System and method for extracting a target portion from a sample using acoustic droplet ejection.

Abstract

A method and system for extracting a target analyte from a sample using acoustic ejection techniques is provided. The method includes applying focused acoustic energy to a fluid reservoir containing a fluid composition containing the target analyte and including an upper region and a lower region, wherein the target analyte concentration in the upper region is different from the target analyte concentration in the lower region. The focused acoustic energy is applied in a manner effective to eject fluid droplets from the fluid composition into a droplet receiver, wherein the analyte concentration in the droplet corresponds to either the analyte concentration in the upper region or the analyte concentration in the lower region, and the analyte concentration is substantially uniform across the droplet. The fluid composition can include an ionic liquid used for extraction of the ionic target analyte. Related methods and acoustic extraction systems are also provided.

Description

[Technical Field]

[0001] (1) Technical field The present invention generally relates to a system and method for extracting a target analyte from a fluid composition using acoustic droplet ejection. The present invention has practical applications in numerous fields, including chemistry and biology. [Background technology]

[0002] (2) Description of related technologies The extraction of molecules from mixtures is an essential step in processes used in numerous technological fields, including organic synthesis chemistry, materials science, pharmaceutical research and development, and molecular biology. The extraction of biomolecules, such as nucleic acids and proteins, is now required in many situations, but is particularly challenging as they are typically contained within complex host environments, such as cells, tissues, and blood. Nevertheless, efficient and effective extraction of DNA, RNA, and proteins is necessary for numerous processes and products. Diagnostic kits, identity and kinship testing, pathogen detection, histological compatibility testing, and genetic research are prime examples.

[0003] DNA purification may involve the isolation and removal of chromosomes and / or mitochondrial DNA from their biological environment, or it may be performed in situations involving the isolation of recombinant DNA constructs, i.e., DNA containing recombinant sequences, such as plasmids. DNA polymerase chain reaction (PCR) amplification, diagnostic testing procedures, and hosts for high-sensitivity assays require high purity. Contaminants in DNA samples may inhibit one or more critical steps in diagnostic or analytical procedures. Some contaminants may, for example, inhibit polymerase chain reaction or restriction enzyme activity.

[0004] Any DNA purification method requires (1) effective disruption of the biological host environment, e.g., cells or tissues containing the target DNA; (2) denaturation of protein and nucleoprotein complexes using proteases and / or denaturants; (3) inactivation of endogenous nucleases; and (4) removal of the target DNA from the sample. The isolated target DNA should not contain any compounds or materials that were originally present, e.g., proteins, lipids, RNA, other nucleic acids, etc., and in most cases, the purification process must avoid DNA fragmentation resulting from mechanical shear or the presence of contaminants. RNA isolation is even more complex, given that RNA is inherently unstable, requires potent denaturants to inhibit endogenous RNAse, RNAse is thermally stable and refolds after thermal denaturation, and RNAse lacking cofactors is difficult to inactivate.

[0005] In nucleic acid purification, cell or tissue disruption is typically performed using a washing agent designed to break down the lipid bilayer of the cell membrane. The washing agent disrupts both lipid-to-lipid and lipid-to-protein interactions within the cell membrane, making the membrane components solubilizable. Organisms containing a cell wall in addition to the cell membrane may require additional treatment; for example, digesting the peptidoglycan cell wall of Gram-positive bacteria requires treatment with lysozyme, and disrupting the polysaccharide cell wall of yeast requires treatment with lithicase or zymolase.

[0006] Protein and nucleoprotein denaturation involves altering the protein structure by disrupting its secondary structure and is carried out using protein denaturants, such as ionic detergents, chaotropic agents, reducing agents, heat, and / or proteases. In mammalian cells, DNA is compressed by the histones (i.e., chromatin) of macromolecular nucleoprotein structures, and denaturation allows for the release of chromosomal DNA from the nucleoprotein complex. Chelating agents, such as ethylenediaminetetraacetic acid (EDTA), are typically used to inactivate nucleases, such as proteinase K. Some commercial systems require stepwise processing, i.e., cell lysis, denaturation, and nuclease inactivation, while others provide a single solution containing components to perform all three of the aforementioned steps.

[0007] Ultimately, the target DNA must be isolated from a treated biological sample that is likely to contain proteins, protein fragments, lipids, carbohydrates, salts, and cell debris. Historically, DNA has been purified via liquid-liquid extraction. Aqueous cell hydrolysates were shaken with a phenol-chloroform mixture containing some isoamyl alcohol as needed to inhibit RNAse activity. This mixture separates into two layers: a hydrophobic chloroform-phenol lower phase containing proteins, lipids, carbohydrates, and cell debris, and an upper aqueous phase with residual nucleic acids. The upper phase of DNA aqueous solution is collected, the DNA precipitates from the supernatant, the DNA precipitate is rinsed, and dissolved with a buffer. However, the technique is cumbersome and time-consuming, and as has been widely pointed out, chloroform is highly toxic, and phenol is flammable, corrosive, and also toxic.

[0008] Liquid-liquid extraction has largely replaced solid-phase nucleic acid purification methods using centrifugation-based columns. In this method, the cell solubilizer is mixed with a buffer and either a chaotropic agent or a short-chain alcohol. The solubilizer is transferred to a column and centrifuged to pass the liquid through a solid phase that has been surface-treated to retain negatively charged nucleic acids. Proteins and other contaminants are washed through the column, while nucleic acids bind to it. After the washing step, the nucleic acids are eluted with water or a buffer (e.g., diluted TRIS-EDTA buffer at approximately pH 8.4). Mixed-bed solid-phase extraction has also been disclosed; see U.S. Patent No. 6,376,194 by Smith et al. Column-based DNA extraction is easier and safer than conventional liquid-liquid extraction using phenol-chloroform techniques, but it is still manual and not readily automated.

[0009] As a variation of conventional solid-phase separation, magnetic bead-based nucleic acid purification methods have been developed. In this process, coated or otherwise surface-treated magnetic beads bind to nucleic acids in the presence of a chaotropic agent. The beads are combined with a biological sample, and the nucleic acids in the sample bind to the bead surface. Magnets are used to attract the beads, which have the nucleic acids bound to their surface, to a stable position in a microplate well, centrifuge tube, or other container. Once the beads are fixed by magnetism, the supernatant containing impurities is removed, the beads are washed with a clean washing buffer, and the nucleic acids are detached from the magnetic beads with a small amount of dilution elution buffer. This technique is generally easier to automate and offers high throughput than other solid-phase purification methods by eliminating centrifugation. However, the cost of purifying each sample is considerably high. Various types of extraction techniques for isolating and purifying DNA, RNA, and proteins are described in Tan et al. (2009) J. Biomed. Biotech., Article ID 574398. As described in that document, there is a continuing need for improved methods for isolating and purifying biomolecules. Tan et al. state that automating the extraction procedure is desirable to reduce working time, lower labor costs, enhance worker safety, and ideally increase both the reproducibility and quality of results. Tan et al. further state that commercially available automated systems intended for use in medium to large laboratories are somewhat limited in that they are large, expensive, and complex, while more recent automated processes adapted to small to medium sample throughput still have a processing time of about 20 to 40 minutes per sample, indicating that the extraction process is still time-consuming. Proper handling of liquids is essential for each extraction step of the automated procedure, as well as for the movement of liquids as needed, and as Tan et al. explain, ideally, a fully automated robotic workstation should be used, thereby eliminating the need for pretreatment steps. Tan et al. further point out that continuous improvements in miniaturization are needed, and that these improvements could address the weaknesses of available extraction systems.

[0010] Acoustic droplet injection (ADE) is a method disclosed as useful for injecting immiscible fluids. See U.S. Patents 6,548,308 and 6,642,061 by Ellson et al. The aforementioned patent documents describe the use of ADE for injecting droplets from immiscible liquids onto a substrate surface, the droplets generally having a first region corresponding to one of the liquids and a second region corresponding to the other liquid. However, ADE has not been performed for the extraction of targeted portions from samples, and it is well known that the development of extraction techniques can be complex and problematic, as described by Tan et al. above.

[0011] An ideal extraction system and method can achieve at least the following objectives:

[0012] To provide isolated target molecules in high purity,

[0013] It can be used to extract any of the wide variety of target molecules.

[0014] To obtain accurate, consistent, and reproducible results,

[0015] To be completely automated,

[0016] It can be used under standard laboratory conditions without requiring high temperatures or an inert atmosphere.

[0017] To minimize processing time per sample and enable high-throughput sample processing,

[0018] To enable effective, efficient, accurate, and reliable processing of very small sample sizes, approximately nanoliters or smaller.

[0019] To eliminate the need for toxic volatile solvents,

[0020] The ability to rely on reagents that can be recycled and reused in subsequent extraction steps, and

[0021] Enabling rapid introduction of the extracted target portion into an analysis device, such as a mass spectrometer.

Prior Art Documents

Patent Documents

[0022]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0023]

Non-Patent Document 1

[0025] The extraction method of the present invention encompasses the partial or complete removal of the target analyte from the initial fluid composition, and also encompasses a separation process in which the final fluid composition contains non-target components at lower concentrations than the initial fluid composition.

[0026] A first embodiment of the present invention provides a method for generating fluid droplets containing a target analyte at a selected concentration. The method includes (a) providing a fluid composition in a fluid reservoir, comprising an upper region and a lower region, wherein the analyte is present at a first concentration in the upper region and at a second concentration in the lower region, the second concentration being different from the first concentration; and (b) applying focused acoustic energy to the fluid reservoir in a manner effective for ejecting fluid droplets from the fluid composition into a droplet receiver, wherein the ejected droplets contain a selected concentration of the target analyte, the selected concentration being (i) substantially equivalent to either the first or second concentration, and (ii) substantially uniform across the droplet. The fluid composition in the fluid reservoir generally includes a sample containing the target analyte, such as a biological sample. The biological sample may include a sample dissolved or suspended in a fluid, or the biological sample may be a fluid itself.

[0027] In one embodiment of the above-described model, the upper region of the fluid composition includes an upper layer of the first liquid, and the lower region of the fluid composition includes a lower layer of the second liquid. Depending on the target analyte, the first liquid, and the second liquid, the analyte may be preferentially distributed to either the first or second liquid. That is, the first and second liquids are selected such that the first liquid has a first affinity for the target analyte and the second liquid has a second affinity for the target analyte, and the first and second affinities are different. For example, when using a hydrophilic, e.g., ionic, target analyte, a hydrophilic upper liquid, and a hydrophobic lower liquid, the target analyte tends to be distributed to the upper hydrophilic liquid. As another example, if the solubility of the target analyte is higher in the lower liquid than in the upper liquid, the target analyte tends to be distributed to the lower liquid. The first and second liquids may differ in volatility, density, viscosity, and / or other physical or chemical properties.

[0028] In the relevant embodiments, the solubility of the target analyte in the lower liquid differs by at least about 50% from its solubility in the upper liquid.

[0029] In another related embodiment, the solubility of the target analyte in the lower liquid differs from its solubility in the upper liquid by at least about 85%.

[0030] In another aspect of the embodiments described above, the target analyte is an ionic target analyte, i.e., an analyte that is ionized at a selected pH, for example, in the range of about 6 to about 8. The ionic target analyte may consist of negatively charged or positively charged portions, each associated with a cationic counterion or an anionic counterion.

[0031] In another embodiment, the target analyte includes a biomolecule. The biomolecule may be a nucleic acid, a peptide or protein, a lipid moiety, etc. In a related embodiment, the biomolecule includes DNA. Peptides, proteins, etc., may have molecular weights in the range of about 100 daltons to about 200 kilodaltons. However, considerably larger target analytes are also conceivable, as long as the present invention is useful in combination with large nucleic acid fragments, unfragmented single-stranded or double-stranded DNA, one whole genome or two or more whole genomes, and intact cells.

[0032] In a further embodiment, the fluid composition comprises an ionic liquid, i.e., a salt that is in liquid form under conditions used for extraction.

[0033] In related embodiments, the method uses an ionic liquid for the acoustic emission of charged biomolecules, such as nucleic acids (e.g., DNA), from a fluid composition.

[0034] In another related embodiment, the ionic liquid acts as the first liquid, the aqueous liquid acts as the second liquid, and the aqueous liquid is buffered to a pH that alters the relative affinity of the ionic analyte to the ionic liquid and the aqueous liquid.

[0035] In another aspect of the embodiments described above, the method further includes, prior to step (a), distributing the fluid composition into an upper and lower region by subjecting the combination of the sample and a miscible mixture of the first and second liquids to conditions that make the two liquids substantially miscible.

[0036] In another embodiment, the droplet receiver includes an analytical instrument. In a related embodiment, the analytical instrument is a mass spectrometer.

[0037] In another embodiment of the method, step (b) of the method is repeated multiple times to inject multiple fluid droplets into a droplet receiver.

[0038] In another embodiment, the droplet receiver includes a reservoir for receiving droplets. In the relevant embodiment, step (b) of the method is repeated multiple times until at least 20 wt.% of the target analyte has been transferred from the fluid reservoir to the droplet-receiving reservoir.

[0039] In a further embodiment of the embodiment, the fluid reservoir is one of a plurality of fluid reservoirs, each containing a fluid composition containing a target analyte, wherein any two of the fluid compositions may be the same or different, and / or any two of the target analytes may be the same or different. The plurality of reservoirs may be arranged in an array and / or contained within a substrate comprising a plurality of integrated reservoir units. In a relevant embodiment, fluid droplets are acoustically ejected from an array of fluid reservoirs to a corresponding array of reservoirs that receive the droplets.

[0040] In another embodiment, the fluid composition in the fluid reservoir has a volume of about 125 μL or less.

[0041] In another embodiment, the ejected fluid droplet has a volume of about 60 nL or less.

[0042] In another embodiment, the fluid droplet has a volume of about 30 nL or less.

[0043] In a relevant aspect of this embodiment, acoustic droplet ejection is performed continuously to multiple fluid reservoirs by rapid reservoir-to-reservoir transitions of, for example, up to about 0.5 seconds, or up to about 0.1 seconds, or up to about 0.001 seconds.

[0044] In a further embodiment of the embodiment, the inner surface of the fluid reservoir is coated with a surface coating composition. In the relevant embodiment, the surface coating is selected to repel or attract the upper fluid layer, thereby altering the shape of the meniscus and the thickness of the central region of the upper fluid layer.

[0045] In another embodiment, the method further includes detecting the presence of a liquid-to-liquid boundary between an upper fluid layer and a lower fluid layer. In the relevant embodiment, fluid droplets are repeatedly ejected from the upper fluid layer until no liquid-to-liquid boundary is detected, meaning that substantially all of the upper layer can be removed from the fluid composition and the acoustic ejection process can be stopped.

[0046] Another embodiment of the present invention provides a method for extracting an ion target analyte from a sample, comprising mixing the sample with an ionic liquid and a nonionic liquid under conditions that facilitate the distribution of the ion target analyte into an ionic liquid, and acoustically removing the nonionic liquid from the mixture.

[0047] In an additional embodiment of the present invention, a method is provided for extracting an ion target analyte from a sample, comprising mixing the sample with an ionic liquid and a nonionic liquid under conditions that facilitate the distribution of the ion analyte into an ionic liquid to provide an ionic liquid solution of the ion analyte, removing the nonionic liquid from the mixture, and acoustically ejecting droplets of the ion analyte solution into a droplet receiver.

[0048] In another embodiment, the present invention provides an extraction method comprising: (a) providing an initial fluid composition in a fluid reservoir comprising an upper layer of a first liquid and a lower layer of a second liquid, wherein the analyte is present in the upper layer at a first concentration and in the lower layer at a second concentration, the second concentration being higher than the first concentration; and (b) repeatedly applying focused acoustic energy to the fluid reservoir in a manner effective for ejecting fluid droplets from the upper layer of the fluid, thereby removing at least a portion of the upper layer while allowing the lower layer to remain in the fluid reservoir.

[0049] A further embodiment of the present invention provides a method for extracting an ionic analyte from a biological sample, comprising the steps of: (a) acoustically injecting droplets of the biological sample containing the ionic analyte and an aqueous medium into an ionic liquid contained in a droplet receiving reservoir; (b) inverting the droplet receiving reservoir to form an upper aqueous layer and a lower ionic liquid layer containing the ionic analyte; and (c) removing the upper aqueous layer to provide an ionic analyte solution containing the ionic analyte in the ionic liquid. The biological sample may be a processed biological sample, such as a sample containing lysed cells.

