG. 7B is similar to FIG. 7A, but shows an auxiliary barcode 799 that is auxiliary to two thermal barcodes of barcoded nucleic acid 711 - first thermal barcode 715 and second thermal barcode 716. The melting profile of the auxiliary barcode may be designed by designing auxiliary barcode 799 to have an appropriate overlap with thermal barcodes 715 and 716, as discussed in greater detail above.

[0118] As illustrated above, one or more recognition species may be used as part of a method described herein. Generally, a recognition species is a species configured to specifically recognize another species (e.g., a target species, another recognition species). A recognition species may be bound to a nucleic acid (e.g., for the purpose of using the nucleic acid to label a target species recognized by the recognition species). For example, in some embodiments, a first nucleic acid is bound to a first recognition species that selectively binds to a first target species and a second nucleic acid is bound to a second recognition species that selectively binds to a second target species. A nucleic acid may be bound to a recognition species covalently. In some embodiments, a nucleic acid is bound to a recognition species non- covalently. For example, as discussed above, in some embodiments an intermediate recognition species is used, and the nucleic acid is configured to bind to a recognition species non-covalently using the intermediate recognition species. Generally, an “intermediate recognition species” refers to a recognition species configured to recognize another recognition species.

[0119] In some embodiments, more than one recognition species is used. A first recognition species and a second recognition species are, in some embodiments, configured to bind to the same species (e.g., the same target species). For example, the first recognition species and the second recognition species may be configured to bind to the same species simultaneously.

[0120] Any of a variety of recognition species may be used in an article or method provided herein. For example, in some embodiments, the recognition species is an antibody. An antibody, as described herein, may comprise a biopolymer, such as a polypeptide. For example, the antibody may comprise a protein. In some embodiments, the antibody is a glycoprotein. However, in some embodiments, the antibody comprising a protein is a glycoprotein. The antibody may be substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Any of a variety of immunoglobulin genes or fragments thereof are known to those of ordinary skill in the art. For example, the antibody may be substantially encoded by immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes and fragments thereof. The antibody may be encoded by a light chain immunoglobulin gene or a fragment thereof. Light chain immunoglobulins may be classified as either kappa or lambda.

[0121] The antibody may be encoded by a heavy chain immunoglobulin gene or a fragment thereof. Heavy chain immunoglobulins may be classified as gamma, mu, alpha, delta, or epsilon(which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively).

[0122] An immunoglobulin structural unit may comprise a tetramer. Each tetramer may be composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. In some embodiments, the light chain has a molecular weight of greater than or equal to 15 kDa, greater than or equal to 20 kDa, greater than or equal to 25 kDa, or greater. In some embodiments, the light chain has a molecular weight of less than or equal to 35 kDa, less than or equal to 30 kDa, less than or equal to 25 kDa, or less. Combinations of these ranges are possible. For example, in some embodiments, the light chain has a molecular weight of greater than or equal to 15 kDa and less than or equal to 35 kDa.

[0123] In some embodiments, the heavy chain has a molecular weight of greater than or equal to 50 kDa, greater than or equal to 55 kDa, greater than or equal to 60 kDa, or greater. In some embodiments, the heavy chain has a molecular weight of less than or equal to 70 kDa, less than or equal to 65 kDa, less than or equal to 60 kDa, or less. Combinations of these ranges are possible. For example, in some embodiments, the heavy chain has a molecular weight of greater than or equal to 50 kDa and less than or equal to 70 kDa.

[0124] The N-terminus of each polypeptide chain may define a variable region of the immunoglobulin structural unit. The variable region may be primarily responsible for antigen recognition. In some embodiments, the variable region comprises greater than or equal to 95, greater than or equal to 98, greater than or equal to 100, greater than or equal to 103, greater than or equal to 105, or more amino acids. In some embodiments, the variable region comprises less than or equal to 115, less than or equal to 113, less than or equal to 110, less than or equal to 108, less than or equal to 105, or fewer amino acids. Combinations of these ranges are possible. For example, in some embodiments, the variable region comprises of greater than or equal to 95 and less than or equal to 115 amino acids.

[0125] Antibodies may exist as intact immunoglobulins. However, in some embodiments, antibodies exist as any of a number of immunoglobulin fragments.

[0126] The immunoglobulin fragment may be produced by digestion with any of a variety of peptidases (e.g., pepsin). For example, in some embodiments immunoglobulin fragments may be formed by digesting an antibody using pepsin. In some, exemplary embodiments, pepsin is used to digest the Fc domain of an antibody, e.g., by degrading disulfide linkages in a hinge region of the Fc domain to produce F(ab)’2. The F(ab)’2 is, according to certain embodiments, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced to break the disulfide linkage in the hinge region, thereby converting the (Fab’)2 dimer into a Fab’ monomer. The Fab’ monomer, according to some embodiments, comprises Fab and a part of the hinge region of the Fc domain.

[0127] In some embodiments, the immunoglobulin fragment is synthesized de novo. The immunoglobulin fragment may be produced by any of a variety of methods known to those of ordinary skill in the art, such as by chemical synthesis, by utilizing recombinant DNA methodology, or by “phage display” methods.

[0128] Examples of antibodies include single chain antibodies, e.g., single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. In one embodiment, the antibody is a monoclonal antibody.

[0129] In some embodiments, a recognition species comprising an antibody is configured such that the antibody or antibody fragment retains a relatively high affinity for a target species or other species (e.g., other recognition species). Without wishing to be bound by theory, the affinity of the antibody or antibody fragment for the target species or other species may be inversely related to the dissociation constant (KD) between the antibody or antibody fragment and the target species or other species, so that a lower KD value corresponds to a higher affinity. In some embodiments, the KD value of the antibody or antibody fragment is less than or equal to 10'6M, less than or equal to IO’7M, less than or equal to 10'8M, less than or equal to 10'9M, less than or equal to 10’10M, or less under physiological conditions. In some embodiments, the KD value of the antibody or antibody fragment is greater than or equal to 1043M, greater than or equal to 1042M, greater than or equal to 101M, or more. Combinations of these ranges are also possible. For example, in some embodiments, the KD value of the antibody or the antibody fragment is greater than or equal to 102M and less than or equal to 10'6M. The KD value of the composition may be determined by a test that would be well-known to one of ordinary skill in the art.