[0050] In one embodiment, the aqueous medium includes a buffering system for maintaining the biological sample at a first pH, the first pH being selected such that at least 60 wt.% of the ion analytes in the biological sample are distributed into the ionic liquid when mixed with the ionic liquid.

[0051] In another embodiment of the embodiment, the method further includes, after step (c), step (d) mixing the ion analyte solution with a second extraction buffer having a pH selected such that at least 60 wt.% of the ion analyte in the ionic liquid is distributed into the extraction buffer.

[0052] In further embodiments, the present invention relates to a method for acoustically extracting DNA from an aqueous biological sample,

[0053] (a) A step of mixing an aqueous biological sample with an ionic liquid in a fluid reservoir under conditions effective for providing a fluid composition comprising an upper aqueous layer and a lower ionic liquid layer,

[0054] (b) A step of treating the fluid composition so that the DNA in the biological sample is distributed into the lower ionic liquid layer,

[0055] (c) The step of removing the upper aqueous layer so that the DNA solution in the ionic liquid remains in the fluid container,

[0056] (d) Mixing the DNA solution with an extraction buffer having a pH selected such that at least 60 wt.% of the DNA in the ionic liquid is distributed into the extraction buffer,

[0057] (e) A method is provided which includes the step of acoustically and continuously ejecting droplets of an extraction buffer containing DNA into a droplet receiver.

[0058] In another embodiment, the present invention provides a method for extracting lipid components from an aqueous biological sample, comprising the steps of: mixing the aqueous biological sample with an organic solvent in a fluid reservoir to provide a distributed fluid composition having an upper organic layer containing a lipid solution and a lower aqueous layer; and acoustically injecting droplets of the lipid solution into a droplet receiver.

[0059] In a further embodiment of the present invention, an acoustic extraction system for extracting an ion target analyte from a sample is provided, comprising: (a) a fluid reservoir containing a fluid composition comprising an ion target analyte and an ionic liquid; and (b) an acoustic droplet ejector having an acoustic connection to the fluid reservoir for generating acoustic radiation in an effective manner for ejecting fluid droplets from the fluid composition into a droplet receiver, the system comprising an acoustic radiation generator and focusing means for focusing the acoustic radiation to a focal point in the reservoir.

[0060] In one aspect of this embodiment, the system further includes a droplet receiver, such as an analytical instrument, such as a mass spectrometer, or a reservoir for receiving droplets.

[0061] In another aspect of this embodiment, the system includes a plurality of fluid reservoirs, each containing a fluid composition comprising an ion target analyte and an ionic liquid, wherein any two of the fluid compositions may be the same or different, and / or any two of the target analytes may be the same or different. The plurality of reservoirs may be arranged in an array and / or contained within a substrate comprising a plurality of integrated reservoir units. In relevant aspects of the embodiment, the system further includes means for positioning an injector in a continuous acoustic connection relationship with respect to each of the fluid reservoirs.

[0062] In relevant aspects of the embodiment, the target analyte includes biomolecules.

[0063] In another embodiment, a method is provided for the synthesis and acoustic extraction of reaction products from a reaction mixture. The method is as follows:

[0064] (a) A step of providing a reaction mixture in a fluid reservoir containing a first reactant, a second reactant, and a fluid medium, having a volume typically in the range of about 1 nL to about 3 mL,

[0065] (b) A step of exposing a reaction mixture to reaction conditions that induce a chemical reaction between a first reactant and a second reactant to produce a reaction product, wherein the fluid medium includes a first liquid having a first solubility for the reaction product,

[0066] (c) A step of mixing a second liquid, which is immiscible with the first liquid and has a second solubility such that the reaction product differs from the first solubility by at least 50%, into the reaction mixture, thereby providing a fluid composition having an upper layer and a lower layer containing reaction products of different concentrations,

[0067] (d) A step of applying focused acoustic energy to a fluid reservoir in a manner effective for ejecting fluid droplets containing reaction products into a droplet receiver, Includes.

[0068] In the relevant aspects of the embodiments described above, the reaction mixture further comprises a reaction catalyst. In another relevant aspect, the method involves partitioning the reaction product and catalyst into different liquids, thereby substantially separating the reaction product and catalyst.

[0069] In another related embodiment of the above-described embodiment, the reaction mixture further comprises a surfactant. In another related embodiment, the method involves partitioning the reaction product and the surfactant into different liquids, thereby substantially separating the reaction product and the surfactant.

[0070] In further embodiments, the present invention relates to a method for the synthesis of reaction products and acoustic transmission,

[0071] (a) Providing a reaction mixture in a fluid reservoir, comprising a first reactant, a second reactant, and a fluid medium, having a volume typically in the range of about 1 nL to about 3 mL;

[0072] (b) The step of exposing the reaction mixture to reaction conditions that induce a chemical reaction between the first reactant and the second reactant to produce a reaction product,

[0073] (c) A method effective for injecting fluid droplets containing reaction products into a droplet receiver, comprising the steps of applying focused acoustic energy to a fluid reservoir and This provides a method that includes [something].

[0074] In another embodiment, the present invention provides a method for determining the partition coefficient D of an analyte in a mixture of two solvents,

[0075] (a) In a fluid reservoir, a known amount X of analyte is combined with a first solvent of a first volume V1 and a second solvent of a second volume V2 that is substantially immiscible with the first solvent, and the analyte has a concentration of X / (V1+V2) in the combined first and second solvents.

[0076] The steps include forming a two-phase fluid composition having an upper layer of a first solvent and a lower layer of a second solvent, wherein the analyte has a concentration C1 in the first solvent and a concentration C2 in the second solvent,

[0077] (b) A step of acoustically ejecting droplets from the upper layer,

[0078] (c) A step of determining C1 in the ejected droplet,

[0079] (d) A step of calculating C2 from C1 according to the relation C2 = (C1V1) / V2,

[0080] (e) The step of determining the distribution coefficient D by confirming the ratio of C1 to C2. This provides a method that includes [something].

[0081] In another embodiment, the present invention relates to an acoustic method for determining the partition coefficient D of an analyte in a mixture of two solvents, where the amount of analyte may be known or unknown.

[0082] (a) In a fluid reservoir, combine an analyte, a first solvent in a first volume V1, and a second solvent in a second volume V2 that is substantially immiscible with the first solvent, thereby forming a distributed fluid composition having an upper layer of the first solvent and a lower layer of the second solvent, wherein the analyte has a concentration C1 in the first solvent and a concentration C2 in the second solvent.

[0083] (b) A step of acoustically ejecting droplets from the upper layer,

[0084] (c)(b) The step of determining C1 in the droplet ejected in (c)(b),

[0085] (d) The step of removing the upper layer from the distributed fluid composition,

[0086] (e) The step of acoustically ejecting droplets from the lower layer,

[0087] (f)(e) The step of determining C2 in the droplet ejected in (f)(e),

[0088] (g) The step of determining the distribution coefficient D by confirming the ratio of C1 to C2. This provides a method that includes [something].

[0089] In a further embodiment of the present invention, an acoustic system for extracting a target analyte from a sample,

[0090] (a) A fluid reservoir containing a fluid composition which is a reaction mixture having a volume in the range of about 1 nL to about 3 mL, comprising a first reactant, a second reactant, and a fluid medium.

[0091] (b) an acoustic droplet ejector having an acoustic connection with a fluid reservoir to generate acoustic radiation in an effective manner for ejecting fluid droplets from a fluid composition into a droplet receiver, the acoustic droplet ejector comprising an acoustic radiation generator and focusing means for focusing acoustic radiation to a focal point in the reservoir A system including this is provided.

[0092] In further embodiments, the present invention relates to a method for removing metal ions from an aqueous sample,

[0093] The steps include adding a metal extraction composition to an aqueous sample containing a target analyte and metal ions, the composition comprising (a) a positively charged crown ether, a positively charged cryptand or a combination thereof, and (b) a negatively charged counterion, and

[0094] The initial two-phase solution is heated until the two phases become miscible, thereby mixing the metal ions with the metal extraction composition in the single-phase solution.

[0095] The steps include: cooling the single-phase solution to prepare a second two-phase solution containing an upper aqueous layer and a lower layer of metal extraction composition and metal ions; This provides a method that includes [something].

[0096] In related embodiments, the present invention provides a metal extraction composition for use in the aforementioned (or other) processes, comprising a positively charged crown ether, a positively charged cryptand or a combination thereof, and an ionic liquid of a negatively charged counterion.

[0097] In another embodiment, the present invention is a method for separating liquids,

[0098] (a) Providing a fluid composition comprising an upper layer and a lower layer, wherein (i) the target analyte is present in the upper layer at a first analyte concentration and in the lower layer at a second analyte concentration, and (ii) the non-analyte components are present in the upper layer at a first component concentration and in the lower layer at a second component concentration.

[0099] (b) A method effective for ejecting fluid droplets from a fluid composition into a droplet receiver, comprising the steps of applying focused acoustic energy to a fluid reservoir and This provides a method that includes [something].

[0100] In a further embodiment, a method for separating liquids,

[0101] (a) A step of providing a sample containing a target analyte and non-analyte components in a first fluid,

[0102] (b) The step of combining the sample with a second fluid to provide a fluid composition,

[0103] (c) The step of exposing the fluid composition to mixing conditions,

[0104] (d) A step of making the fluid composition a separated fluid composition comprising an upper layer and a lower layer, wherein the target analyte has an upper analyte concentration in the upper layer and a lower analyte concentration in the lower layer, and the non-analyte components have an upper concentration in the upper layer and a lower component concentration in the lower layer, wherein (i) the lower analyte concentration and the upper analyte concentration are different, (ii) the lower component concentration and the upper component concentration are different, or (i) and (ii) are both. A method for separating liquids is provided, including the following. [Brief explanation of the drawing]

[0105] [Figure 1] Figure 1 schematically illustrates a typical method of the present invention, in which lipid components are extracted from a biological sample into an upper organic layer, and then the lipid layer is acoustically ejected.

[0106] [Figure 2] Figures 2 and 3 schematically illustrate another representative method of the present invention, in which DNA is extracted from an aqueous buffer into an ionic liquid, the buffer is then acoustically removed (Figure 2), and the DNA in the ionic liquid is then treated with an extraction buffer for further analysis (Figure 3). [Figure 3] Same as above.

[0107] [Figure 4] Figure 4 schematically illustrates the present invention's method for removing DNA from a biological sample using an ionic liquid in a two-step extraction process. [Modes for carrying out the invention]

[0108] Detailed description of the invention 1. Definitions and Terms

[0109] Unless otherwise defined, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which this invention relates. Specific terms that are particularly important to the description of this invention are defined below.

[0110] In this specification and the appended claims, the singular forms “a,” “an,” and “it” include multiple referents unless otherwise indicated by the context. Thus, for example, “a fluid” refers not only to a single fluid but also to a combination of two or more different fluids, which may be combined or not; and “a solvent” or “a liquid,” for example “a liquid ionic,” refers to a single solvent or liquid, as well as two or more solvents and two or more liquids, which may be individual or combined.

[0111] The term "radiation," used in its ordinary sense, refers to the emission and propagation of energy in the form of disturbance waveforms moving through a medium, so that energy moves from one particle in the medium to another without causing any permanent replacement of the medium itself. The radiation used in conjunction with the acoustic-based extraction method and system of the present invention is acoustic radiation.

[0112] The terms “acoustic emission” and “acoustic energy” are used interchangeably herein and refer to the emission and propagation of energy in the form of sound waves. Like other waveforms, acoustic emission can be focused using focusing means, as will be discussed below.

[0113] The terms “focusing means” and “acoustic focusing means” refer to means for focusing acoustic waves to a focal point, either by a device separated from the acoustic energy source acting like a lens, or by a spatial arrangement of acoustic energy sources that focus acoustic energy to the focal point through constructive and destructive interference. Focusing means may be as simple as a solid member with a curved surface, or they may include complex structures such as those found in Fresnel lenses that use diffraction to direct acoustic radiation. Suitable focusing means include phased array methods, which are known in the art and are described, for example, in U.S. Patent No. 5,798,779 by Nakayasu et al. and in Amemiya et al. (1997) Proceedings of the 1997 IS&T NIP13 International Conference on Digital Printing Technologies, pp. 698-702.

[0114] As used herein, the terms “acoustic connection” and “acoustically connected” refer to a state in which objects are placed in direct or indirect contact with another object in such a way that acoustic radiation can be transmitted between the objects without substantial loss of acoustic energy. When two items are acoustically indirectly connected, an “acoustic connection medium” is required to provide a medium through which acoustic radiation can be transmitted. Thus, an injector can be acoustically connected to a fluid, for example, by immersing the injector in a fluid, or by inserting an acoustic connection medium between the injector and the fluid, thereby transmitting the acoustic radiation generated by the injector into the fluid through the acoustic connection medium.

[0115] The terms “fluid reservoir” and “reservoir,” as used herein, refer to a container, chamber, or surface area for holding or containing a fluid. Thus, a fluid in a reservoir necessarily has a free surface, i.e., a surface from which droplets can be ejected. In one of its simplest forms, a reservoir may simply be a location on a solid surface that has sufficient wetting properties to hold a fluid within a localized area as a result of contact between the fluid and the surface, and the localized area acts as a reservoir.

[0116] As used herein, the term "fluid" refers to a substance that is at least partially liquid. Fluids may contain solids that are minimally, partially, or completely solvated, dispersed, or suspended. Examples of fluids, but not limited to, include aqueous liquids (including water itself and brine), aqueous solutions, non-aqueous liquids such as organic solvents, non-aqueous solutions, colloids, suspensions, emulsions, and gels. A fluid may be a biological sample fluid in which the analyte of interest is just one component of many components.

[0117] As used herein, the term “part” refers to any particular composition of a substance, such as a molecular fragment, an intact molecule (including monomeric molecules, oligomeric molecules, or polymers), or a mixture of intact molecules or other materials (for example, a mixture of DNA of different lengths and / or sequences).

[0118] Where used herein, the term “nearby” refers to the distance from the focal point of focused acoustic radiation to the fluid surface from which the droplet is ejected, and that this distance should be such that the focused acoustic radiation directed at the fluid ejects the droplet from the fluid surface, and thus those skilled in the art can select a suitable distance for any given fluid using simple, routine experimental methods. However, generally, a suitable distance between the acoustic radiation focal point and the fluid surface is in the range of about 1 to about 15 times the wavelength of the acoustic radiation in the fluid (i.e., the acoustic radiation used to eject the droplet), more typically in the range of about 1 to about 10 times that wavelength, preferably in the range of about 1 to about 5 times that wavelength.

[0119] For example, the term "substantially identical reservoirs" refers to reservoirs whose acoustic properties do not substantially deviate from each other. For instance, the acoustic attenuation of "substantially identical reservoirs" deviates from each other by no more than 10%, preferably no more than 5%, more preferably no more than 1%, and most preferably by a maximum of 0.1%. Other uses of the term "substantially" include similar definitions.

[0120] The “target analyte” (sometimes simply referred to herein as the “analyte”) in a fluid sample may be any part that is the analyte of interest. The analyte may be an atom, ion, salt, molecule, or a class of molecules having common characteristics (e.g., lipids or salts), and such molecules and molecular classes include organic compounds, inorganic compounds, and organometallic compounds. The analyte may be relevant to environmental research (e.g., water quality assessment), the pharmaceutical context, the chemical industry, the energy sector, and numerous other fields. Representative examples of analytes, but not limited to, include drugs, metabolites, inhibitors, ligands, receptors, catalysts, synthetic polymers, metals, metal ions, dyes, biocides (e.g., DDT, aldrin, tetrachlorodibenzodioxin [TCDD], etc.), carcinogens (e.g., polycyclic aromatic hydrocarbons [PCAHs]), allosteric effectors, antigens, and viruses (e.g., HIV, HPV, hepatitis A, B, C, D, E, F, or G, cytomegalovirus, Epstein-Barr virus, yellow fever, etc.). The target analyte may also be a reaction product or intermediate in a multi-step reaction. Furthermore, the target analyte may be the portion of interest in which the extraction process of the present invention involves the transfer of a fraction of the analyte from a first fluid to a second fluid. The target analyte may also be a component removed from the fluid, such as an impurity.

[0121] Often, the analytes are “biomolecules,” also referred to herein as “biological molecules,” and these terms refer to any molecular entities commonly found in cells and tissues, which exist naturally, are produced by recombination, are biologically derived, can be chemically synthesized, or can be chemically or biologically modified, in whole or in part. The terms include, for example, nucleic acids; amino acids; peptides, including oligopeptides, polypeptides, proteins, and their conjugates with non-peptide portions, such as nucleoproteins and glycoproteins; sugars, including monosaccharides, disaccharides, and polysaccharides; lipid portions; and any two or more of the aforementioned covalent or non-covalent conjugates, such as nucleoproteins, glycoproteins, lipoproteins, peptidoglycans, mucopolysaccharides, etc. Representative examples of biomolecules include enzymes, receptors, glycosaminoglycans, neurotransmitters, hormones, cytokines, cell response modifiers, such as growth factors and chemotactic factors, antibodies, vaccines, haptens, toxins, interferons, ribozymes, antisense drugs, plasmids, DNA, and RNA.

[0122] "Nucleic acids" may be nucleosides or nucleotides themselves, but may also include nucleosides and nucleotides containing, for example, protected forms thereof, as well as conventional purine and pyrimidine bases, namely adenine (A), thymine (T), cytosine (C), guanine (G), and uracil (U), and the bases are protected with protecting groups, such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, or benzoyl, as well as purine and pyrimidine analogs. Suitable analogs are known to those skilled in the art and are described in relevant documents and literature. Common analogs include, but are not limited to, 1-methyladenine, 2-methyladenine, N 6 -Methyladenine, N 6 --Isopentyl-adenine, 2-methylthio-N 6-Isopentyl adenine, N,N-dimethyl adenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil This includes uracil, 5-(carboxyhydroxymethyl)uracil, 5-(methyl-aminomethyl)uracil, 5-(carboxymethylaminomethyl)uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1-methylpsoiduracil, queosine, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, and 2,6-diaminopurine. Furthermore, the terms "nucleoside" and "nucleotide" include portions that contain not only conventional ribose and deoxyribose sugars but also other sugars. Modified nucleosides or nucleotides include modifications on the sugar moiety, for example, one or more hydroxyl groups may be replaced with halogen atoms or aliphatic groups, or functionalized with ethers, amines, etc.

[0123] Nucleic acids also include oligonucleotides, and for the purposes of this invention, the term “oligonucleotide” is a comprehensive term encompassing polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone. Therefore, oligonucleotide analytes as used herein may include substitutions of one or more naturally occurring nucleotides by oligonucleotide modifications, e.g., analogs; internucleotide modifications, e.g., those having uncharged links (e.g., methylphosphonates, phosphotriesters, phosphoramidic acids, carbamimates, etc.); those having negatively charged links (e.g., phosphorothioates, phosphorodithioates, etc.); and those having positively charged links (e.g., aminoalkylphosphoramides and aminoalkylphosphotriesters); those containing pendant moieties, e.g., proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those containing intercalators (e.g., acridine, psoralens, etc.); and those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.). No lengthy distinction is intended between the terms "polynucleotide" and "oligonucleotide," and these terms are used interchangeably. These terms refer only to the primary structure of the molecule. Where used herein, nucleotide and polynucleotide symbols follow the IUPAC-IUB Commission of Biochemical Nomenclature recommendations (Biochemistry 9:4022, 1970).

[0124] A "peptide" analyte (or "peptidogenic" analyte) encompasses any structure composed of one or more amino acids, and therefore includes peptides, dipeptides, oligopeptides, polypeptides, and proteins. The amino acids that form all or part of a peptide analyte are the 20 conventional naturally occurring amino acids, namely alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y), as well as non-conventional amino acids, such as isomers and modifications of conventional amino acids, e.g., D-A The peptide analyte may be any amino acid, non-protein amino acid, post-translationally modified amino acid, enzymatically modified amino acid, β-amino acid, constructs or structures designed to mimic amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and norleucine), and any other unconventional amino acid as described, for example, in U.S. Patent No. 5,679,782 by Rosenberg et al. The peptide analyte may also contain non-peptide backbone linkages in which naturally occurring amide-CONH- linkages are replaced at one or more sites in the peptide backbone by unconventional linkages, such as N-substituted amides, esters, thioamides, retropeptides (-NHCO-), retrothioamides (-NHCS-), sulfonamides (-SO2NH-), and / or peptoid (N-substituted glycine) linkages. Therefore, peptide analytes may include pseudo-peptides and peptide mimetic drugs.Peptide analytes may be produced by (a) naturally occurring, (b) chemically synthesized, (c) recombinant DNA technology, (d) biochemical or enzymatic fragmentation of larger molecules, (e) by methods obtained from combinations of the methods (a) to (d) listed above, or (f) by any other means for producing peptides.

[0125] "Sugar" or "sugar analyte" includes, but is not limited to, monosaccharides, disaccharides, oligosaccharides, polysaccharides, mucopolysaccharides, or peptidoglycans (peptide-polysaccharides), pseudopeptidoglycans, etc. Monosaccharides, including monosaccharide units in disaccharides, oligosaccharides, polysaccharides, etc., include hexoses, pentoses, and tetroses, which may be in D- or L-form, and the glycosidic linkages between monosaccharide units may be either α-glycosidic linkages or β-glycosidic linkages. Exemplary examples of sugar analytes include the monosaccharides fructose, glucose, dextrose, galactose, mannose, ribose, deoxyribose, allose, fucose, rhamnose, erythrose, threose, and glyceraldehyde; the disaccharides sucrose, lactose, maltose, lactulose, trehalose, and cellobiose; the polysaccharides amylose, amylopectin, glycogen, cellulose, chitin, callose, laminarin, chrysolaminarin, xylan, and galactomannan; and the mucopolysaccharides (also called glycosaminoglycans) chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and hyaluronan.

[0126] "Lipids" or "lipid analytes" refer to hydrophobic or amphiphilic molecules, which include a broad classification of fatty acids, phospholipids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids (saccharolipids), and polyketides. Representative examples of lipid materials, though not limited to them, include phospholipids, such as phosphorylated diacylglycerides, particularly diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylserine, diacylphosphatidylinositol, diacylphosphatidylglycerol, diacylphosphatidic acid, and mixtures thereof (each acyl group contains about 10 to about 22 carbon atoms and is saturated or unsaturated); fatty acids, such as isovaleric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid; and lower fatty acid esters, including esters of the aforementioned fatty acids (where the carboxylic acid group -(CO)-OH of the fatty acid is replaced by the ester moiety -(CO)-OR). Oils include: a C1-C3 alkyl moiety where R is optionally substituted with one or two hydroxyl groups; fatty alcohols corresponding to the aforementioned fatty acids (where the carboxylic acid group of the fatty acid is replaced by a -CH2OH group); glycolipids, e.g., cerebrosides and gangliosides; animal oils, e.g., liver oil and herring oil; and vegetable oils, e.g., babassu oil, castor oil, corn oil, cottonseed oil, linseed oil, mustard oil, olive oil, palm oil, palm kernel oil, peanut oil, poppy seed oil, rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower seed oil, tung oil, or wheat germ oil; and waxes, e.g., animal waxes, e.g., beeswax, lanolin and shellac wax; mineral waxes, e.g., montan wax; petroleum-derived waxes, e.g., microcrystalline wax and paraffin wax; and vegetable waxes, e.g., carnauba wax and candelilla wax.

[0127] "Extraction" and "to extract," as used herein, refer to a process involving the migration of a target analyte from a first fluid to a second fluid, and thus encompass the separation process as described in the preceding section. The term typically refers to the enhancement of a target analyte, which is one component of a composition, to other components of the composition, for example, an impurity in one of the two fluid phases. Extraction may be complete, meaning that the target analyte is completely separated from the other components of the sample, and so that after extraction one of the fluid phases contains 100% of the target analyte. Extraction may also be partial, in which case a fraction less than 100% of the target analyte is isolated from the other components of the composition. Accordingly, extraction using the method of the present invention may aim to increase or decrease the concentration of a target analyte in one of the fluids, remove some or all of the analyte from one of the fluids, concentrate the amount of analyte in one of the fluids, isolate the analyte, purify the analyte, remove components initially associated with the target analyte, such as impurities, or a combination of two or more of the above. “Liquid-liquid extraction,” as the term is used herein, refers to an extraction process in which the first fluid and the second fluid are independently selected from a fluid containing a liquid, and thus liquid-liquid extraction includes gel-liquid extraction, suspension-liquid extraction, etc. The extraction process is connected to an acoustic injection process such that fluid droplets are acoustically ejected from a fluid having an increased or decreased concentration of the target analyte (relative to the initial concentration of the target analyte in the sample or in the fluid layer or fluid composition prior to the initial extraction) or an increased or decreased concentration of a non-target component (relative to the initial concentration of the non-target component in the sample or in the fluid layer or fluid composition prior to the initial extraction).

[0128] References to samples that "contain" or "include" an analyte include both samples that are known to contain an analyte but whose identification may be unknown, and samples that are suspected to contain an analyte.

[0129] As used herein, the term "array" refers to a two-dimensional arrangement of features, such as an arrangement of reservoirs (e.g., wells in a well plate), or an arrangement of fluid droplets or molecular portions on a substrate surface (such as an oligonucleotide or peptide array). Arrays generally consist of features regularly aligned, for example, in a linear grid, parallel stripes, spiral, etc., but unaligned arrays can also be used to their advantage. Arrays differ from patterns in that patterns do not necessarily contain regularly aligned features. Furthermore, as provided herein, arrays and patterns formed by the deposition of ejected droplets onto a surface are usually substantially invisible to the naked eye. Arrays typically contain, but not necessarily, at least about 4 to about 10,000,000 features, and generally in the range of about 4 to about 1,000,000 features.

[0130] 2.Extraction method

[0131] The present invention utilizes acoustic droplet injection (ADE) for the extraction of a target analyte from a fluid composition. In one embodiment, ADE is performed in the extraction process to generate fluid droplets containing a target analyte at a selected concentration. The fluid composition containing the target analyte is provided in a fluid reservoir, and the fluid composition consists of two or more phases. That is, the fluid composition comprises an upper region or upper layer and one or more lower regions or lower layers, and for example, the fluid composition may consist of two, three, four, or five or more layers. For brevity, a method for a two-phase system is described in which the fluid composition comprises an upper layer of a first fluid and a lower layer of a second fluid, and the first and second fluids are substantially immiscible under the conditions used for extraction. The target analyte in the fluid composition is present in the first fluid at a first concentration and in the second fluid at a second concentration different from the first concentration. Generally, the first and second concentrations differ by at least about 50%, for example, at least about 85%, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Focused acoustic energy is applied to the fluid reservoir in a manner effective in ejecting fluid droplets from the fluid composition, generally toward a droplet receiver. The selected concentration in the ejected fluid droplet, i.e., the concentration of the target analyte, is substantially equivalent to either the first or second concentration. Furthermore, the selected concentration is substantially uniform across the droplet. In one example, a fraction of the target analyte moves from one fluid (e.g., a fluid containing a biological sample, e.g., a cell solubilizer) to the second fluid. In another example, the concentrations of the target analyte in the two fluids may change or not change, but the target analyte and related components in one fluid may not be present in the second fluid after the extraction process. For example, after extraction, one fluid may contain the target analyte and several impurities, while the second fluid may contain the target analyte and impurities that are absent or substantially low in concentration. Another example is an extraction process that involves varying the concentrations of the target analyte or other components in one or more fluid layers.In a variation of the example described above, the extraction involves two target analytes, and the process increases the concentration of one target analyte in one or more fluid layers while decreasing the concentration of the other target analyte.

[0132] Since the method of the present invention is effective when using very small sample sizes, the total fluid composition in the reservoir generally occupies a volume of about 125 μL or less, for example, about 60 μL or less, about 45 μL or less, about 30 μL or less, etc.

[0133] The fluid composition may include a sample containing the target analyte, such as a biological sample. The biological sample may include a sample dissolved or suspended in the fluid, or the biological sample itself may be a fluid. Examples of biological samples include tissues, tissue homogenates, cells, cell suspensions, cell extracts, whole blood, plasma, serum, saliva, sputum, nasal secretions, cerebrospinal fluid, interstitial fluid, lymph, semen, vaginal fluid, or feces. More typical biological samples consist of tissues, cells, or blood. The biological sample may be processed in a certain manner before extraction and may not be well-processed. Preliminary processing methods are known in the art and include, for example, incorporating anticoagulants into blood samples, separating blood into plasma and serum, alternating centrifugation and resuspension procedures according to different sample types, and incorporating preservatives or transport media containing preservatives into the sample. However, the present invention is not limited thereto and can be easily carried out using biological samples and non-biological samples that have not undergone any preliminary processing.

[0134] Target analytes in biological samples include, but are not limited to, proteins, peptides, peptide fragments, lipid compounds, and nucleic acids, particularly DNA. Other biological target analytes and other types of target analytes are described in Part (1) “Definitions and Terminology” of this section.

[0135] The extraction of a target analyte using two or more fluids depends on the difference in affinity of the analyte to one fluid for the other. The term "affinity" includes any factor or combination of factors that cause the analyte to be distributed to one fluid for the other. Examples of such factors, but not limited to, include the compatibility of the analyte and fluid in terms of polarity, ionic interactions between the analyte and fluid, hydrogen bonding between the analyte and fluid, the relative hydrophilicity or hydrophobicity of the analyte and solvent, and, more generally, the degree of solubility of the analyte in the fluid, which is not related to the factors mentioned above.

[0136] In some embodiments, the extraction process of the present invention uses two liquids with different solubility of the target analyte, as previously described, where the difference in solubility corresponds to a difference between a first and a second concentration. The difference in solubility may be at least about 50%, for example, at least about 85%, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. In some embodiments, the difference in solubility may be less than about 50%. In other embodiments, as previously mentioned, the difference in solubility arises from the use of a relatively hydrophobic first liquid and a relatively hydrophilic second liquid, so that the hydrophilic portion is preferentially distributed to the hydrophilic liquid, while the hydrophobic portion is preferentially distributed to the hydrophobic liquid. Such a system is useful, for example, when using a fluid composition containing a sample that includes multiple components having different hydrophobicities, for example, a target analyte having a different hydrophobicity than other components also present in the sample.

[0137] In other embodiments, the target analyte may be a polar molecule or salt, in which case the relatively polar solvent may be either protic or aprotic and is combined with a relatively nonpolar solvent in the extraction process. When a target that undergoes hydrogen bonding is used, a protic solvent is useful, in which case the protic solvent is combined with an aprotic solvent which may or may not be nonpolar.

[0138] Furthermore, some analytes, including, but not limited to, certain ionic analytes, can be readily extracted with ionic liquids, as described below. In some cases, ionic liquids are used to extract ionic analytes from another fluid composition. In other cases, ionic liquids are used to preferentially distribute the ionic analyte into a second fluid composition that is not an ionic liquid. In some cases, both of these processes are combined into a multi-stage extraction process, as described in more detail below.

[0139] A wide variety of solvents can be used in combination with the present invention. In fact, virtually any solvent can be used, provided that it does not have a harmful effect on the extraction process of the present invention. Naturally, non-toxic solvents are preferred. The liquid used in a single extraction step or process may vary in viscosity, volatility, and other chemical and physical properties. Extraction of a target analyte from a viscous material is facilitated by using a relatively non-viscous fluid. If one of the solvents is volatile, concerns about evaporation can be eliminated by using a second solvent with a lower density and lower volatility, where the less volatile, lower-density solvent forms an upper layer over the more volatile lower solvent, preventing its evaporation.

[0140] Solvents that can be used in the methods and systems of the present invention include aqueous and organic solvents, protic and aprotic solvents, ionic and nonionic liquids, polymers and nonpolymeric liquids, and the like. The fluids, in combination, can form single-phase, two-phase, three-phase, or more multi-phase fluid compositions, and when a combination of two fluids is used, the resulting fluid composition is single-phase or two-phase. In some embodiments, the present invention uses the degree to which the selected fluids are miscible and the conditions under which the selected combination of liquids can be made more or less miscible.

[0141] Examples of solvents useful in combination with the present invention include, but are not limited to, acetic acid, acetone, acetonitrile, ammonia, benzene, n-butanol, i-butanol, 2-butanol, t-butanol, 2-butanone, butyl acetate, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, cyclohexanone, cyclopentane, 1,2-dichloroethane, dichloromethane, diethyl ether, diethylene glycol, diethylene glycol dimethyl ether (diglym), di-isopropyl ether, 1,2-dimethoxyethane (grim, DME), dimethyl ether, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, ethyl formate, formic acid, furan, glycerin, and heptane. Examples include hexamethylphosphoramide (HMPA), hexamethylphosphite triamide (HMPT), n-hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, methyl ethyl ketone (MEK), methyl formate, N-methylpyrrolidinone (NMP), nitromethane, 1-octanol, n-pentane, petroleum ether (ligroin), piperidine, polyethylene glycol, n-propanol, i-propanol, pyridine, tetrahydrofuran (THF), toluene, trichloroethylene, triethylamine (TEA), water, heavy water, o-xylene, m-xylene, p-xylene, and other solvents described in Ellson et al.'s U.S. Patents 6,548,308 and 6,642,061, which are incorporated herein by reference. Preferred aqueous solvents include water, aqueous solutions of buffer compounds and / or salts, such as phosphate buffers, Tris buffers, MES buffers, HEPES buffers, and ammonium bicarbonate.