[0130] In some embodiments, the antibody is a nanobody or a minibody. In some embodiments, an antibody fragment is used as a recognition species. In this context, the term “antibody fragment” can refer to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) an epitope of a target species or other species.

[0131] Of course, the recognition species need not be an antibody, and in general any of a variety of other recognition species may be used. For example, other peptides (e.g., affibodies or short-chain peptides) may be used. As another example, the recognition species may be a nucleic acid (e.g., an aptamer). In some embodiments, the recognition species is a molecularly imprinted polymer. Other sorts of recognition species (e.g., recognition species configured for forming covalent linkages to targeted species) are also possible as the disclosure is not so limited.

[0132] Although the methods described above may be implemented in any of a variety of contexts, in some embodiments it is advantageous to perform one or more of the methods in a plurality of compartments, e.g., to take advantage of the multiplex labeling of target species to process large numbers of samples and / or target species simultaneously. As a specific example, in some embodiments, a method comprises using a plurality of compartments including a first compartment and a second compartment, wherein the first compartment comprises a first target species and the second compartment comprises a second target species. In some embodiments, the first target species is determined by selectively amplifying a first nucleic acid and a second nucleic acid bound to the first target species in the first compartment to produce a third nucleic acid. Likewise, according to some embodiments, the second target species is determined by selectively amplifying a fourth nucleic acid and a fifth nucleic acid bound to the second target species in the second compartment to produce a sixth nucleic acid.

[0133] In some embodiments, the method comprises determining a plurality of target species in a plurality of compartments. According to some embodiments, the method comprises determining a single target species in a plurality of compartments comprising samples drawn from a plurality of subjects.

[0134] An article comprising a plurality of compartments may be used to perform one or more of the methods provided herein, depending on the embodiment. For example, according to some embodiments, the article is a well-plate comprising a plurality of wells configured to define compartments comprising the target species and / or barcoded nucleic acids. As another example, in some embodiments the article is a microfluidic chip configured to define compartments comprising target species and / or barcoded nucleic acids using droplets, as discussed in greater detail below. Any of a variety of other articles comprising compartmentalized fluids may also be used, depending on the embodiment. It should generally be understood that methods using a solid substrate may be performed in any of a variety of articles comprising a plurality of compartments, depending on the embodiment. For example, in embodiments where compartments are defined by wells or other solid-liquid interfaces, the solid-liquid interface (e.g., the well wall) may be the solid substrate. However, in some embodiments beads may be suspended in compartments such that solid-liquid interfaces are not required to provide a substrate suitable for performance of a method.

[0135] Articles and / or fluidic systems may be used to perform one or more of the methods described herein within a plurality of droplets (or other compartments). Droplet-based fluidic systems and articles may offer several advantages for determining nucleic acids. For example, microfluidic droplet-based fluidic systems and articles may facilitate high-throughput processing of samples of nucleic acids. In the context of the present disclosure, it has been recognized that the throughput and accuracy of dropletbased methods may be advantageously improved by thermal barcoding, which can help to distinguish the presence of distinct target species, and / or to help identify a subject from which a target species originated, as discussed in greater detail above.

[0136] Some aspects of the present disclosure are generally directed to systems and methods for containing or encapsulating nucleic acids and / or target species such as those discussed herein within microfluidic droplets or other suitable compartments, for example, microwells of a microwell plate, individual spots on a slide or other surface, or the like. Thus, it should be understood that as discussed herein, when nucleic acids and / or target species are contained within droplets, this is by way of example only, and in other embodiments, the nucleic acids may be contained within micro wells, spots on a slide, or other suitable compartments.

[0137] A plurality of compartments (e.g., a plurality of a droplets) generally comprises a first compartment (e.g., a first droplet) and a second compartment (e.g., a second droplet), but can comprise any of an appropriate number of compartments. In some embodiments, a plurality of compartments contains greater than or equal to 5, greater than or equal to 10, greater than or equal to 102, greater than or equal to 103, greater than or equal to 104, greater than or equal to 105, greater than or equal to 106, greater than or equal to 107, or more compartments. In some embodiments, a plurality of compartments contains less than or equal to 108, less than or equal to 107, less than or equal to 106, less than or equal to 105, or less compartments. Combinations of these ranges are possible. For example, in some embodiments, a plurality of compartments contains greater than or equal to 5 and less than or equal to 108compartments. Other ranges are also possible.

[0138] Nucleic acids, barcodes, primers, reagents and / or target species may be allocated to the compartments (e.g., droplets) of the plurality in any of a variety of suitable ways. In some embodiments, it is advantageous to limit the number of target species per compartment, while including a plurality of nucleic acids in the compartment, such that the plurality of nucleic acids may be used to uniquely identify the target species present in each compartment. This may reduce or eliminate confusion that could result from simultaneous melting of distinct thermal barcodes, amplified in the presence of distinct target species. In some embodiments, a first plurality of compartments (e.g., droplets) is formed such that greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 98%, or more of the compartments contain either only one target species or no target species. In some embodiments, a first plurality of compartments is formed such that less than or equal to 100%, less than or equal to 98%, less than or equal to 95%, less than or equal to 90%, or less of the compartments contain either only one target species or no target species. Combinations of these ranges are possible. For example, in some embodiments, a first plurality of compartments is formed such that greater than or equal to 80% and less than or equal to 100% of the compartments contain either only one target species or no target species. Other ranges are also possible.

[0139] The compartments (e.g., droplets) may comprise any of a variety of appropriate number of barcoded nucleic acids. In some embodiments, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 98%, or more of the compartments of a plurality of compartments comprise one or more barcoded nucleic acids. In some embodiments, less than or equal to 100%, less than or equal to 98%, less than or equal to 95%, less than or equal to 90%, less than or equal to 85%, or less of the compartments of a plurality of compartments comprise one or more barcoded nucleic acids. Combinations of these ranges are possible. For example, in some embodiments, greater than or equal to 80% and less than or equal to 100% of the compartments of a plurality of compartments comprise one or more barcoded nucleic acids. Other ranges are also possible. For example, the compartments may comprise no barcoded nucleic acids.