[0142] Specific examples of useful solvent systems and solvent combinations described herein, but not limited to, include aqueous solvents, e.g., water or buffer and organic solvents, e.g., cyclohexane, dichloromethane, 1-octanol, n-pentane, n-butanol, or totally fluorinated or hemifluorinated alkane solvents, e.g., perfluoroheptane, 1,1,1,2,3,3-hexafluoropropane, pentafluoropentane, etc.; aqueous solvents, e.g., water or buffer and lipid solvents, e.g., oil; and two-component systems using two aqueous layers (aqueous liquids containing different solutes and being immiscible).For example, see Partitioning in Aqueous Two-Phase System: Theory, Methods, Uses, and Applications to Biotechnology, Eds. Harry Walter et al. (Academic Press, 1985), Hamta et al. (2017), "Application of polyethylene glycol based aqueous two-phase systems for extraction of heavy metals," Journal of Molecular Liquids 231:20-24, and Eiden et al. (2016), "Two-Phase System Rehydration of Antibody-Polymer Microarrays Enables Convenient Compartmentalized Multiplex Immunoassays," Analytical Chemistry 88(23). (Also see Tavana et al. (2010) Adv. Mater. 22(24):2628-2631 and Fang et al. (2012) Tissue Engineering Part C: Methods.) See 18)9):647-657; for example, combinations of two aqueous liquids having the same solute but different pH levels, such as two aqueous liquids having the same buffering composition but the first and second aqueous liquids being buffered to different pH levels; as well as two-component systems using two organic layers in which the organic liquids are immiscible, such as ethanol / cyclohexane, hexane / dichloromethane, ether / chloroform, etc.

[0143] The two fluids selected for the extraction process should be immiscible under the conditions used for extraction. Miscibility, as understood in the art, refers to the degree to which one fluid is soluble in the other. This solubility may vary with temperature, hydrostatic pressure, or other factors, and this variable can be advantageously incorporated into the process of the present invention. That is, the fluid composition can be subjected to conditions that induce a phase transition and make the two fluids miscible, so that the target analyte is thoroughly mixed with both fluids. Subsequently, the fluid composition can be subjected to conditions that return the two fluids to an immiscible state, so that acoustic energy can target a focal zone in one of the two fluid layers and eject a fluid droplet formed from and containing primarily only one of the two layers. The miscibility of solvents under various conditions can be determined by referring to relevant documents and literature, and / or empirically by combining two solvents and observing the degree of miscibility at various temperatures, pressures, etc. Most commonly, the miscibility of two fluids as described herein changes with temperature (and thus, their miscibility changes with an increase or decrease in the temperature of a fluid composition containing the two fluids) and / or is chemically altered by the addition of, for example, salt, such as sodium chloride, or by a change in pH. In some cases, the two fluids used in the extraction process of the present invention are miscible between about 40°C and about 90°C.

[0144] It is understood that the target analyte and the two fluids may be mixed before extraction using other techniques, such as repeated inversion of the container holding the analyte and fluids, stirring, sonication, agitation, temperature relaxation of the fluids, or injection of droplets of one fluid through the other fluid (e.g., acoustic injection) (as illustrated in Figure 2 discussed below).

[0145] In one embodiment, the extraction method uses an ionic liquid. As shown in Part (1) of this section, an ionic liquid contains a salt in liquid form. That is, an ionic liquid is composed mostly or entirely of ions, in contrast to ordinary liquids which are made up mainly from electronically neutral species (i.e., “substantially ionic” means that substantially all ionic liquids are ionic). Preferred ionic liquids for use with the present invention are those that are purely ionic or substantially ionic and are in liquid form under extraction conditions. A more preferred ionic liquid is a “room temperature ionic liquid” (RTIL). An RTIL consists of a salt that has a relatively low melting point and is in liquid form at temperatures below 100°C, e.g., in the range of about 0°C to about 100°C. RTILs are preferred herein insofar as they provide the convenience of facilitating an extraction process that can be carried out without changing the temperature of the fluid composition. Ionic liquids are useful for the extraction of ionic analytes, such as negatively charged analytes, such as DNA, but are equally useful for the extraction of other types of analytes. Common ionic liquids include, but are not limited to, imidazolium salts, pyrrolidinium salts, piperidinium salts, pyridinium salts, morpholinium salts, ammonium salts, phosphonium salts, sulfonium salts, and guanidium salts. Suitable ionic liquids are listed, for example, in the Ionic Fluid Catalog published by Sigma-Aldrich (October 2012) and EMD Chemicals Inc., and can be obtained as commercially available products. Specific examples of ionic liquids are provided below. In the following list, the anion abbreviations are as follows:

[0146] Acetate (CH3COO - ), OAc;

[0147] Bis[oxalate(2-)]borate, bob;

[0148] Bis(trifluoromethylsulfonyl)imide, Tf2N;

[0149] Dicyanamide, DCA;

[0150] Formate (HCO2 - ), HCOO;

[0151] Hexafluorophosphate, PF6 - ;

[0152] Bisulfate, HSO4 - ;

[0153] Hydroxyacetate (CH2(OH)COO - ), HOOAc;

[0154] Methanesulfonate (mesylate), CH3SO3 - ;

[0155] Bisulfate of 2-(methoxyethoxy)ethyl, CH3(OCH2CH2)2OSO4 - ;

[0156] Methyl sulfate (CH3-O-SO3 - ), MeSO4;

[0157] Octyl sulfate (C8H7SO4 - ), OcSO4;

[0158] Nitrate, NO3 - ;

[0159] Sulfamate, H2NSO3<着 - ;

[0160] Tetracyanoborate, B(CN)4 - ;

[0161] Tetrafluoroborate, BF4 - ;

[0162] Thiocyanate, SCN - ;

[0163] p-Toluenesulfonate, or tosylate, Tos;

[0164] It should be noted that there seems to be an incorrect tag "着0000014" in the original text which is maintained as is in the translation. Hope this meets your requirements. If you have any further questions, feel free to ask.Tricyanomethane (C(CN)3 - ), TCM;

[0165] Trifluoroacetate (CF3COO - ), TFA;

[0166] Trifluoromethanesulfonate (Triflate, CF3SO3) - ), OTf; and

[0167] Tris(pentafluoroethyl)trifluorophosphate, FAP.

[0168] Representative ionic liquids (the first species listed are cations, the second species are anions, and the plus and minus signs are omitted to conform to standard ionic liquid nomenclature) include the following:

[0169] 1-benzylmethylimidazolium (Zmim) salts, e.g., [Zmim][Cl];

[0170] N,N-bis(2-hydroxyethyl)butylammonium (HEBA) salts, e.g., [HEBA][Tf2N] and [HEBA][HCOO];

[0171] Bis(2-hydroxyethyl)ammonium (HEA) salts, e.g., [HEA][TFA] and [HEA][OAc];

[0172] Bis(2-methoxyethyl)ammonium (MEA) salts, e.g., MEA sulfamate;

[0173] 1-butyl-2,3-dimethylimidazolium (Bmmim) salts, e.g., [Bmmim][Cl], [Bmmim][I], [Bmmim][PF6], [Bmmim][BF4], and [Bmmim][OTf];

[0174] 1-butyl-3-methylimidazolium (Bmim) salts, e.g., [Bmim][Tf2N], [Bmim][Cl], [Bmim][Br], [Bmim][I], [Bmim][DCA], [Bmim][PF6], [Bmim][HSO4], [Bmim][MeSO4], [Bmim][OcSO4], [Bmim][BF4], [Bmim][B(CN)4], [Bmim][Tos], [Bmim][TCM], [Bmim][TFA], [Bmim][NO3], and [Bmim][OAc];

[0175] 1-butyl-3-methylpyridinium (B3mpy) salts, e.g., [B3mpy][Cl], [B3mpy][DCA], [B3mpy][MeSO4], and [B3mpy][BF4];

[0176] 1-butyl-4-methylpyridinium (B4mpy) salts, e.g., [B4mpy][Cl] and [B4mpy][BF4];

[0177] 1-butyl-1-methylpyrrolidinium (Bmpyr) salts, e.g., [Bmpyr][bob], [Bmpyr][Tf2N], [Bmpyr][Cl], [Bmpyr][DCA], [Bmpyr][OTf], [Bmpyr][FAP], and [Bmpyr][B(CN)4];

[0178] N-butylpyridinium chloride (Bpy) salts, e.g., [Bpy][Cl], [Bpy][PF6], [Bpy][BF4], and [Bpy][OTf];

[0179] N,N-dimethyl(2-hydroxyethyl)ammonium (MMHEA) salts, e.g., [MMHEA][HOOAc], [MMHEA][Tf2N], and [MMHEA][TFA];

[0180] 1,3-dimethylimidazolium (Mmim) salts, e.g., [Mmim][Cl], [Mmim][Br], and [Mmim][MeSO4],

[0181] 1,1-dimethylpyrrolidinium (MMpyr) salts, e.g., [MMpyr][I] and [MMpyr][Tf2N];

[0182] N-dodecyl-N,N-dimethyl-3-sulfopropylammonium Tf2N and OTf;

[0183] 1-(2-ethoxyethyl)-1-methylpyrrolidinium (EOEMpyr) salts, e.g., [EOEMpyr][Tf2N], [EOEMpyr][Br], [EOEMpyr][BCN4,], [EOEMpyr][Tf2N], and [EOEMpyr][FAP];

[0184] 1-ethyl-2,3-dimethylimidazolium (Emmim) salts, e.g., [Emmim][Br], [Emmim][Cl], [Emmim][MeSO4], and [Emmim][BF4];

[0185] N-ethyl-N,N-dimethyl-2-methoxyethylammonium Tf2N, Br, B(CN)4, and FAP;

[0186] N-ethyl-N,N-dimethylpropylammonium Tf2N, Br, DCA, bis(trifluoromethylsulfonyl)imide (Nemmp tfn), bromide (Nemmp Br), dicyanamide (Nemmp DCN), B(CN)4, and FAP;

[0187] 1-ethyl-3-methylimidazolium (Emim) salts, e.g., [Emim][bob], [Emim][Tf2N], [Emim][Br], [Emim][Cl], [Emim][DCA], [Emim][HSO4], [Emim][MeSO4], [Emim][OcSO4], [Emim][B(CN)4], [Emim][BF4], [Emim][TFA], and [Emim][OTf];

[0188] Guanidium (gua) salts, e.g., [gua][OTf] and [gua][FAP];

[0189] 1-Hexadecyl-2,3-dimethylimidazolium (Cmmim) salt, e.g., [Cmmim][Cl];

[0190] 1-Hexadecyl-3-methylimidazolium (Cmim) salts, e.g., [Cmim][Cl] and [Cmim][FAP];

[0191] 1-Hexyl-1-methylpyrrolidinium (Hmpyr) salts, e.g., [Hmpyr][Tf2N], [Hmpyr][FAP], and [Hmpyr][Cl];

[0192] 1-Hexyl-2,3-dimethylimidazolium (Hmmim) salts, e.g., [Hmmim][Cl] and [Hmmim][FAP];

[0193] 1-Hexyl-3-methylimidazolium (Hmim) salts, e.g., [Hmim][Tf2N], [Hmim][Cl], [Hmim][PF6], [Hmim][BF4][Hmim][OTf], and [Hmim][FAP];

[0194] N-hexylpyridinium (HPy) salts, e.g., [HPy][Cl], [HPy][Tf2N], [HPy][OTf], and [HPy][FAP];

[0195] 1-(2-hydroxyethyl)-3-methylimidazolium (HOE-Mim) salts, e.g., [HOE-Mim][Tf2N], [HOE-Mim][Cl], [HOE-Mim][Br], [HOE-Mim][OTf], and [HOE-Mim][FAP];

[0196] N-(3-hydroxypropyl)pyridinium (HOP-Py) salts, e.g., [HOP-Py][Tf2N], [HOP-Py][Cl], [HOP-Py][Br], [HOP-Py][B(CN4)], and [HOP-Py][FAP];

[0197] 1-(3-methoxypropyl)-1-methylpiperidinium (MOPMpi) salts, e.g., [MOPMpi][Tf2N], [MOPMpi][Cl], [MOPMpi][Br], [MOPMpi][B(CN4)], and [MOPMpi][FAP];

[0198] 1-Methylimidazolium (Mim) salts, e.g., [Mim][BF4];

[0199] Methyltrioctylammonium [MOc3A] salts, e.g., [MOc3A][Tf2N], [MOc3A][TFA], and [MOc3A][OTf];

[0200] 1-Octyl-3-methylimidazolium (Omim) salts, e.g., [Omim][Cl], [Omim][I], [Omim][BF4], and [Omim][FAP];

[0201] 1-Octyl-1-methylpyrrolidinium (OMpyr) salts, e.g., [OMpyr][Cl];

[0202] 1-Propyl-3-methylimidazolium (Pmim) salt, e.g., [Pmim][I];

[0203] 1-(3-sulfopropyl)-3-butylimidazolium Tf2N and OTf;

[0204] N-(3-sulfopropyl)-pyridinium Tf2N and FAP;

[0205] Tetrabutylammonium (NB4) salt, e.g., [NB4][Tf2N];

[0206] Tetramethylammonium (Nm4) salts, e.g., [Nm4][bob] and [Nm4][FAP];

[0207] Trihexyl(tetradecyl)phosphonium(P(h3)t) salts, e.g., [P(h3)t][bob], [P(h3)t][Tf2N], [P(h3)t][PF6], [P(h3)t][BF4], [P(h3)t][DCA], and [P(h3)t][FAP];

[0208] 1,2,3-trimethylimidazolium (Mmmi) salts, e.g., [Mmmi][I];

[0209] 2-amino-1,6-dimethylimidazo[4,5-b]-pyridine salts, e.g., 2-amino-1,6-dimethylimidazo[4,5-b]-pyridineTf2N; and

[0210] Triethyl-hexadecylphosphonium (THP) salts, e.g., [THP][DCN]. In this specification, [Bmim][Tf2N], [Bmim][OAc], [B3mpy][Tf2N], [B4mpy][Tf2N], [MOc3A][Tf2N], [MMpyr][Tf2N], and [P(h3)t][DCA] are of particular interest.

[0211] Polymeric ionic liquids can also be used in combination with the present invention. Polymeric ionic liquids are known in the art and are described, for example, in Shaplov et al. (2011), "Polymeric Ionic Liquids: Comparisons of Polycations and Polyanions," Macromolecules 44(24):9792-9803, Wu et al. (2017), "Polymerizable ionic liquids and polymeric ionic liquids: facile synthesis of ionic liquids containing ethylene oxide repeating unit via methanesulfonate and their electrochemical properties," RSC Advances 7: 5394-5401, and Mecerreyes et al. (2011), "Polymeric ionic liquids: Broadening the properties and applications of polyelectrolytes," Progress in Polymer Science 36(12): 1629-1648. The thermoresponsive poly(ionic liquid)-based nanogels described in Zhang et al. (2015) Molecules 20:17378-17392 are also suitable for use herein, and their preparation is described in that publication.

[0212] It should be understood that the above list of ionic liquids is merely illustrative and not intended to be limiting. Other ionic liquids useful herein include those described in Plechkova et al. (2008) Chem. Soc. Rev. 37:123-150, Branco et al., "Physico-Chemical Properties of Task-Specific Ionic Liquids," in Ionic Liquids: Theory, Properties, New Approaches, Ed. A. Korkorin (Intech, 2011), and Plechkova et al. (2015), in Ionic Liquids Completely Uncoiled (Wiley, 2015). Further ionic liquids useful in conjunction with the methods of the present invention are described elsewhere in the literature and / or will be apparent to those skilled in the art. The ionic liquids generally preferred for use herein provide a fluid composition that allows for the extraction of a significant fraction of the target analyte from a sample (about 50% or more, e.g., 50%–100%, 50%–95%, 50%–85%, 50%–75%, etc.) and / or the concentration of at least one non-target component (e.g., at least one impurity) originally associated with the target analyte is reduced in one of the fluid layers (e.g., reduced by at least 50%, e.g., 50%–100%, 50%–95%, 50%–85%, 50%–75%, etc.). The preferred ionic liquids are relatively non-toxic, easy to use in the laboratory, and have a high affinity for the fluid reservoir surface to concentrate the aqueous extraction fluid towards the reservoir center. In some cases, it may be desirable that the selected ionic liquid has an auditable acoustic impedance. Furthermore, in some cases, an ionic liquid that exhibits extraction bias based on analyte size is preferred, while in other cases, the preferred ionic liquid is one that does not exhibit extraction bias related to analyte size (typically when using nucleic acid analytes, such as DNA).

[0213] In some embodiments, the ionic liquid used in the extraction process of the present invention is a magnetic ionic liquid. The magnetic ionic liquid acts as a liquid form of magnetic beads, and the magnetic ionic liquid containing the target analyte of interest can be attracted to one side of the fluid reservoir so that the depleted non-ionic (e.g., aqueous) layer can be easily removed. Magnetic ionic liquids (MILs) are known in the art and described in the literature. See, for example, Clark et al. (2015) Anal. Chem. 87:1552-1559. As described in that literature, examples of MILs include benzyltrioctylammonium bromotrichloroferrate(III) and 1,12-di-(3-hexadecyl-benzimidazolium)dodecambis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III).

[0214] In one embodiment, the method is used for the extraction of biomolecules from a biological sample, the biomolecules being preferentially distributed to a first liquid relative to a second liquid, and after the mixing and subsequent distribution are complete, one of the two phases is removed using an acoustic injection technique. For example, as will be described in more detail below, the acoustic injection technique can be implemented to rapidly and continuously eject droplets from the upper fluid layer containing the analyte from a fluid reservoir, and the fluid droplets containing the analyte are ejected into a droplet reservoir for further processing and / or analysis. The acoustic injection technique can also be implemented to repeatedly eject droplets from the upper fluid layer that do not contain the analyte, thereby removing the upper fluid containing the non-analyte from the fluid reservoir, leaving the analyte in the lower fluid layer.

[0215] An example of such a method is illustrated in Figure 1. In Figure 1, a biological sample is provided in a fluid container. The sample is a mixture of multiple components in an aqueous fluid. A second fluid is added to remove a component from the sample because at least one component preferentially selects the second fluid. The upper layer can then be removed from the fluid container by decanting, or more preferably by repetitive acoustic ejection using a focused acoustic ejection system, for example, as described below. In the specific example illustrated in Figure 1, it is desirable to remove lipid components from the biological sample. Therefore, the “first fluid” is the aqueous sample itself, for example, 50 μL of a cell assay as shown in the figure, containing nucleic acids, proteins, lipids and other components (the term “cell assay” is used herein to refer to a cell sample that may be partially or untreated, e.g., a cell solubilizer). Since lipids are much more soluble in non-polar or low-polarity solvents, the selected “second fluid” is an organic solvent, for example, 10 μL of diethyl ether. After this extraction process, the lipid-containing ether layer at the top of the aqueous layer can be removed as described above.

[0216] Interestingly, as can be seen from Figure 1, combining two solvents with different properties in a selected fluid reservoir can result in meniscus inversion. That is, the first fluid has a concave meniscus before the addition of the second fluid, but upon addition of the second fluid, this concave meniscus transforms into a convex shape at the fluid-fluid boundary. This can be desirable in several cases. For example, using acoustic radiation focused to a central point within the fluid container, the lower fluid can be ejected through a central "liquid opening" without (or with minimal) contact with the upper fluid layer. This allows for the omission of the upper layer removal step, streamlining the overall extraction process. Meniscus inversion in this manner can be achieved by using a fluid reservoir with an inner surface that attracts or repels specific types of fluids, such as organic solvents, aqueous liquids, ionic liquids, etc. Fluid reservoirs with such inner surfaces can be commercially available, for example, as microwell plates with various types of coatings obtained from numerous sources.

[0217] Another example of the extraction process of the present invention relates to the extraction of biomolecules that are preferentially distributed to an ionic liquid over a nonionic liquid. Such important biomolecules are DNA, which may or may not be double-stranded DNA (dsDNA). The first step of such a method is schematically shown in Figure 2. A biological sample can be initially treated by lysing the cells and / or tissue material in the sample and then resuspending the sample in a suitable aqueous buffer (e.g., Tris buffer), which is referred to herein as the “initial buffer.” The biological sample may also contain circulating cell-free DNA. When performed in a microwell plate, e.g., a 384-well plate, each well acts as a fluid reservoir in which the extraction takes place, so a known amount of ionic liquid is added to each of the multiple wells. The ionic liquid is selected to have a strong affinity for DNA, and the initial buffer is selected so that DNA is preferentially distributed to the ionic liquid, i.e., preferentially compared to the buffer. As illustrated in Figure 2, the method may involve adding 5 to 30 μl, for example 25 μl, of an ionic liquid, such as [Bmim][PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) to each individual well. The wells are preferably filled with the ionic liquid by acoustically ejecting droplets upward into the well using focused acoustic ejection technique, so the individual wells illustrated in the figure are shown in an inverted configuration. Alternatively, wells containing a small amount of ionic liquid can simply be inverted, and surface tension will hold the ionic liquid in place. Next, an aqueous buffer containing DNA is introduced into each well, preferably again using focused acoustic droplet ejection. The volumes of the two liquids may or may not be the same; in the figure, both volumes, i.e., the volume of the ionic liquid and the volume of the aqueous buffer containing DNA, are shown as 25 μl.

[0218] As shown in Figure 2, the densities of the two liquids may be different. Figure 2 provides typical non-limiting density values, where the ionic liquid density is generally shown to be greater than 1.3 g / mL, and the density of the DNA aqueous solution is shown to be approximately 1.1 g / mL. As a result, the two liquids in the inverted fluid reservoir are distributed as an aqueous buffer, which is the upper layer above the "bottom" of the well, and as the ionic liquid containing DNA, which is the lower layer below the buffer. The well plate can then be inverted to swap the relative positions of the two layers, as shown. That is, after inversion, the ionic liquid containing DNA is at the bottom of the well, and the aqueous buffer layer has risen above it through the ionic liquid layer.

[0219] At this point, a further step is taken to remove the aqueous buffer layer. This step uses a second aqueous buffer, referred to herein as the “extraction buffer.” The extraction buffer is selected so that, in contrast to the initial buffer, DNA is preferentially distributed from the ionic liquid to the extraction buffer. The initial buffer and the extraction buffer may contain different buffer components, or they may contain the same buffer components but have different pH levels. The process is schematically illustrated in Figure 3. In this case, the extraction buffer is introduced into a fluid reservoir containing the ionic liquid containing the DNA, and the two liquids are mixed. Since the microwells in a microwell plate can be readily used as fluid reservoirs, mixing can be achieved simply by rapidly inverting the well plate several times. After mixing and allowing time for the two phases to separate, each reservoir contains the ionic liquid as the lower layer and the extraction buffer containing the DNA as the upper layer. The upper layer, i.e., the aqueous DNA layer, can be removed using any suitable means, but it is preferable to remove the layer acoustically. Using acoustic ejection, layers can be transferred to individual inverted containers as described above, or acoustically ejected directly to analytical instruments, such as mass spectrometers. Transfer of DNA aqueous solutions to analytical instruments using acoustic ejection can be carried out as described in Sinclair et al. (2016) Journal of Laboratory Automation 21(1):19-26 and U.S. Patent No. 7,405,395 by Ellson et al. (Labcyte Inc., San Jose, CA), both of which are incorporated by reference. The use of acoustic droplet ejection to transfer DNA-containing aqueous liquids to analytical instruments can be carried out using very small sample sizes, approximately a few nanoliters, as needed. Furthermore, acoustic transfer to analytical instruments can be carried out very rapidly, producing more than 10,000 data points per hour, and is therefore ideally suited for high-throughput processes.

[0220] In another embodiment, an extraction method is provided that uses three different fluids, in a first step using a first and second fluid, the target analyte is distributed into the second fluid, while in a second step using a second and third fluid, the target analyte is distributed into the third fluid. That is, the target analyte is preferentially distributed into the second fluid if the alternative is the first fluid, but preferentially distributed into the third fluid relative to the second fluid. The first, second, and third fluids are selected to remove multiple types, such as impurities or analytes, from the sample for separate processing, thereby providing a purer solution of the target analyte in the third fluid. An example of this method is as follows: An aqueous biological sample (i.e., a first fluid containing DNA as the target analyte) can be mixed with an ionic liquid (i.e., a second fluid into which the target analyte is distributed) and distributed into an upper aqueous layer and a lower ionic liquid layer. The ionic liquid is selected so that the DNA is distributed into its layer in this first step. Next, preferably as described elsewhere herein, the upper layer is removed using acoustic injection to leave the fluid composition containing the target analyte, DNA, in the ionic liquid. In a subsequent step, the ionic liquid containing DNA is mixed with an aqueous extraction buffer having a pH selected such that at least 60 wt.% (preferably at least 75 wt.%, e.g., at least 85 wt.%, at least 90 wt.%, at least 95 wt.%, or 100%) of the DNA moves from the ionic liquid to the extraction buffer, thereby forming an upper fluid layer on top of the ionic liquid. The aqueous layer containing DNA can then be acoustically injected as described above.

[0221] Figure 4 schematically illustrates the aforementioned double extraction method using 35 μL of a biological sample containing DNA as the target analyte (shown in the figure as a bacterial solubilizer). The biological sample is contained in a reservoir which may be a standalone single reservoir, a standalone reservoir which is one of a group of other such reservoirs (e.g., a tube in a rack of tubes containing other tubes), a well in a microwell plate, e.g., a 384-well plate. The “first fluid” may be an aqueous buffer containing DNA, as shown in the figure. When the ionic liquid is added to the fluid reservoir, a lower layer is formed below the aqueous phase. The two fluid layers are mixed using a suitable method, but heating is preferred, as shown in Figure 4. Heating not only mixes the layers and makes them miscible (provided that a suitable ionic liquid is selected that can be made miscible with the aqueous fluid at a relatively mild temperature), but also lyses the cells in the biological sample and releases the cellular contents. The DNA still preferentially remains in the ionic liquid, however the first fluid is buffered to an appropriate pH, i.e., a pH at which the DNA is distributed into the ionic liquid without remaining in the aqueous phase. The upper fluid containing undesirable cellular components is removed by decanting, aqueous injection, or any other suitable method, but the preferred method is again with focused acoustic injection. After removing the aqueous phase, a single phase of the ionic liquid containing the target analyte remains in the fluid reservoir. Next, an extraction buffer is added and the two phases are mixed. In one embodiment, as previously described, the extraction buffer is buffered to a selected pH so that at least 60 wt.% of the DNA (preferably at least 75 wt.% of the DNA, as previously stated) can move from the ionic liquid to the extraction buffer, thereby forming an upper aqueous base. Next, the aqueous fluid layer containing the DNA is acoustically injected as previously described.

[0222] 3. Reaction Products

[0223] As described earlier in this specification, the target analyte is not limited to an analyte contained in or derived from a biological sample. The target analyte may be an inorganic compound, an organometallic compound, or even an atom or ion; see Part (1) of this section. In further embodiments, the target analyte may be a reaction product in a multi-step reaction. That is, the present invention provides a method for the synthesis and acoustic extraction of a reaction product, further comprising the steps of: providing a reaction mixture in a fluid reservoir comprising a first reactant, a second reactant, and a fluid medium constituting a first liquid; exposing the reaction mixture to conditions that induce a chemical reaction between the first reactant and the second reactant to produce a reaction product having a first solubility; mixing a second liquid immiscible with the first liquid and having a second solubility such that the reaction product differs from the first solubility by at least 50%, with the reaction mixture, thereby providing a fluid composition having an upper layer and a lower layer containing reaction products of different concentrations; and applying focused acoustic energy to the fluid reservoir in a manner effective for injecting fluid droplets containing the reaction product into a droplet receiver. The reaction mixture may further comprise a reaction catalyst, a surfactant, or additional useful components. When a reaction catalyst and / or surfactant is used, the two liquids can be selected such that the reaction product is distributed into one layer and the catalyst and / or surfactant into the other layer, allowing for the removal of these other components from the reaction product. In a variation of the process described above, the reaction product is not necessarily distributed into one of the two layers, but the other components, namely the catalyst and surfactant (which may be considered impurities to the reaction product), actually have an affinity for one layer compared to the other. In this case, the extraction process moves the impurities from one layer to the other without simultaneously moving the target analyte, so that both layers contain the target analyte, but one of those layers has a considerably lower concentration of impurities.

[0224] The reaction conditions are generally, but not necessarily, selected from the group consisting of allowing the reaction to proceed in the reaction mixture for a predetermined reaction time, mixing the reactants, changing the temperature of the reaction mixture, adding at least one catalyst to the reaction mixture, adding at least one surfactant to the reaction mixture, introducing at least one additional reactant to the reaction mixture, and two or more combinations thereof.

[0225] In related aspects of the present invention, methods for the synthesis of reaction products and for acoustic transmission are provided. The method is

[0226] (a) Providing a reaction mixture in a fluid reservoir having a volume in the range of about 1 nL to about 3 mL, comprising a first reactant, a second reactant, and a fluid medium,

[0227] (b) The step of exposing the reaction mixture to reaction conditions that induce a chemical reaction between the first reactant and the second reactant to produce a reaction product,

[0228] (c) A method effective for injecting fluid droplets containing reaction products into a droplet receiver, comprising the steps of applying focused acoustic energy to a fluid reservoir and Includes.

[0229] The reaction conditions are as described above, namely, allowing the reaction to proceed in the reaction mixture for a predetermined reaction time, mixing the reactants, changing the temperature of the reaction mixture, adding at least one catalyst to the reaction mixture, adding at least one surfactant to the reaction mixture, introducing at least one additional reactant to the reaction mixture, and being selected from the group consisting of two or more combinations of the above.

[0230] In related embodiments, an acoustic system for extracting a target analyte from a sample is provided, comprising: (a) a fluid reservoir containing a fluid composition which is a reaction mixture having a volume in the range of about 1 nL to about 3 mL, comprising a first reactant, a second reactant, and a fluid medium; and (b) an acoustic droplet ejector, which is acoustically connected to the fluid reservoir to generate acoustic radiation in an effective manner for ejecting fluid droplets from the fluid composition into a droplet receiver, and includes an acoustic radiation generator and focusing means for focusing the acoustic radiation to a focal point in the reservoir. In one embodiment, the droplet receiver is an inverted fluid reservoir, for example, a well in an inverted microwell plate. In another embodiment, the droplet receiver is an analytical instrument, for example, a mass spectrometer, and the droplets ejected into the mass spectrometer are analyzed directly or indirectly by the instrument.

[0231] 4. Extraction of metal ions

[0232] In another embodiment, an extraction method is provided for removing metal ions, such as alkali metal ions and alkaline earth metal ions, from an aqueous sample containing a target analyte and at least one alkali metal ion, typically a lithium cation, sodium cation, or potassium cation, and / or at least one alkaline earth metal ion, such as calcium or magnesium ions. This is particularly useful in mass spectrometry situations where the aqueous fluid containing the target analyte of interest is transferred to a mass spectrometer for analysis (e.g., via acoustic emission). Ion suppression, i.e., ionization suppression of the target analyte, is a well-known problem in mass spectrometry, and one of the most common causes of ion suppression is a significant amount (about 10%). -5The presence of alkali metal cations (M or more) and / or alkaline earth metal cations is the cause. See Volmer et al. (2006) LCGC North America 24(5): 498-510. Sodium and potassium are the two alkali metals of greatest concern because they are biologically related. Also, while polyvalent cations can generally be removed from solution by precipitation to form insoluble salts (similar to the precipitation of iron or copper by phosphates), monovalent alkali metal cations are more difficult to remove from solution. Crown ethers bind tightly to alkali metal ions, but to date, they have not been effectively used to remove alkali metals from aqueous solutions because no effective extraction process has yet been developed to separate crown ethers from the rest of the sample.