[0140] A plurality of droplets may be broken to the amplified nucleic acids (and amplified thermal barcodes) of the droplets. Breaking the droplets may be advantageous for any of a variety of reasons. For example breaking the droplets may allow the melting profile of a sample to be determined using a larger overall quantity of the amplified thermal barcode, which may improve detection efficiency. Of course, breaking the droplets can complicate the measurement of the temperature profile of amplified thermal barcodes, when multiple thermal barcodes are amplified within the plurality of droplets. As discussed in greater detail above, one advantage to the use of thermal barcodes with distinct melting profiles is that the thermal barcodes may be distinguished by their melting profiles, even when mixed together.

[0141] In some embodiments, a method comprises forming more than one plurality of compartments. For example, a method may comprise forming a first plurality of compartments, suspected of containing a target species from a first subject, and a second plurality of compartments, suspected of containing a target species from a second subject. Alternatively, different pluralities of compartments may be formed from the same source, but including different pluralities of barcoded nucleic acids. The use of multiple pluralities of compartments may permit the re-use of thermal barcodes for barcoding different target species, since a barcoded nucleic acid formed after amplification could be uniquely identified by knowing the barcode in combination with knowing the plurality of compartments in which the amplification of the barcoded nucleic acid took place.

[0142] One or more methods provided herein may comprise amplifying a nucleic acid (e.g., based on its specific binding to a target species, as discussed in greater detail above). Nucleic acids may be amplified by any of a variety of suitable protocols that would be known to one of ordinary skill in the art. This may be useful, for example, to produce a larger number or concentration of nucleic acids, e.g., for subsequent analysis, sequencing, or the like. Those of ordinary skill in the art will be familiar with various amplification methods that can be used, including, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase (RT) PCR amplification, in vitro transcription amplification (IVT), multiple displacement amplification (MDA), multiplex ligationdependent probe amplification (MLPA), isothermal amplification, or quantitative realtime PCR (qPCR).

[0143] As examples, PCR (polymerase chain reaction) or other amplification techniques may be used to amplify nucleic acids, e.g., contained within droplets (or other compartments). Typically, in PCR reactions, the nucleic acids are heated (e.g., to a temperature of at least about 50 °C, at least about 70 °C, or least about 90 °C in some cases) to cause dissociation of the nucleic acids into single strands, and a heat-stable DNA polymerase (such as Taq polymerase) is used to amplify the nucleic acid. This process is often repeated multiple times to amplify the nucleic acids.

[0144] Thus, in one set of embodiments, PCR amplification may be performed within the droplets (or other compartments). For example, the droplets may contain a polymerase (such as Taq polymerase), and DNA nucleotides (deoxyribonucleotides), and the droplets may be processed (e.g., via repeated heated and cooling) to amplify the nucleic acid within the droplets. Suitable reagents for PCR or other amplification techniques, such as polymerases and / or deoxyribonucleotides, may be added to the droplets (or other compartments) during their formation, and / or afterwards (e.g., via merger with droplets containing such reagents, and / or via direct injection of such reagents, e.g., contained within a fluid). Various techniques for droplet injection or merger of droplets will be known to those of ordinary skill in the art. See, e.g., U.S. Pat. Apl. Pub. No. 2012 / 0132288, incorporated herein by reference. In some embodiments, primers may be added to the droplets (or other compartments), or the primers may be present on one or more of the nucleic acids within the droplets. Those of ordinary skill in the art will be aware of suitable primers, many of which can be readily obtained commercially. In some cases, the nucleic acids may be amplified within droplets (or other compartments). This may allow amplification to occur “evenly” in some embodiments, e.g., such that the distribution of nucleic acids is not substantially changed after amplification, relative to before amplification. For example, according to certain embodiments, the nucleic acids within a plurality of droplets (or other compartments) may be amplified such that the number of nucleic acid molecules for each type of nucleic acid may have a distribution such that, after amplification, no more than about 5%, no more than about 2%, or no more than about 1% of the nucleic acids have a number less than about 90% (or less than about 95%, or less than about 99%) and / or greater than about 110% (or greater than about 105%, or greater than about 101%) of the overall average number of amplified nucleic acid molecules per droplet (or other compartment). In some embodiments, the nucleic acids within the droplets (or other compartments) may be amplified such that each of the nucleic acids that are amplified can be detected in the amplified nucleic acids, and in some cases, such that the mass ratio of the nucleic acid to the overall nucleic acid population changes by less than about 50%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% after amplification, relative to the mass ratio before amplification.

[0145] In some cases, certain primers are contained within the droplets (or other compartments) to promote amplification. Such primers may be present during formation of the droplets, and / or added to the droplets after formation of the droplets (or other compartments). Such primers may be barcoded or not, depending on the embodimetn. It should be noted that the manner in which the primers are added to the droplets (or other compartments) may be the same or different from the manner in which the nucleic acids are added to the droplets.

[0146] In certain embodiments, a plurality of different types of primers may be added to the droplets (or other compartments). For instance, the primers may be distinguishable due to their having different sequences, and / or such that they are able to amplify different potential targets. In some cases, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 5,000, or at least 10,000, etc., different primers may be used. This may allow, for example, a variety of different target species to be recognized by amplifying a plurality of nucleic acids within different droplets (or other compartments), e.g., depending on their specific binding to a target species.

[0147] In many embodiments, primers are not barcoded. However, as discussed above, some or all of the primers may be barcoded (e.g., thermally barcoded). For example, in some embodiments, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, or more of the primers comprise thermal barcodes. In some embodiments, less than or equal to 100%, less than or equal to 90%, less than or equal to 80%, less than or equal to 70%, less than or equal to 60%, less than or equal to 50%, or less of the primers comprise thermal barcodes. Combinations of these ranges are possible. For example, in some embodiments, greater than or equal to 20% and less than or equal to 100% of the primers comprise thermal barcodes. Combinations of these ranges are possible. Other ranges are also possible.

[0148] The nucleic acids may be amplified to any suitable extent. The degree of amplification may be controlled, for example, by controlling factors such as the temperature, cycle time, or amount of enzyme and / or deoxyribonucleotides contained within the compartments. For instance, in some embodiments, a plurality of compartments may have at least about 50,000, at least about 100,000, at least about 150,000, at least about 200,000, at least about 250,000, at least about 300,000, at least about 400,000, at least about 500,000, at least about 750,000, at least about 1,000,000 or more molecules of the amplified nucleic acid per compartment. Of course, it should be understood that amplification may occur even without multiplication of the number of molecules within the compartment. For example, the nucleic acids may be amplified using isothermal amplification, wherein the amplified nucleic acid(s) are amplified multiple times within a nucleic acid molecule, with the result that successive amplification steps increase the length of the amplified nucleic acids.

[0149] In one set of embodiments, the droplets are broken down after amplification, e.g., to allow the amplified nucleic acids to be pooled together. A wide variety of methods for “breaking” or “bursting” droplets are available to those of ordinary skill in the art. For example, droplets contained in a carrying fluid may be disrupted using techniques such as mechanical disruption, chemical disruption, or ultrasound. Droplets may also be disrupted using chemical agents or surfactants, for example, 1H,1H,2H,2H- perfluorooctanol .

[0150] In some embodiments, a method comprises purifying nucleic acids (e.g., nucleic acids pooled from a plurality of droplets). Purification may be used, for example, to extract the nucleic acids from unwanted reagents used in earlier steps. Any of a variety of appropriate techniques may be used to purify the nucleic acids. For example, the nucleic acids may be purified using any of a variety of suitable methods, such as column- or gel-based methods (including electrophoretic and centrifuge-based methods). For example, nucleic acids may be purified using a PCR clean-up kit.

[0151] A binding detection agent may be a compound that is able to indicate whether DNA present in solution is double stranded (bound) or single stranded (unbound). A binding detection agent may be non-specific, and may be used to detect DNA binding in general, without significant dependence on the sequence of the DNA. Any of a variety of binding detection agents may be used, many of which are readily available commercially. For example, in some embodiments, a double stranded binding detection agent is used. The double stranded DNA binding detection agent may be configured such that, in the presence of double stranded DNA, the binding detection agent is activated and generates a detectable signal. The binding detection agent may be an optical detection agent, such as a dye. For example, in some embodiments, the binding detection agent is a non-specific double-strand DNA binding dye (e.g., a binSYBR Green Dye or Eva Green Dye), which is able to fluoresce upon encountering doublestranded DNA, but not upon encountering single- stranded DNA.

[0152] Additional details regarding systems and methods for manipulating droplets in a microfluidic system follow, in accordance with certain aspects. For example, various systems and methods for screening and / or sorting droplets are described in U.S. Patent Application Serial No. 11 / 360,845, filed February 23, 2006, entitled “Electronic Control of Fluidic Species,” by Link, et al., published as U.S. Patent Application Publication No. 2007 / 000342 on January 4, 2007, incorporated herein by reference. As a non-limiting example, in some aspects, by applying (or removing) a first electric field (or a portion thereof), a droplet may be directed to a first region or channel; by applying (or removing) a second electric field to the device (or a portion thereof), the droplet may be directed to a second region or channel; by applying a third electric field to the device (or a portion thereof), the droplet may be directed to a third region or channel; etc., where the electric fields may differ in some way, for example, in intensity, direction, frequency, duration, etc.

[0153] As mentioned, certain embodiments comprise a droplet contained within a carrying fluid. For example, there may be a first phase forming droplets contained within a second phase, where the surface between the phases comprises one or more proteins. For example, the second phase may comprise oil or a hydrophobic fluid, while the first phase may comprise water or another hydrophilic fluid (or vice versa). It should be understood that a hydrophilic fluid is a fluid that is substantially miscible in water and does not show phase separation with water at equilibrium under ambient conditions (typically 25 °C and 1 atm). Examples of hydrophilic fluids include, but are not limited to, water and other aqueous solutions comprising water, such as cell or biological media, ethanol, salt solutions, saline, blood, etc. In some cases, the fluid is biocompatible.

[0154] Similarly, a hydrophobic fluid is one that is substantially immiscible in water and will show phase separation with water at equilibrium under ambient conditions. As previously discussed, the hydrophobic fluid is sometimes referred to by those of ordinary skill in the art as the “oil phase” or simply as an oil. Non-limiting examples of hydrophobic fluids include oils such as hydrocarbons oils, silicon oils, fluorocarbon oils, organic solvents, perfluorinated oils, perfluorocarbons such as perfluoropoly ether, etc. Additional examples of potentially suitable hydrocarbons include, but are not limited to, light mineral oil (Sigma), kerosene (Fluka), hexadecane (Sigma), decane (Sigma), undecane (Sigma), dodecane (Sigma), octane (Sigma), cyclohexane (Sigma), hexane (Sigma), or the like. Non-limiting examples of potentially suitable silicone oils include 2 cst polydimethylsiloxane oil (Sigma). Non-limiting examples of fluorocarbon oils include FC3283 (3M), FC40 (3M), Krytox GPL (Dupont), etc. In addition, other hydrophobic entities may be contained within the hydrophobic fluid in some embodiments. Non-limiting examples of other hydrophobic entities include drugs, immunologic adjuvants, or the like. Thus, the hydrophobic fluid may be present as a separate phase from the hydrophilic fluid. In some embodiments, the hydrophobic fluid may be present as a separate layer, although in other embodiments, the hydrophobic fluid may be present as individual fluidic droplets contained within a continuous hydrophilic fluid, e.g. suspended or dispersed within the hydrophilic fluid. This is often referred to as an oil / water emulsion. The droplets may be relatively monodisperse, or be present in a variety of different sizes, volumes, or average diameters. In some cases, the droplets may have an overall average diameter of less than about 1 mm, or other dimensions as discussed herein. In some cases, a surfactant may be used to stabilize the hydrophobic droplets within the hydrophilic liquid, for example, to prevent spontaneous coalescence of the droplets. Non-limiting examples of surfactants include those discussed in U.S. Pat. Apl. Pub. No. 2010 / 0105112, incorporated herein by reference. Other non-limiting examples of surfactants include Span80 (Sigma), Span80 / Tween-20 (Sigma), Span80 / Triton X-100 (Sigma), Abil EM90 (Degussa), Abil we09 (Degussa), polyglycerol polyricinoleate “PGPR90” (Danisco), Tween-85, 749 Fluid (Dow Coming), the ammonium carboxylate salt of Krytox 157 FSL (Dupont), the ammonium carboxylate salt of Krytox 157 FSM (Dupont), or the ammonium carboxylate salt of Krytox 157 FSH (Dupont). In addition, the surfactant may be, for example, a peptide surfactant, bovine serum albumin (BSA), or human serum albumin.