[0233] Accordingly, this embodiment provides a novel method for effectively extracting alkali metals and / or alkaline earth metals from biological samples and other aqueous compositions. The method comprises the steps of: adding an ionic liquid and a metal extraction composition comprising a metal-binding compound selected from crown ethers, cryptands and combinations thereof to an aqueous sample containing a target analyte and alkali metal ions and / or alkaline earth metal ions, thereby forming an initial two-phase solution; applying conditions (e.g., heating) to the two-phase solution such that the two phases become miscible, thereby mixing the metal salt with the metal extraction composition in a single-phase solution; and re-creating the two-phase solution by, for example, cooling, which comprises an upper aqueous layer and a lower layer of the metal extraction composition and metal ions. The aqueous layer can then be injected into a mass spectrometer (or other type of droplet receiver) for analysis of the target analyte. The metal extraction composition contains the ionic liquid and the metal-binding compound in a weight ratio ranging from about 1:100 to about 100:1, more typically from about 1:10 to about 10:1.

[0234] Preferred metal-bonded compounds are crown ethers, such as 12-crown-4, 15-crown-5, and 18-crown-6. Particularly preferred metal-bonded compounds include crown ethers that have been converted into ionic liquids by functionalization with a cationic moiety, which is typically but not necessarily so, a cationic moiety corresponding to a cation contained in the liquid salt enumerated in Part 2, “Extraction Methods,” of this section. For example, but not limited to, modification of a crown ether in this manner can be achieved by replacing a hydrogen atom (in the CH group) with a functional group containing a positively charged nitrogen atom associated with a negatively charged counterion. One such crown ether salt has the structure of formula (I). [ka]

[0235] [In the formula, R can be selected from a tertiary amino group and a nitrogen heterocycle, and X is an anionic species, such as a halide ion.] A specific example of such a crown ether salt has the structure of formula (II). [ka]

[0236] This crown ether salt can be synthesized from chloromethyl 18-crown-6 and N-methylimidazole using a technique similar to that described by Dharaskar et al. (2016), "Synthesis, characterization and application of 1-butyl-3-methylimidazolium tetrafluoroborate for extractive desulfurization of liquid fuel" Arabian Journal of Chemistry 9:578-587.

[0237] In related embodiments, the present invention provides metal extraction compositions for use in the aforementioned extraction (or other) processes, comprising ionic liquids and metal-binding compounds selected from crown ethers, cryptands, and combinations thereof. It should be noted that these functionalized crown ethers and cryptands can serve a dual purpose in the extraction processes of the present invention, being ionic liquids with selective affinity for certain types of target analytes and chelating agents for removing alkali metal ions and alkaline earth metal ions.

[0238] 5. Determination of the partition coefficient of the analyte in two solvents

[0239] In any extraction process, including the processes described and claimed herein, it is extremely useful to know the partition coefficient of a particular target analyte in two solvents. One of the solvents may be water, such as in an aqueous biological sample or aqueous buffer, and the other solvent may be a candidate solvent considered for use in the extraction process. Therefore, in further embodiments, the present invention relates to a method for determining the partition coefficient D of an analyte in a mixture of two solvents,

[0240] (a) In a fluid reservoir, combine a known amount X of analyte with a first solvent in a first volume V1 and a second solvent in a second volume V2 substantially immiscible with the first solvent, thereby forming a distributed fluid composition in which the analyte has a concentration X / (V1+V2) in the combined first and second solvents, and thereby having an upper layer of the first solvent and a lower layer of the second solvent, wherein the analyte has a concentration C1 in the first solvent and a concentration C2 in the second solvent.

[0241] (b) A step of acoustically ejecting droplets from the upper layer,

[0242] (c) A step of determining C1 in the ejected droplet,

[0243] (d) A step of calculating C2 from C1 according to the relation C2 = (C1V1) / V2,

[0244] (e) The step of determining the distribution coefficient D by confirming the ratio of C1 to C2. This provides a method that includes [something].

[0245] In a related embodiment, a method for determining the partition coefficient D in two solvents,

[0246] (a) In a fluid reservoir, combine an analyte, a first solvent in a first volume V1, and a second solvent in a second volume V2 that is substantially immiscible with the first solvent, thereby forming a distributed fluid composition having an upper layer of the first solvent and a lower layer of the second solvent, wherein the analyte has a concentration C1 in the first solvent and a concentration C2 in the second solvent.

[0247] (b) A step of acoustically ejecting droplets from the upper layer,

[0248] (c)(b) The step of determining C1 in the droplet ejected in (c)(b),

[0249] (d) The step of removing the upper layer from the distributed fluid composition,

[0250] (e) The step of acoustically ejecting droplets from the lower layer,

[0251] (f)(e) The step of determining C2 in the droplet ejected in (f)(e),

[0252] (g) The step of determining the distribution coefficient D by confirming the ratio of C1 to C2. A method is provided that includes this.

[0253] 6. Acoustic Emission and High-Throughput Processes

[0254] Acoustic ejection enables the rapid processing and production of nanoliter-sized droplets of a predetermined, consistent size. See U.S. Patent No. 6,416,164 by Stearns et al., which is incorporated herein by prior reference. The aforementioned patent describes how the size of droplets acoustically ejected from a fluid surface can be carefully controlled by varying the acoustic output, acoustic frequency, tone burst duration, and / or the F-number of the focusing lens, with lenses generally having an F-number greater than approximately 2 being preferred. Thus, ADE enables the ejection of "ultra-monodisperse" droplets, which in the context of the present invention means that the ejected particles have a consistent diameter with a coefficient of variation of about 1%. Furthermore, ADE allows for the introduction of a precise predetermined volume of fluid sample into the system for analysis. A further advantage of using acoustic ejection is that droplets can be ejected from very small sample sizes of approximately 5 μl or less. This is particularly advantageous when sample availability is limited and small fluid samples must be analyzed out of necessity. Regarding processing capacity, Mutz et al.'s U.S. Patent No. 6,938,995 explains that acoustic injection technology, used in conjunction with acoustic assessment of fluid samples in multiple reservoirs, can achieve analysis of 5, 10, or even more than 25 reservoirs per second, which translates to well over 50,000 fluid samples per day.

[0255] The system of the present invention can be used to acoustically eject very small sample fluid droplets thanks to the precision made possible by using acoustic ejection technology. However, the present invention is not limited thereto, and the volume of the acoustically ejected droplet may be in the range of about 0.5 pL to about 3 mL. The system of the present invention is used in many applications to produce nanoliter-sized fluid droplets for analysis, where “nanoliter-sized” droplets generally contain fluid samples up to about 30 nL, typically about 10 nL or less, preferably about 5.0 nL or less, more preferably about 3.0 nL or less, for example, 1.0 nL or less, about 50 pL or less, about 25 pL or less, and about 1 pL or less, including ranges such as about 0.5 pL to 2.0 nL, about 0.5 pL to 1.5 nL, about 0.5 pL to 1.0 nL, about 1.0 pL to 2.0 nL, about 1.0 pL to 1.5 nL, about 1.0 pL to 1.0 nL, etc. In a typical operating range, droplets ranging from approximately 1 nL to approximately 30 nL are generated. Acoustic ejection of droplets from the fluid sample surface is performed using an acoustic ejector, as described in more detail below. Acoustic ejection is particularly suitable for high-throughput processing, and especially for high-throughput mass spectrometry (HTMS) insofar as HTMS is hindered by the lack of easy automation of sample preparation and loading, the need to preserve samples, the need to eliminate cross-contamination, the inability to transfer fluids directly from the fluid reservoir to the analytical device, and the inability to produce droplets of appropriate size.

[0256] Next, in one embodiment, the system and method of the present invention uses an acoustic ejector as a fluid sample droplet preparation device for ejecting droplets from a fluid composition in a liquid-liquid extraction situation. The acoustic ejector directs acoustic energy into a reservoir containing the fluid composition containing the analyte in a manner that causes the ejection of fluid droplets upward from the fluid surface.

[0257] The system may also include means for positioning the reservoir and acoustic ejector in an acoustic connection relationship. Typically, a single ejector is used, comprising an acoustic emission generator and focusing means for focusing the acoustic emission generated by the acoustic emission generator. However, multiple ejectors can also be used advantageously. Similarly, a single reservoir can be used, but the device typically includes multiple reservoirs, for example, as an array. When the system is used to eject droplets of a fluid sample containing an analyte from each of multiple reservoirs, positioning means are incorporated to move the substrate containing the reservoir (which may be located on a mobile stage, for example) relative to the acoustic ejector, or vice versa. Rapid and continuous acoustic ejection of fluid droplets from each of the series of reservoirs is thus easily facilitated. Either type of positioning means, i.e., positioning means for the ejector, or positioning means for the reservoir or reservoir substrate, can be constructed from, for example, a motor, lever, pulley, gear, a combination thereof, or other electromechanical or mechanical means.

[0258] Any acoustic droplet ejection system can be used in conjunction with the systems and methods of the present invention, but preferred ADE systems are those described in the following U.S. Patents, all of which are incorporated herein by reference: Stearns et al. No. 6,416,164, Ellson et al. No. 6,666,541, Ellson et al. No. 6,603,118, Ellson et al. No. 6,746,104, Ellson et al. No. 6,802,593, Ellson et al. No. 6,938,987, Mutz et al. No. 7,270,986, Ellson et al. No. 7,405,395, and Mutz et al. No. 7,439,048. Preferred ADE systems for use in this specification are those available from Labcyte Inc., particularly the Echo® 500-series liquid handler systems, including the Echo® 525, Echo® 550, and Echo® 555 liquid handlers, as well as the Echo® 600-series liquid handler systems, including the Echo® 600 and Echo® 655 liquid handlers, all of which are capable of injecting a wide range of fluid classes with high accuracy, precision, and speed.

[0259] As described in the aforementioned patent documents, acoustic ejection devices can be constructed to eject fluid droplets from a single reservoir or multiple reservoirs. To provide modularity and interchangeability of components, the device may sometimes be preferred to be used in conjunction with multiple removable reservoirs, such as tubes in a rack. Generally, the reservoirs are arranged in a pattern or array to provide individual systematic addressing capability to each reservoir. Furthermore, each reservoir may be provided as a separate or independent container in environments requiring many reservoirs, but it is preferable that the reservoirs are contained within an integrated reservoir unit. As an example, the reservoir unit may be a solid surface on which separate fluid-containing regions are maintained in place thanks to surface wetting properties, and each fluid-containing localized region constitutes a reservoir. As another example, the reservoir unit may be a well plate having individual wells that act as reservoirs. Many well plates suitable for use with devices are commercially available, for example, each well plate can contain 96, 384, 1536, or 3456 wells, and may have a full skirt, a half skirt, or no skirt at all. Well plates, or microtiter plates, have become commonly used laboratory items. The Society for Laboratory Automation and Screening (SLAS), in cooperation with the American National Standards Institute, has established and maintains standards for microtiter plates. The wells in such well plates are generally in the form of a linear array.

[0260] The availability of such commercially available well plates does not preclude the manufacture and use of custom well plates of other geometric structures containing at least approximately 10,000 wells, or as many as 100,000 to 500,000 wells, or even more. Furthermore, the materials used to construct the reservoir must be compatible with the fluid sample contained within. Therefore, if the reservoir or well is intended to contain an organic solvent, such as acetonitrile, polymers that dissolve in or swell with acetonitrile should not be suitable for use in forming the reservoir or well plate. Similarly, a reservoir or well intended to contain DMSO must be compatible with DMSO. For water-based fluids, several materials are suitable for constructing reservoirs, including, but are not limited to, ceramics such as silicon oxide and aluminum oxide, metals such as stainless steel and platinum, and polymers such as polyester, polypropylene, cyclic olefin copolymers (commercially available, e.g., Zeonex® from Nippon Zeon and Topas® from Ticona), polystyrene, and polytetrafluoroethylene. For photosensitive fluids, reservoirs may be constructed from optically opaque materials that have sufficient acoustic transparency so as not to substantially impair the function of the device.

[0261] Furthermore, to reduce the amount of movement and time required to align the acoustic emission generator with each reservoir or reservoir well during operation, the center of each reservoir is preferably located about 1 centimeter or less, more preferably about 1.5 millimeters or less, even more preferably about 1 millimeter or less, and optimally about 0.5 millimeters or less, from the center of an adjacent reservoir. These dimensions tend to limit the size of the reservoir to its maximum volume. The reservoirs are constructed to contain typically about 1 mL or less, preferably about 100 μL or less, more preferably about 1 μL or less, and optimally about 1 nL or less of fluid. To facilitate handling of multiple reservoirs, it is also preferable that the reservoirs are acoustically substantially indistinguishable.

[0262] Acoustic ejection devices used in conjunction with the system and method of the present invention enable acoustic ejection of droplets at speeds of at least about 250 Hz, but higher ejection speeds including 500 Hz, 1 kHz or higher are also possible, with smaller droplets enabling higher repeating speeds. The device can also rapidly eject droplets from each of a plurality of reservoirs that can be arranged in an array, such as in a well plate or a rack of individual tubes. That is, a substrate positioning means or an ejector positioning means continuously acoustically connects the ejector to each of a series of fluid reservoirs, thereby enabling rapid and controlled ejection of fluid sample droplets from different reservoirs. With current commercially available technology, the substrate can move relative to the ejector and / or the ejector can move from one reservoir to another within the same substrate, and the repeatable and controlled acoustic connection of each reservoir can be moved in less than about 0.1 seconds with high-performance positioning means and less than about 1 second with conventional positioning means. As described in U.S. Patent No. 6,666,541 by Ellson et al., a custom-designed system can reduce the reservoir-to-reservoir transition time (equivalent to the time between acoustic injection events) to less than approximately 0.001 seconds. To provide a custom-designed system, it is important to always keep in mind that there are two basic types of motion: pulsed and continuous. Pulsed motion involves moving the substrate or injector to a position where the injector is acoustically connected to a reservoir within the substrate, acoustically ejecting droplets from the sample fluid in the reservoir, and then repositioning the substrate and / or injector so that the injector is acoustically connected to the next reservoir. Using high-performance positioning means in conjunction with such a method allows for repeatable, controlled acoustic connection of each reservoir in less than 0.1 seconds. On the other hand, a continuous motion design moves the substrate and / or injector continuously, though not at the same speed, providing injection during the movement. Because the pulse width is very short, this type of process allows for reservoir transitions exceeding 10 Hz, and even exceeding 1000 Hz.

[0263] Therefore, the method of the present invention is ideal for carrying out the extraction process of the present disclosure in high-throughput situations. Extraction can be carried out as described herein in each of the wells in a series of fluid reservoirs, for example, in a microwell plate, along with very rapid transitions from reservoir to reservoir and acoustic droplet injection into any type of droplet reservoir, for example, an inverted microwell plate or an analytical instrument.

[0264] A typical focused acoustic ejection system that can be advantageously used herein is illustrated in Figure 1 of U.S. Patent No. 6,666,541 by Ellson et al., whose disclosure is incorporated by reference. As described in that document, an acoustic droplet ejection device includes an acoustic ejector, which includes an acoustic emission generator and focusing means for focusing the generated acoustic emission to a focal point near the fluid surface in a fluid sample. Thus the acoustic ejector is adapted to generate and focus acoustic emission to eject droplets of fluid from a fluid composition in a fluid reservoir. The acoustic emission generator and focusing means can function as a single unit controlled by a single controller or can be controlled independently. Any of the various focusing means known in the art, including curved lenses or Fresnel lenses, can be used in conjunction with the present invention. Such focusing means are described in U.S. Patent No. 4,308,547 by Lovelady et al., U.S. Patent No. 5,041,849 by Quate et al., and U.S. Patent Application Publication No. 2002037579. Furthermore, there are several ways in which the injector is acoustically connected to each individual reservoir and thus to the fluid within it. Acoustic connection can be achieved by direct contact with the fluid contained in the reservoir, but a preferred method is to acoustically connect the injector to the reservoir and the fluid in the reservoir without any part of the injector (e.g., the focusing means) coming into contact with any of the fluids being injected.

[0265] Acoustic droplet injectors can come into direct or indirect contact with the outer surface of each reservoir. For acoustic connection of the injector to the reservoir using direct contact, it is preferable that the direct contact be entirely isometric to ensure efficient acoustic energy transfer. That is, the injector and reservoir should have corresponding surfaces adapted to the joint contact. Therefore, when acoustic connection is achieved between the injector and reservoir via a focusing means, it is desirable that the reservoir has an outer surface corresponding to the surface profile of the focusing means. If the contact is not isometric, the efficiency and accuracy of acoustic energy transfer may be impaired. Furthermore, since many focusing means have curved surfaces, the direct contact method may require the use of reservoirs having specially formed curved surfaces.