[0155] The droplets may have any suitable shape and / or size. In some cases, the droplets may be microfluidic, and / or have an average diameter of less than about 1 mm. For instance, the droplet may have an average diameter of less than about 1 mm, less than about 700 micrometers, less than about 500 micrometers, less than about 300 micrometers, less than about 100 micrometers, less than about 70 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 1 micrometer, etc. The average diameter may also be greater than about 1 micrometer, greater than about 3 micrometers, greater than about 5 micrometers, greater than about 7 micrometers, greater than about 10 micrometers, greater than about 30 micrometers, greater than about 50 micrometers, greater than about 70 micrometers, greater than about 100 micrometers, greater than about 300 micrometers, greater than about 500 micrometers, greater than about 700 micrometers, or greater than about 1 mm in some cases. Combinations of any of these are also possible; for example, the diameter of the droplet may be between about 1 mm and about 100 micrometers. The diameter of a droplet, in a non- spherical droplet, may be taken as the diameter of a perfect mathematical sphere having the same volume as the non- spherical droplet.

[0156] In some embodiments, the droplets may be of substantially the same shape and / or size (i.e., “monodisperse”), or of different shapes and / or sizes, depending on the particular application. In some cases, the droplets may have a homogenous distribution of cross-sectional diameters, i.e., in some embodiments, the droplets may have a distribution of average diameters such that no more than about 20%, no more than about 10%, or no more than about 5% of the droplets may have an average diameter greater than about 120% or less than about 80%, greater than about 115% or less than about 85%, greater than about 110% or less than about 90%, greater than about 105% or less than about 95%, greater than about 103% or less than about 97%, or greater than about 101% or less than about 99% of the average diameter of the microfluidic droplets. Some techniques for producing homogenous distributions of cross-sectional diameters of droplets are disclosed in International Patent Application No. PCT / US2004 / 010903, filed April 9, 2004, entitled “Formation and Control of Fluidic Species,” by Link, el al., published as WO 2004 / 091763 on October 28, 2004, incorporated herein by reference. In addition, in some instances, the coefficient of variation of the average diameter of the droplets may be less than or equal to about 20%, less than or equal to about 15%, less than or equal to about 10%, less than or equal to about 5%, less than or equal to about 3%, or less than or equal to about 1%. However, in other embodiments, the droplets may not necessarily be substantially monodisperse, and may instead exhibit a range of different diameters.

[0157] Those of ordinary skill in the art will be able to determine the average diameter of a population of droplets, for example, using laser light scattering or other known techniques. The droplets so formed can be spherical, or non- spherical in certain cases. The diameter of a droplet, in a non- spheric al droplet, may be taken as the diameter of a perfect mathematical sphere having the same volume as the non-spherical droplet. In some embodiments, one or more droplets may be created within a channel by creating an electric charge on a fluid surrounded by a liquid, which may cause the fluid to separate into individual droplets within the liquid. In some embodiments, an electric field may be applied to the fluid to cause droplet formation to occur. The fluid can be present as a series of individual charged and / or electrically inducible droplets within the liquid. Electric charge may be created in the fluid within the liquid using any suitable technique, for example, by placing the fluid within an electric field (which may be AC, DC, etc.), and / or causing a reaction to occur that causes the fluid to have an electric charge.

[0158] The electric field, in some embodiments, is generated from an electric field generator, i.e., a device or system able to create an electric field that can be applied to the fluid. The electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc. Techniques for producing a suitable electric field (which may be AC, DC, etc.) are known to those of ordinary skill in the art. For example, in one embodiment, an electric field is produced by applying voltage across a pair of electrodes, which may be positioned proximate a channel such that at least a portion of the electric field interacts with the channel. The electrodes can be fashioned from any suitable electrode material or materials known to those of ordinary skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as well as combinations thereof.

[0159] In another set of embodiments, droplets of fluid can be created from a fluid surrounded by a liquid within a channel by altering the channel dimensions in a manner that is able to induce the fluid to form individual droplets. The channel may, for example, be a channel that expands relative to the direction of flow, e.g., such that the fluid does not adhere to the channel walls and forms individual droplets instead, or a channel that narrows relative to the direction of flow, e.g., such that the fluid is forced to coalesce into individual droplets. In some cases, the channel dimensions may be altered with respect to time (for example, mechanically or electromechanically, pneumatically, etc.) in such a manner as to cause the formation of individual droplets to occur. For example, the channel may be mechanically contracted (“squeezed”) to cause droplet formation, or a fluid stream may be mechanically disrupted to cause droplet formation, for example, through the use of moving baffles, rotating blades, or the like.

[0160] Some embodiments generally relate to systems and methods for fusing or coalescing two or more droplets into one droplet, e.g., where the two or more droplets ordinarily are unable to fuse or coalesce, for example, due to composition, surface tension, droplet size, the presence or absence of surfactants, etc. In certain cases, the surface tension of the droplets, relative to the size of the droplets, may also prevent fusion or coalescence of the droplets from occurring.

[0161] As a non-limiting example, two droplets can be given opposite electric charges (i.e., positive and negative charges, not necessarily of the same magnitude), which can increase the electrical interaction of the two droplets such that fusion or coalescence of the droplets can occur due to their opposite electric charges. For instance, an electric field may be applied to the droplets, the droplets may be passed through a capacitor, a chemical reaction may cause the droplets to become charged, etc. The droplets, in some cases, may not be able to fuse even if a surfactant is applied to lower the surface tension of the droplets. However, if the droplets are electrically charged with opposite charges (which can be, but are not necessarily of, the same magnitude), the droplets may be able to fuse or coalesce. As another example, the droplets may not necessarily be given opposite electric charges (and, in some cases, may not be given any electric charge), and are fused through the use of dipoles induced in the droplets that causes the droplets to coalesce. Also, the two or more droplets allowed to coalesce are not necessarily required to meet “head-on.” Any angle of contact, so long as at least some fusion of the droplets initially occurs, is sufficient. See also, e.g., U.S. Patent Application Serial No. 11 / 698,298, filed January 24, 2007, entitled “Fluidic Droplet Coalescence,” by Ahn, et al., published as U.S. Patent Application Publication No. 2007 / 0195127 on August 23, 2007, incorporated herein by reference in its entirety.

[0162] In one set of embodiments, a fluid may be injected into a droplet. The fluid may be microinjected into the droplet in some cases, e.g., using a microneedle or other such device. In other cases, the fluid may be injected directly into a droplet using a fluidic channel as the droplet comes into contact with the fluidic channel. Other techniques of fluid injection are disclosed in, e.g., International Patent Application No. PCT / US2010 / 040006, filed June 25, 2010, entitled “Fluid Injection,” by Weitz, et al., published as WO 2010 / 151776 on December 29, 2010; or International Patent Application No. PCT / US2009 / 006649, filed December 18, 2009, entitled “Particle- Assisted Nucleic Acid Sequencing,” by Weitz, et al., published as WO 2010 / 080134 on July 15, 2010, each incorporated herein by reference in its entirety.

[0163] The following documents are each incorporated herein by reference in its entirety for all purposes: Int. Pat. Apl. Pub. No. WO 2016 / 168584, entitled “Barcoding System for Gene Sequencing and Other Applications,” by Weitz et al. Int. Pat. Apl. Pub. No. WO 2015 / 161223, entitled “Methods and Systems for Droplet Tagging and Amplification,” by Weitz, et al. U.S. Pat. Apl. Ser. No. 61 / 980,541, entitled “Methods and Systems for Droplet Tagging and Amplification,” by Weitz, et al.; U.S. Pat. Apl. Ser. No. 61 / 981,123, entitled “Systems and Methods for Droplet Tagging,” by Bernstein, et al.; Int. Pat. Apl. Pub. No. WO 2004 / 091763, entitled “Formation and Control of Fluidic Species,” by Link et al.; Int. Pat. Apl. Pub. No. WO 2004 / 002627, entitled “Method and Apparatus for Fluid Dispersion,” by Stone et al.; Int. Pat. Apl. Pub. No. WO 2006 / 096571, entitled “Method and Apparatus for Forming Multiple Emulsions,” by Weitz et al.; Int. Pat. Apl. Pub. No. WO 2005 / 021151, entitled “Electronic Control of Fluidic Species,” by Link et al.; Int. Pat. Apl. Pub. No. WO 2011 / 056546, entitled “Droplet Creation Techniques,” by Weitz, et al.; Int. Pat. Apl. Pub. No. WO 2010 / 033200, entitled “Creation of Libraries of Droplets and Related Species,” by Weitz, et al.; U.S. Pat. Apl. Pub. No. 2012-0132288, entitled “Fluid Injection,” by Weitz, et al.; Int. Pat. Apl. Pub. No. WO 2008 / 109176, entitled “Assay And Other Reactions Involving Droplets,” by Agresti, et al.; and Int. Pat. Apl. Pub. No. WO 2010 / 151776, entitled “Fluid Injection,” by Weitz, et al.; and U.S. Pat. Apl. Ser. No. 62 / 072,944, entitled “Systems and Methods for Barcoding Nucleic Acids,” by Weitz, et al.

[0164] In addition, the following are incorporated herein by reference in their entireties: U.S. Pat. Apl. Ser. No. 61 / 981,123 filed April 17, 2014; PCT Pat. Apl. Ser. No. PCT / US2015 / 026338, filed April 17, 2015, entitled “Systems and Methods for Droplet Tagging”; U.S. Pat. Apl. Ser. No. 61 / 981,108 filed April 17, 2014; U.S. Pat. Apl. Ser. No. 62 / 072,944, filed October 30, 2014; PCT Pat. Apl. Ser. No. PCT / US2015 / 026443, filed on April 17, 2015, entitled “Systems and Methods for Barcoding Nucleic Acids”; U.S. Pat. Apl. Ser. No. 62 / 106,981, entitled “Systems, Methods, and Kits for Amplifying or Cloning Within Droplets,” by Weitz, et al. U.S. Pat. Apl. Pub. No. 2010-0136544, entitled “Assay and Other Reactions Involving Droplets,” by Agresti, et al. ; U.S. Pat.

[0165] Apl. Ser. No. 61 / 981,108, entitled “Methods and Systems for Droplet Tagging and Amplification,” by Weitz, et al. Int. Pat. Apl. Pub. No. PCT / US2014 / 037962, filed May 14, 2014, entitled “Rapid Production of Droplets,” by Weitz, et al.; and U.S. Provisional Patent Application Serial No. 62 / 133,140, filed 03 / 13 / 15, entitled “Determination of Cells Using Amplification,” by Weitz, et al.

[0166] A fluidic system may be used to perform some or all of the method steps described above. In one aspect of the present invention, emulsions are formed by flowing two, three, or more fluids through a system of channels of a fluidic system. The fluidic system may be or comprise an article. The system or article may be a microfluidic system or article. "Microfluidic," as used herein, refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension (measured perpendicular to the direction of fluid flow) of less than about 1 millimeter (mm), and in some cases, a ratio of length to largest cross-sectional dimension of at least 3:1.

[0167] A "channel," as used herein, means a feature on or in a system or article that at least partially directs flow of a fluid. The channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. One or more of the channels may (but not necessarily), in cross section, have a height that is substantially the same as a width at the same point.

[0168] In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and / or outlet(s). A channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2:1, more typically at least 3:1, 5:1, 10:1, 15:1, 20:1, or more. An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and / or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid. The fluid within the channel may partially or completely fill the channel. In some cases where an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus).

[0169] The channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. In some cases the dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flowrate of fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, positioned to intersect with each other, etc.

[0170] The fluidic droplets within the channels may have a cross-sectional dimension smaller than about 100% of an average cross-sectional dimension of the channel, and in certain embodiments, smaller than about 90%, smaller than about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 3%, about 1%, about 0.5%, about 0.3%, about 0.1%, about 0.05%, about 0.03%, or about 0.01% of the average cross-sectional dimension of the channel.

[0171] During use, at least some processing of the droplets may be performed on an article. Thus, in some embodiments, an article comprises at least some of a plurality of droplets described above. For example, the article may comprise all droplets of a plurality of droplets. The droplets may be fluidic ally connected to one or more reservoirs of the fluidic system (e.g., to a pool used to form droplets, to a hydrophobic fluid used to form droplets, to a supply of a target substrate, to a supply of a signaling entity, to a supply of in vitro transcription and translation reagents, or any of a variety of other fluids described herein) via the article. For example, the droplets may be connected to one or more reservoirs of a fluidic system via the microchannel.

[0172] In some embodiments, the fluidic system comprises one or more additional components, such as a pressure source (for example, a pump), a detection tool (e.g., a sensor that may be used to detect fluorescence, luminescence, and / or colorimetric changes resulting from activity of a target substrate); and / or a waste stream.