[0266] Optimally, acoustic connection is achieved through indirect contact between the injector and the reservoir, provided that an acoustic connection medium is placed between the injector and the base of the fluid reservoir. The acoustic connection medium may be an acoustic connection fluid, preferably an acoustically homogeneous material, in equiangled contact with both the acoustic focusing means and the lower surface of the reservoir. The system may include a single acoustic injector or multiple injectors. Generally, a single injector design is preferred over a multiple injector design because accuracy of droplet placement, as well as consistency of droplet size and velocity, is more easily achieved with a single injector. However, the present invention is not limited to a single injector design.

[0267] When two or more fluid reservoirs are used in the method of the present invention, the reservoirs are preferably substantially identical and acoustically indistinguishable, but identical construction is not a requirement. As described earlier in this section, the reservoirs may be individual, removable components in a tray, rack, or other such structure, or they may be fixed in a plate, such as a microwell plate, or other substrate. Each reservoir is preferably substantially axially symmetric as shown and has vertical walls extending upward from a circular reservoir base, but other reservoir shapes and reservoir base shapes can also be used. The material and thickness of each reservoir base should be such that acoustic radiation can be transmitted through its base to the fluid sample contained within each reservoir.

[0268] In the procedure, as previously described, a fluid reservoir is filled with a fluid composition containing a sample that includes the target analyte. The target analyte is generally in an extracted form in a solvent or solvent mixture, or may be present in the lower fluid layer as described earlier herein. The acoustic ejector is positioned directly below the fluid reservoir using an acoustic connection between the ejector and the provided reservoir. Once the ejector and reservoir are properly positioned relative to each other, the acoustic emission generator is activated to generate acoustic emission, which is then directed by a focusing means to a focal point near the fluid surface in the reservoir (the fluid surface may be either a liquid-air interface or a liquid-liquid interface). As a result, fluid droplets are ejected from the fluid surface toward a droplet receiver, such as a substrate, an inverted reservoir, a well in an inverted microwell plate, a droplet transfer device, or an analytical instrument. In a multiple reservoir system, for example, a multiwell plate or tube rack can then be rearranged relative to the acoustic ejector, so that another reservoir aligns with the ejector and droplets of the next fluid composition can be ejected.

[0269] The analytical instrument to which the ejected droplet can be directed may be any instrument used to detect a target analyte, determine the amount or concentration of the target analyte in a sample, or determine the chemical composition of the target analyte. If the analytical instrument is a mass spectrometer or other type of device that requires the analyte to be in an ionized form, the ejected droplet passes through an ionization region before entering the mass spectrometer or other analytical instrument that requires the analyte to be in an ionized form. In the ionization region, a selected ionization source, such as an electrospray ion source, converts the analyte to an ionized form. When using an ejected fluid droplet containing the target analyte in an ionized form, exposure to an ionization source is not required. See, for example, Datwani et al., U.S. Provisional Patent Application No. 62 / 590,079, filed November 22, 2017, “System and Method for the Acoustic Loading of an Analytical Instrument Using a Continuous Flow Sampling Probe,” the disclosure of which is incorporated herein by reference. Exemplary analytical instruments include, but are not limited to, mass spectrometers, spectroscopic devices, separation systems, and combinations thereof. Exemplary ionization techniques include, but are not limited to, chemical ionization, electron impulse ionization, desorption chemical ionization, inductively coupled plasma ionization, and atmospheric pressure ionization including electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization. Exemplary separation methods include, but are not limited to, liquid chromatography, solid-phase extraction, HPLC, capillary electrophoresis, or any other liquid-phase sample purification or separation process. Exemplary mass spectrometers include, but are not limited to, sector-type mass spectrometers, time-of-flight mass spectrometers, quadrupole mass filter mass spectrometers, three-dimensional quadrupole ion trap mass spectrometers, linear quadrupole ion trap mass spectrometers, donut-type ion trap mass spectrometers, and Fourier transform ion cyclotron resonance mass spectrometers.

[0270] Furthermore, the present invention as described herein includes modifications of the acoustic droplet ejector process to optimize the results, as already described. For example, as described in U.S. Patent Nos. 6,932,097, 6,938,995, 7,354,141, 7,899,645, 7,900,505, 8,107,319, 8,453,507, and 8,503,266 of Ellson et al., the aforementioned acoustic droplet ejectors. To characterize the fluid composition within the reservoir, for example, the height of the fluid meniscus, as well as other properties such as the fluid's volume, viscosity, density, surface tension, composition, acoustic impedance, acoustic attenuation, and speed of sound in the fluid, can be measured. These can then be used, in one or all form, to determine the optimal parameters for droplet ejection, including acoustic output, acoustic frequency, tone burst duration, and / or the F-number of the focusing lens. As another example, an acoustic matching process can be used to optimize the relative positions of the acoustic ejector and the fluid-containing reservoir in a focused-activated acoustic ejection system, as described in Ellson's U.S. Patents 8,544,976 and 8,882,226. A further example is a method for optimizing the amplitude of acoustic radiation used to eject fluid droplets by analyzing the waveform of acoustic radiation reflected from surfaces within the reservoir before ejection. See Stearns et al., U.S. Patents 7,717,544 and 8,770,691. Droplet size and consistency can be ensured using the method of Hadimioglu et al., U.S. Patent No. 6,383,115, and variations in reservoir characteristics can be controlled using the methods of Mutz et al., U.S. Patent No. 7,481,511 and Ellson et al., U.S. Patent No. 7,784,331.

[0271] 7. Dynamic tracking of the boundary between liquids

[0272] In a preferred embodiment, the combination of the aforementioned methods and systems for optimizing the acoustic injection process can be advantageously used in the context of the present invention. More specifically, the vertical position of the boundary between the two fluid layers within the fluid reservoir can be tracked prior to each acoustic injection event, such as the height of the total fluid composition within the reservoir. This can be done using the acoustic matching techniques described in the aforementioned patent documents. Next, during the extraction process of the present invention, the height of the upper fluid (i.e., the distance from the liquid-liquid boundary at the center point to the meniscus) can be calculated from the combination of the overall (central) height of the fluid composition and the vertical position of the center of the liquid boundary. That is, the height of the upper fluid is equal to the overall fluid height minus the height of the identified boundary. This then facilitates a process by which acoustic injection can be stopped after the upper layer has been fully ejected without ejecting the lower layer.

[0273] Although the present invention has been described with some specific embodiments, it should be understood that the foregoing description and the following examples are intended to illustrate and not limit the scope of the present invention. In this regard, there is no intention to show the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention, and the description, together with the figures and / or examples, will clarify to those skilled in the art how the present invention can actually be embodied. This disclosure includes any modifications and equivalent forms of the subject matter recited in the appended claims as permitted by applicable law. Further, any combination of the elements of the present invention described herein is included by this disclosure, unless otherwise specified herein or clearly inconsistent with the context.

[0274] All patent documents, patent publications, references, and other materials cited herein are incorporated by reference in their entirety.

[0275] Experiment

[0276] The Echo® 555 liquid handler (Labcyte Inc., San Jose, CA) functions as an acoustic droplet ejector (ADE) system, capable of ejecting a wide range of fluid classes with high accuracy, precision, and speed. Fluid samples are loaded into the wells of a 384-well polypropylene source plate, which is then mounted on a motorized stage system to automate sampling from any source well. The Echo 555 system is calibrated for aqueous solutions containing up to 50% methanol and up to 50% acetonitrile. The system's acoustic transducer can also be used for automated characterization of the fluid in the reservoir to determine optimal parameters for droplet ejection, including acoustic output, acoustic frequency, tone burst duration, and / or the f-number of the focusing lens, by measuring the height of the fluid meniscus and other properties (e.g., fluid volume, viscosity, density, surface tension, acoustic impedance, acoustic attenuation, speed of sound in the fluid, etc.).