[0173] U.S. Provisional Patent Application Serial No. 63 / 652,645, filed May 28, 2024, entitled “Highly Multiplexed Target Species Determination Using Thermal Barcodes,” by Weitz, et al., is incorporated herein by reference in its entirety.

[0174] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0175] EXAMPLE 1

[0176] This example describes the use of thermal barcoding to determine a target protein in solution, according to some non-limiting embodiments. FIG. 8 schematically illustrates the method used. At step (a), a sample comprising a plurality of target proteins was incubated with “barcoded antibodies” — nucleic acids comprising thermal barcodes and covalently bound to recognition species in the form of antibodies configured to recognize the target proteins. High temperature (high CG) and low temperature (low CG) barcodes were partnered to each target protein by choice of appropriate antibodies, such that selective amplification produced a third nucleic acid comprising both a high-melting and a low-melting barcode. After incubation, the incubated solution was partitioned into a plurality of droplets for subsequent analysis. Then, selective amplification was performed on the droplets as illustrated schematically at step (b). At step (c), an imaging system and a heater were used to monitor the melting profiles of the amplified barcodes. Finally, the melting profiles of the droplets were analyzed in order to determine the target molecules present in the droplets.

[0177] FIGS. 9A-9D are micrographs of droplets, some of which contain a single target protein, human IL10. The protein was partitioned from solutions with a plurality of distinct protein concentrations. FIG. 9A is a control, illustrating the intensity of a plurality of droplets without any human IL 10. FIGS. 9B-9D were prepared by partitioning human IL 10 concentrations of 1 pg / mL, 10 pg / mL and 50 pg / mL, respectively. The droplets included a binding detection agent, and FIGS. 9A-9D illustrate that selective amplification of nucleic acid barcodes occurred in a proportion of the droplets determined by the concentration of the protein solution, as expected. Generally, the bright droplets illustrated in FIGS. 9B-9D correspond to droplets comprising the human IL 10, which resulted in amplification of nucleic acids and determination of the human IL 10 based on the visible activity of the binding detection agent, which was detected most effectively in the presence of melted nucleic acids.

[0178] FIG. 10 compares the total intensity of the droplets pictured in FIGS. 9A-9D with the concentration of the human IL 10 solution used to prepare them. As expected, signal intensity increased with the concentration of the human IL 10. The trendline included in FIG. 10 is the linear fit to the four data points.

[0179] EXAMPLE 2

[0180] This example reports detection of multiple target proteins — human IL 10, human IL la, human IL 6 and human IL 7 — from a pooled sample of the proteins using thermal barcodes. The method schematized in FIG. 8 was used to prepare pluralities of droplets, some of which comprised one or more of the target proteins, and some of which comprised barcoded antibodies configured to recognize and be selectively amplified in the presence of exactly one of the target proteins. FIG. 11 illustrates the distinct melting profiles of barcoded nucleic acids produced in the presence of each target molecule. The median melting temperature of the low-melting barcode and the high-melting barcode are reported for each profile below the x-axis. Using clustering, relative concentrations of each target protein in the droplets were determined. FIG. 12 reports the determined concentrations for each protein type.

[0181] EXAMPLE 3

[0182] This example compares detection of IL 10 (as in Example 1) with comparative results from an enzyme-linked immunosorbent assay (ELISA). IL- 10 was measured across a series of input concentrations ranging from ~10 fM to 1400 fM. FIG. 13A presents detection measurements (modified from FIG. 10 to show copies per microliter vs input concentration) using the barcoded nucleic acid method, while FIG. 13B presents comparative results from ELISA (in optical density (OD, arbitrary units) vs input concentration). The barcode assay (FIG. 13A) yielded copy number estimates with a linear response (Y=0.00834*X + 0.3993; R2=0.8408; P value <0.0001), minimal background, and narrow confidence intervals. In contrast, ELISA (FIG. 13B) also demonstrated a linear trend (Y=0.008304*X + 0.3993; R2=0.9894; P value <0.0001) but exhibited higher background signals and broader variability at elevated concentrations, resulting in reduced dynamic range and sensitivity. These results highlight the superior performance of barcode-based measurements for low-abundance protein detection.

[0183] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0184] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0185] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0186] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0187] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0188] Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and / or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

[0189] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0190] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. A method of determining a target species, comprising: selectively amplifying a first nucleic acid and a second nucleic acid to produce a third nucleic acid when both the first nucleic acid and the second nucleic acid are bound to the target species, wherein the first nucleic acid comprises a first barcode, wherein the second nucleic acid comprises a second barcode, wherein the third nucleic acid comprises:(a) the first barcode or a sequence complementary to the first barcode, and(b) the second barcode or a sequence complementary to the second barcode, wherein the first barcode is a thermal barcode sequence exhibits a first peak melting point and the second thermal barcode sequence exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1 °C.

2. The method of claim 1, wherein third nucleic acid does not comprise the target species,3. The method of any one of the preceding claims, wherein determining the target species comprises determining the presence or absence of the target species.

4. The method of any one of the preceding claims, wherein the target species is a first target species, and wherein determining the first target species comprises determining binding of the first target species to a second species.

5. The method of any one of the preceding claims, further comprising determining the target species by determining the first peak melting point and the second peak melting point.

6. The method of any one of the preceding claims, wherein the first thermal barcode sequence has a first ratio of total nearest-neighbor binding enthalpy to total nearest- neighbor binding entropy and the second thermal barcode sequence has a second ratio of total nearest-neighbor binding enthalpy to total nearest-neighbor binding entropy, wherein the first ratio differs from the second ratio by at least 1% of the second ratio.

7. The method of any one of the preceding claims, wherein determining the target species comprises determining the presence or the absence of the target species within a sample.

8. The method of any one of the preceding claims, wherein determining the target species comprises using one or more auxiliary barcodes configured to bind to at least a portion of the thermal barcode sequence or at least a portion of the target species.

9. The method of any one of the preceding claims, wherein the presence of the target species is indicative of a disease or condition.

10. The method of any one of the preceding claims, wherein the absence of the target species is indicative of a disease or condition.

11. The method of any one of the preceding claims, wherein determining the target species comprises determining a subject from which the target species originated.

12. The method of any one of the preceding claims, wherein the target species is a first target species of a plurality of target species.

13. The method of any one of the preceding claims, wherein a compartment comprises the target species.

14. The method of any one of the preceding claims, wherein a droplet comprises the target species.

15. The method of any one of the preceding claims, wherein a micro well comprises the target species.

16. The method of any one of the preceding claims, wherein the target species is a first target species and wherein the method further comprises determining a second species by selectively amplifying a fourth nucleic acid and a fifth nucleic acid to produce a sixth nucleic acid when both the fourth nucleic acid and the fifth nucleic acid are bound to the target species, and wherein at least one of the fourth nucleic acid and the fifth nucleic acid comprises a third barcode.

17. The method of any one of the preceding claims, further comprising determining a plurality of target species including the first target species and a second species by selectively amplifying a plurality of pairs of barcodes when the pairs of barcodes are bound together via binding to a common target species.

18. The method of any one of the preceding claims, wherein the first nucleic acid is bound to a first recognition species that selectively binds to the target species, and wherein the second nucleic acid is bound to a recognition species that selectively binds to the target species.

19. The method of claim 18, wherein the first recognition species and the second recognition species can simultaneously bind to the same target species.

20. The method of any one of claims 18-19, wherein the first recognition species and / or the second recognition species are antibodies or aptamers configured to bind to the target species.

21. The method of any one of the preceding claims, wherein the target species is a first target species, wherein the first nucleic acid is bound to a first recognition species that selectively binds to the first target species, and wherein the second nucleic acid is bound to a second recognition species that selectively binds to a second species.

22. The method of claim 21, wherein the first recognition species and / or the second recognition species are antibodies or aptamers configured to bind to the first target species and / or the second species, respectively.

23. The method of any one of claims 18-22, wherein the first nucleic acid is bound to a first recognition species covalently.

24. The method of any one of claims 18-23, wherein the second nucleic acid is bound to the second recognition species covalently.

25. The method of any one of claims 18-24, wherein the first nucleic acid is only bound to a first recognition species non-covalently.

26. The method of any one of claims 18-25, wherein the second nucleic acid is only bound to the second recognition species non-covalently.

27. The method of any one of claims 20 and 22 wherein the first nucleic acid and / or the second nucleic acid is covalently bound to an intermediate recognition species configured to bind to the first recognition species and / or the second recognition species.

28. The method of any one of the preceding claims, wherein the method is performed in a plurality of droplets.

29. The method of any one of the preceding claims, wherein determining the nucleic acid further comprises detecting the species, the first barcode, and / or the second barcode using an optical signaling entity.

30. The method of any one of the preceding claims, wherein the optical signaling entity comprises a fluorophore.

31. The method of any one of the preceding claims, wherein the optical signaling entity comprises a fluorescent quencher.

32. The method of any one of the preceding claims, wherein the target species is a target protein or a target nucleic acid.

33. Use of the method of any one of the preceding claims to at least partially determine the proteome, genome, or metabolome of an organism.

34. An article, comprising: a plurality of compartments comprising a first compartment comprising a first target species, a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first target species is bound to the first nucleic acid and the second nucleic acid, wherein the first nucleic acid comprises a first barcode, wherein the second nucleic acid comprises a second barcode, and wherein the third nucleic acid comprises:(a) the first barcode or a sequence complementary to the first barcode, and(b) the second barcode or a sequence complementary to the second barcode, wherein the first barcode is a thermal barcode sequence exhibits a first peak melting point and the second thermal barcode sequence exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1 °C.

35. The article of claim 34, wherein third nucleic acid does not comprise the target species.

36. The article of any one of the preceding claims, further comprising a second compartment comprising a second target species, a fourth nucleic acid, a fifth nucleic acid, and a sixth nucleic acid, wherein the second target species is bound to the fourth nucleic acid and the fifth nucleic acid, wherein the fourth nucleic acid comprises a third barcode, wherein the fifth nucleic acid comprises a fourth barcode, andwherein the sixth nucleic acid comprises:(a) the third barcode or a sequence complementary to the third barcode, and(b) the fourth barcode or a sequence complementary to the fourth barcode.

37. The article of any one of the preceding claims, wherein the plurality of compartments comprises a plurality of droplets.

38. The article of any one of the preceding claims, wherein the plurality of compartments comprises a plurality of micro wells.

39. A method of determining a target species, comprising: selectively amplifying a first nucleic acid and a second nucleic acid to produce a third nucleic acid when both the first nucleic acid and the second nucleic acid are bound to the target species, wherein the first nucleic acid comprises a first barcode, wherein the second nucleic acid comprises a second barcode, wherein the third nucleic acid comprises:(a) the first barcode or a sequence complementary to the first barcode, and(b) the second barcode or a sequence complementary to the second barcode.

40. The method of claim 39, wherein the third nucleic acid does not comprise the target species.

41. A method of determining a target species, comprising: binding the target species to a solid substrate and a nucleic acid in a solution and selectively amplifying the nucleic acid bound to the target species, wherein the nucleic acid comprises a first barcode, wherein the first barcode exhibits a peak melting point differing by at least 0.1 °C from a second peak melting point of a second barcode present in the solution.

42. The method of claim 41, wherein the nucleic acid further comprises the second barcode.

43. An article, comprising: a plurality of compartments comprising a first compartment comprising a first target species bound to a first nucleic acid and a first solid substrate and a second compartment comprising a second target species, a second nucleic acid, and a second solid substrate, wherein the first nucleic acid comprises a first barcode, and wherein the first thermal barcode exhibits a first peak melting point differing by at least 0.1 °C from a second peak melting point of a second barcode present in the first compartment.

44. The method of claim 43, wherein the nucleic acid further comprises the second barcode.

45. A method of determining a target species, comprising: amplifying a first nucleic acid to produce a second nucleic acid, wherein during the amplification: the first nucleic acid is bound to a recognition species; the recognition species is bound to a target species; and the target species is bound to a substrate, wherein the first barcode is a thermal barcode sequence exhibits a first peak melting point and the second thermal barcode sequence exhibits a second peak melting point, the first peak melting point and the second peak melting point differing by at least 0.1 °C.

46. The method of claim 45, wherein the recognition species is a first recognition species, and wherein the target species is bound to the substrate via a second recognition species.

47. The method of any one of claims 45-46, wherein the substrate is a bead.

48. The method of any one of claims 45-47, wherein the substrate is the wall of a well.

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