[0277] The fluid reservoir consisted of wells in a 384-well microplate. A suitable biocompatible buffer, such as 20 μL of Tris / EDTA buffer and 620 μL of BMIM PF as an ionic liquid, were added to the fluid reservoir, i.e., to the wells loaded with the sample in the microwell plate. Some wells were coated with a hydrophilic amine coating, while others were left uncoated. A surfactant was added to some wells, while others were left uncoated. The fluid droplets were injected into the inverted microwell plate or into an analytical instrument, such as a mass spectrometer, according to the extraction / separation protocol of the present invention. In certain embodiments, for example, the following items are provided: (Item 1) A method for generating fluid droplets containing a target analyte at a selected concentration, (a) Providing a fluid composition comprising an upper region and a lower region, wherein the analyte is present in the upper region at a first concentration and in the lower region at a second concentration, the second concentration being different from the first concentration; (b) A step of applying focused acoustic energy to the fluid reservoir in a manner effective for ejecting fluid droplets from the fluid composition into a droplet receiver, wherein the ejected droplets contain the target analyte at a selected concentration, and the selected concentration is (i) substantially equivalent to either the first concentration or the second concentration, and (ii) substantially uniform across the droplet. Methods that include... (Item 2) The method according to item 1, wherein the fluid composition comprises a sample containing the target analyte. (Item 3) The method according to item 2, wherein the sample includes a biological sample. (Item 4) The method according to item 3, wherein the biological fluid sample includes tissue, tissue homogenate, cells, cell suspension, cell extract, whole blood, plasma, serum, saliva, sputum, nasal secretions, cerebrospinal fluid, interstitial fluid, lymph, semen, vaginal fluid, or feces. (Item 5) The method according to item 3, wherein the biological sample includes tissue, cells, or blood. (Item 6) The method according to any one of items 1 to 5, wherein the upper region is an upper layer containing a first liquid, the lower region is a lower layer containing a second liquid, and a liquid-liquid boundary exists between the upper and lower layers. (Item 7) The method according to item 6, wherein the first liquid and the second liquid are selected such that the target analyte has a first solubility in the first liquid and a second solubility in the second liquid. (Item 8) The method according to item 7, wherein the first solubility differs from the second solubility by at least about 50%. (Item 9) The method according to item 8, wherein the first solubility differs from the second solubility by at least about 85%. (Item 10) The method according to item 6, wherein the first liquid and the second liquid are selected such that the first liquid has a first affinity for the target analyte and the second liquid has a second affinity for the target analyte, and the first and second affinities are different. (Item 11) The method according to item 9, wherein one of the first liquid and the second liquid is hydrophilic, and the other of the first liquid and the second liquid is hydrophobic. (Item 12) The method according to item 10, wherein the fluid composition comprises a plurality of components having different hydrophobic properties. (Item 13) The method according to item 11, wherein the target analyte has a hydrophobicity different from that of the other component in the fluid composition. (Item 14) The method according to item 6, wherein one of the first liquid and the second liquid contains an ionic species that ionically bonds to the target analyte. (Item 15) The method according to item 14, wherein the target analyte is negatively charged. (Item 16) The method according to item 15, wherein the second liquid includes an ionic liquid. (Item 17) The method according to item 16, wherein the target analyte includes nucleic acids. (Item 18) The method according to item 17, wherein the nucleic acid includes DNA. (Item 19) The method according to item 6, wherein the first liquid and the second liquid have different degrees of volatility. (Item 20) The method according to item 19, wherein the first liquid is less volatile than the second liquid. (Item 21) The method according to item 6, wherein the first liquid and the second liquid have different viscosities. (Item 22) The method according to item 6, wherein, prior to step (b), the first liquid and the second liquid are subjected to mixing conditions. (Item 23) The method according to item 22, wherein the mixing conditions include stirring, agitation, ultrasonic treatment, inversion, or a combination thereof. (Item 24) The method of item 6, comprising, prior to step (a), exposing the combination of the sample and the miscible mixture of the two liquids to conditions that make the two liquids substantially immiscible. (Item 25) The method according to item 24, wherein the conditions include heating or cooling. (Item 26) The method according to item 24, wherein the condition includes adding a salt to the fluid composition. (Item 27) The method according to item 6, wherein the first concentration and the second concentration differ by at least 50%. (Item 28) The method according to item 27, wherein the first concentration and the second concentration differ by at least 85%. (Item 29) The method according to item 27, wherein the droplet receiver includes an analytical instrument. (Item 30) The method according to item 29, wherein the analytical instrument includes a mass spectrometer. (Item 31) The method according to item 29, further comprising repeating step (b) to inject a plurality of fluid droplets into the droplet receiver. (Item 32) The method according to item 28, further comprising acoustically detecting the presence of the liquid-liquid boundary before each iteration of step (b). (Item 33) The method according to item 32, further comprising suspending the iteration of step (b) if the boundary between the liquids cannot be detected. (Item 34) The method according to item 1, wherein the droplet receiver is a reservoir that receives droplets. (Item 35) The method according to item 34, further comprising repeating step (b) to inject a plurality of fluid droplets into a reservoir receiving the droplets. (Item 36) The method of item 35, wherein step (b) is repeated until at least 20 wt.% of the target analyte is transferred from the fluid reservoir to the reservoir receiving the droplets. (Item 37) The method of item 36, wherein step (b) is repeated until at least 50 wt.% of the target analyte is transferred from the fluid reservoir to the reservoir receiving the droplets. (Item 38) The method of item 37, wherein step (b) is repeated until at least 80 wt.% of the target analyte is transferred from the fluid reservoir to the reservoir receiving the droplets. (Item 39) The method according to any one of items 34 to 38, wherein the analyte is present at an initial concentration in the fluid composition and at an extraction concentration in the reservoir receiving the droplet, the extraction concentration being higher than the initial concentration. (Item 40) The method of item 35, further comprising acoustically detecting the presence of the liquid-liquid boundary before each iteration of step (b). (Item 41) The method according to item 39, further comprising acoustically detecting the presence of the liquid-liquid boundary before each iteration of step (b). (Item 42) The method according to item 1, wherein the fluid reservoir is one of a plurality of fluid reservoirs, each containing a fluid composition containing a target analyte. (Item 43) The method according to item 42, wherein any two of the fluid compositions may be the same or different. (Item 44) The method according to item 42, wherein any two of the target analytes may be the same or different. (Item 45) The method according to item 43, wherein any two of the target analytes may be the same or different. (Item 46) The method according to item 42, wherein the fluid reservoirs are arranged in an array. (Item 47) The method according to item 46, wherein the fluid reservoir is contained within a substrate comprising a plurality of integrated reservoir units. (Item 48) The method according to item 47, wherein the integrated plurality of reservoir units are microwell plates, and the fluid reservoir is a well therein. (Item 49) The method according to item 34, wherein the reservoir that receives the droplets is contained within an integrated structure that includes a plurality of reservoirs that receive droplets. (Item 50) The method according to item 49, wherein the reservoirs that receive the droplets are arranged in an array. (Item 51) The method according to item 50, wherein the droplet receiver is a well in an inverted microwell plate. (Item 52) The method according to item 1, wherein step (b) is performed by acoustically connecting an acoustic droplet injector to the fluid reservoir, activating the injector to generate acoustic radiation in the fluid composition toward the reservoir, the acoustic droplet injector comprising an acoustic radiation generator and focusing means for focusing the acoustic radiation to a focal point in the reservoir. (Item 53) The method according to any one of items 42 to 51, wherein step (b) is performed by acoustically connecting an acoustic droplet injector to the reservoir, activating the injector to generate acoustic radiation in the fluid composition toward the reservoir, the acoustic droplet injector comprising an acoustic radiation generator and focusing means for focusing the acoustic radiation to a focal point in the reservoir. (Item 54) The method according to item 53, further comprising the step of (c) positioning the second reservoir and the acoustic droplet ejector in an acoustic connection relationship after ejecting the fluid droplets, and repeating step (b). (Item 55) The method of item 54, further comprising repeating steps (b) and (c) in multiple reservoirs using additional fluid reservoirs. (Item 56) The method of item 55, wherein step (c) is repeated with a maximum reservoir-to-reservoir transition time of approximately 0.5 seconds. (Item 57) The method according to item 56, wherein the transition time from one reservoir to the other is a maximum of approximately 0.1 seconds. (Item 58) The method according to item 57, wherein the transition time from reservoir to reservoir is a maximum of approximately 0.001 seconds. (Item 59) The method according to item 56, wherein the transition from one reservoir to another is performed by moving the reservoir relative to the acoustic droplet ejector. (Item 60) The method according to item 56, wherein the transition from one reservoir to another is performed by moving the acoustic droplet ejector relative to the reservoir. (Item 61) The method according to item 1, wherein the target analyte comprises atoms, ions, salts, molecules, or a class of molecules having common characteristics. (Item 62) The method according to item 1, wherein the target analyte includes an organic compound, an organometallic compound, or an inorganic compound. (Item 63) The method according to item 62, wherein the target analyte is selected from pharmacologically active drugs, metabolites, enzyme inhibitors, ligands, receptors, catalysts, polymers, metals, metal ions, dyes, pesticides, carcinogens, allosteric effectors, antigens, and viruses. (Item 64) The method according to item 62, wherein the target analyte includes a biomolecule. (Item 65) The method according to item 64, wherein the biomolecule is DNA. (Item 66) The method according to item 1, wherein the target analyte is a reaction product. (Item 67) The method according to item 1, wherein the fluid composition in the fluid reservoir occupies a volume of approximately 125 μL or less. (Item 68) The method according to item 67, wherein the fluid composition in the fluid reservoir occupies a volume of approximately 60 μL or less. (Item 69) The method according to item 68, wherein the fluid composition in the fluid reservoir occupies a volume of approximately 30 μL or less. (Item 70) The method according to item 1, wherein the ejected fluid droplet has a volume of approximately 60 nL or less. (Item 71) The method according to item 67, wherein the ejected fluid droplet has a volume of approximately 60 nL or less. (Item 72) The method according to item 68, wherein the ejected fluid droplet has a volume of approximately 60 nL or less. (Item 73) The method according to item 1, wherein the fluid reservoir has an inner surface coated with a surface coating composition. (Item 74) The method according to item 73, wherein the surface coating composition repels the upper layer. (Item 75) The method according to item 73, wherein the surface coating attracts the upper layer. (Item 76) A method for extracting an ion target analyte from a sample, comprising mixing the sample with the ionic liquid and a nonionic liquid under conditions that promote the distribution of the ion analyte into the ionic liquid, and acoustically removing the nonionic liquid from the mixture. (Item 77) A method for extracting an ion target analyte from a sample, comprising mixing the sample with the ionic liquid and a nonionic liquid under conditions that facilitate the distribution of the ion analyte into the ionic liquid to provide an ionic liquid solution of the ion analyte, removing the nonionic liquid from the mixture, and acoustically ejecting droplets of the ion analyte solution into a droplet receiver. (Item 78) The method according to item 76 or item 77, wherein the sample includes a biological fluid sample. (Item 79) The method according to item 78, wherein the biological fluid sample includes tissue, tissue homogenate, cells, cell suspension, cell extract, whole blood, plasma, serum, saliva, sputum, nasal secretions, cerebrospinal fluid, interstitial fluid, lymph, semen, vaginal fluid, or feces. (Item 80) The method according to item 79, wherein the biological sample includes tissue, cells, or blood. (Item 81) The method according to item 76 or 77, wherein the ionic analyzer is negatively charged. (Item 82) The method according to item 81, wherein the target analyte includes nucleic acids. (Item 83) The method according to item 82, wherein the nucleic acid includes DNA. (Item 84) The method according to item 83, wherein the DNA includes double-stranded DNA. (Item 85) The method according to item 76 or 77, wherein the nonionic liquid comprises an extraction buffer. (Item 86) (a) Providing an initial fluid composition in a fluid reservoir comprising a target analyte, an upper layer of a first liquid, and a lower layer of a second liquid, wherein the analyte is present in the upper layer at a first concentration and in the lower layer at a second concentration, the second concentration being higher than the first concentration; (b) An extraction method comprising the step of repeatedly applying focused acoustic energy to the fluid reservoir in a manner effective for ejecting fluid droplets from the upper layer of the fluid, thereby removing at least a portion of the upper layer while allowing the lower layer to remain in the fluid reservoir. (Item 87) The method according to item 86, wherein the initial fluid composition comprises a sample containing the target analyte. (Item 88) The method according to item 87, wherein the sample includes a biological sample. (Item 89) The method according to item 88, wherein the biological fluid sample includes tissue, tissue homogenate, cells, cell suspension, cell extract, whole blood, plasma, serum, saliva, sputum, nasal secretions, cerebrospinal fluid, interstitial fluid, lymph, semen, vaginal fluid, or feces. (Item 90) The method according to item 89, wherein the biological sample includes tissue, cells, or blood. (Item 91) The method of item 86, comprising, prior to step (a), exposing a miscible mixture of the sample and the two liquids to conditions that make the two liquids substantially immiscible. (Item 92) The method according to item 91, wherein the aforementioned conditions include heating. (Item 93) The method according to item 91, wherein the conditions include cooling. (Item 94) A method for extracting ion analytes from biological samples, (a) The step of acoustically ejecting droplets of the biological sample containing the ion analyte and the aqueous medium into an ionic liquid contained in a reservoir that receives the droplets, (b) The step of inverting the reservoir that receives the droplets, thereby forming an upper aqueous layer and a lower ionic liquid layer containing the ion analyte, (c) The step of removing the upper aqueous layer and providing an ionic analyte solution containing the ionic analyte in the ionic liquid. Methods that include... (Item 95) The method according to item 94, wherein the biological sample includes a processed biological sample. (Item 96) The method according to item 95, wherein the processed biological sample contains lysed cells. (Item 97) The method according to item 95 or 96, wherein the aqueous medium includes a buffering system for maintaining the biological sample at a first pH, the first pH being selected such that at least 60 wt.% of the ion analyte in the biological sample is distributed into the ionic liquid when it comes into contact with the ionic liquid. (Item 98) The method according to item 97, wherein the first pH is selected such that at least 75 wt.% of the ion analyte in the biological sample is distributed into the ionic liquid when it comes into contact with the ionic liquid. (Item 99) The method according to item 97, further comprising the step of (d) after step (c), mixing the ion analyte solution with a second extraction buffer having a pH selected such that at least 60 wt.% of the ion analyte in the ionic liquid is distributed into the extraction buffer. (Item 100) The method according to item 99, wherein the second pH is selected such that at least 75 wt.% of the ionic analyte in the ionic liquid is distributed to the extraction buffer. (Item 101) The method according to item 98, further comprising mixing the ion analyte solution with a second extraction buffer having a pH selected such that at least 60 wt.% of the ion analyte in the ionic liquid is distributed to the extraction buffer. (Item 102) The method according to item 101, wherein the second pH is selected such that at least 75 wt.% of the ionic analyte in the ionic liquid is distributed to the extraction buffer. (Item 103) A method for acoustically extracting DNA from an aqueous biological sample, (a) The step of mixing the aqueous biological sample with the ionic liquid in a fluid reservoir under conditions effective for providing a fluid composition comprising an upper aqueous layer and a lower ionic liquid layer, (b) The step of treating the fluid composition so that the DNA in the biological sample is distributed into the lower ionic liquid layer, (c) The step of removing the upper aqueous layer so that the DNA solution in the ionic liquid remains in the fluid container, (d) The step of mixing the DNA solution with an extraction buffer having a pH selected such that at least 60 wt.% of the DNA in the ionic liquid is distributed into the extraction buffer, (e) A step of acoustically and continuously ejecting droplets of the extraction buffer containing the DNA into a droplet receiver. Methods that include... (Item 104) The method according to item 103, wherein the DNA includes double-stranded DNA. (Item 105) A method for extracting lipid components from an aqueous biological sample, A method comprising the steps of: mixing the aqueous biological sample with an organic solvent in a fluid reservoir to provide a distributed fluid composition having an upper organic layer containing a lipid solution and a lower aqueous layer; and acoustically and continuously ejecting droplets of the lipid solution into a droplet receiver. (Item 106) An acoustic system for extracting ion target analytes from a sample, (a) A fluid reservoir containing the fluid composition comprising the ion target analyte and the ionic liquid, (b) an acoustic droplet ejector having an acoustic connection with the fluid reservoir to generate acoustic radiation in an effective manner for ejecting fluid droplets from the fluid composition into a droplet receiver, the acoustic droplet ejector comprising an acoustic radiation generator and focusing means for focusing the acoustic radiation to a focal point in the reservoir. A system that includes this. (Item 107) The system according to item 106, further comprising the aforementioned droplet receiver. (Item 108) The system described in item 107, wherein the droplet receiver includes an analytical instrument. (Item 109) The analytical instrument is the system described in item 108, including a mass spectrometer. (Item 110) The system according to item 107, wherein the droplet receiver is a reservoir that receives droplets. (Item 111) The system according to item 107, comprising a plurality of fluid reservoirs, each containing the ion target analyte and the fluid composition containing the ionic liquid. (Item 112) The system according to item 107, wherein any two of the fluid compositions may be the same or different. (Item 113) The system described in item 107, wherein any two of the target analytes may be the same or different. (Item 114) The system according to item 112, wherein any two of the target analytes may be the same or different. (Item 115) The system according to item 111, wherein the fluid reservoirs are arranged in an array. (Item 116) The system according to item 115, wherein the reservoir is contained within a substrate comprising a plurality of integrated reservoir units. (Item 117) The system according to item 116, wherein the integrated plurality of reservoir units are microwell plates, and the fluid reservoir is a well contained therein. (Item 118) The method according to item 117, wherein the droplet receiver is a well in an inverted microwell plate. (Item 119) The system according to item 111, further comprising means for positioning the injector in a continuous acoustic connection relationship with respect to each of the fluid reservoirs. (Item 120) The system according to item 106, wherein the fluid composition further comprises an aqueous fluid. (Item 121) The system according to item 120, wherein the aqueous fluid contains a buffering system. (Item 122) The system according to item 106, wherein the fluid composition in the fluid reservoir occupies a volume of approximately 125 μL or less. (Item 123) The system according to item 122, wherein the fluid composition in the fluid reservoir occupies a volume of about 60 μL or less. (Item 124) The system according to item 123, wherein the fluid composition in the fluid reservoir occupies a volume of about 30 μL or less. (Item 125) The system according to item 124, wherein the fluid composition occupies a volume of approximately 2.5 μL or less. (Item 126) The system according to item 106, wherein the ion target analyte includes nucleic acids. (Item 127) The system according to item 126, wherein the nucleic acid includes DNA. (Item 128) The system described in item 127, wherein the DNA includes double-stranded DNA. (Item 129) The system according to item 106, wherein the fluid composition contains a biological sample. (Item 130) The system according to item 129, wherein the ion target analyte includes nucleic acids. (Item 131) The system according to item 130, wherein the nucleic acid includes DNA. (Item 132) The system described in item 131, wherein the DNA includes double-stranded DNA. (Item 133) A method for the synthesis and acoustic extraction of reaction products, (a) Providing a reaction mixture in a fluid reservoir, comprising a first reactant, a second reactant, and a fluid medium, having a volume in the range of about 1 nL to about 5 mL, (b) The step of subjecting the reaction mixture to reaction conditions that induce a chemical reaction between the first reactant and the second reactant to produce a reaction product, wherein the fluid medium comprises a first liquid having a first solubility for the reaction product, (c) Mixing a second liquid into the reaction mixture, which is immiscible with the first liquid and has a second solubility such that the reaction product differs from the first solubility by at least 50%, thereby providing a fluid composition having an upper layer and a lower layer containing the reaction product at different concentrations; (d) The step of applying focused acoustic energy to the fluid reservoir in a manner effective for injecting the fluid droplets containing the reaction product into the droplet receiver. Methods that include... (Item 134) The method according to item 133, wherein the upper layer contains the first liquid and the lower layer contains the second liquid. (Item 135) The method according to item 133 or 134, wherein the first solubility is higher than the second solubility. (Item 136) The method according to item 133, wherein the reaction mixture further comprises a reaction catalyst. (Item 137) The method according to item 136, wherein in step (c), the reaction product and the catalyst are preferentially partitioned into different liquids, and by step (c), the reaction product and the catalyst are substantially separated. (Item 138) The method according to item 133, wherein the reaction mixture further comprises a surfactant. (Item 139) The method according to item 138, wherein in step (c), the reaction product and the surfactant are preferentially partitioned into different liquids, and by step (c), the reaction product and the catalyst are substantially separated. (Item 140) A method for the synthesis of reaction products and for acoustic transmission, (a) Providing a reaction mixture in a fluid reservoir, comprising a first reactant, a second reactant, and a fluid medium, having a volume in the range of about 1 nL to about 3 mL, (b) The step of exposing the reaction mixture to reaction conditions that induce a chemical reaction between the first reactant and the second reactant to produce a reaction product, (c) A step of applying focused acoustic energy to the fluid reservoir in a manner effective for injecting the fluid droplets containing the reaction product into the droplet receiver. Methods that include... (Item 141) The aforementioned reaction conditions are, To enable the reaction to proceed in the reaction mixture for a predetermined reaction time, Mixing the aforementioned reactants, Changing the temperature of the reaction mixture, Adding at least one catalyst to the reaction mixture, Adding at least one surfactant to the reaction mixture, Introducing at least one additional reactant into the reaction mixture, and These two or more combinations A method described in item 140, selected from the group consisting of the following. (Item 142) A method for determining the partition coefficient D of an analyte in a mixture of two solvents, (a) In a fluid reservoir, a known amount X of analyte is placed in a first volume V 1 A first solvent, and a second volume V substantially immiscible with the first solvent. 2 When combined with the second solvent, the analyte is mixed with the first and second solvents, and the concentration X / (V) in the combined first and second solvents. 1 +V 2 ) has, The step of thereby forming a distributed fluid composition having an upper layer of the first solvent and a lower layer of the second solvent, wherein the analyte is concentrated in the first solvent at a concentration of C 1 It has a concentration C in the second solvent. 2 Steps having, (b) A step of acoustically ejecting the droplets of the upper layer, (c) C in the ejected droplet 1 The steps to determine, (d) Relational expression C 2 =(C 1 V 1 ) / V 2 According to C 1 From C 2 The steps to calculate, (e)C 2 C for 1 The step of determining the distribution coefficient D by confirming the ratio of Methods that include... (Item 143) An acoustic method for determining the partition coefficient D of an analyte in a mixture of two solvents, (a) In the fluid reservoir, the analyte, first volume V 1 A first solvent, and a second volume V substantially immiscible with the first solvent. 2 Add the second solvent, The step of thereby forming a distributed fluid composition having an upper layer of the first solvent and a lower layer of the second solvent, wherein the analyte is concentrated in the first solvent at a concentration of C 1 It has a concentration C in the second solvent. 2 Steps having, (b) A step of acoustically ejecting the droplets of the upper layer, (c)(b) C in the droplet ejected in (c)(b) 1 The steps to determine, (d) the step of removing the upper layer from the distributed fluid composition, (e) The step of acoustically ejecting the droplets from the lower layer, C in the droplet ejected in (f)(e) 2 The steps to determine, (g)C 2 C for 1 The step of determining the distribution coefficient D by confirming the ratio of Methods that include... (Item 144) An acoustic system for extracting a target analyte from a sample, (a) A fluid reservoir containing a fluid composition which is a reaction mixture having a volume in the range of about 1 nL to about 3 mL, comprising a first reactant, a second reactant, and a fluid medium. (b) an acoustic droplet ejector having an acoustic connection with the fluid reservoir to generate acoustic radiation in an effective manner for ejecting fluid droplets from the fluid composition into a droplet receiver, the acoustic droplet ejector comprising an acoustic radiation generator and focusing means for focusing the acoustic radiation to a focal point in the reservoir. A sound system, including (Item 145) A method for removing metal ions from an aqueous sample, The process involves adding a metal ion extraction composition containing a metal ion-binding compound, which includes an ionic liquid containing a metal ion, a positively charged crown ether associated with a negatively charged counterion, a cryptand, or a combination thereof, to an aqueous sample containing the target analyte, thereby forming an initial two-phase solution. The initial two-phase solution is heated until the two phases become miscible, thereby mixing the metal ions with the metal extraction composition in the single-phase solution. The steps include: cooling the single-phase solution to prepare a second two-phase solution containing an upper aqueous layer and a lower layer of the metal extraction composition and metal ions; Methods that include... (Item 146) The method according to item 1, further comprising acoustically ejecting the aqueous layer into a droplet receiver. (Item 147) The method according to item 146, wherein the droplet receiver includes an analytical instrument. (Item 148) The method according to item 147, wherein the analytical instrument includes a mass spectrometer. (Item 149) The method according to item 145, wherein the metal ion is selected from alkali metal ions, alkaline earth metal ions, and iron ions. (Item 150) The method according to item 149, wherein the metal ion is an alkali metal ion. (Item 151) The method according to item 150, wherein the alkali metal ion is selected from lithium cations, sodium cations, potassium cations, and combinations thereof. (Item 152) The method according to item 149, wherein the metal ion is an alkaline earth metal ion. (Item 153) The method according to item 152, wherein the alkaline earth metal ion is selected from calcium ions and magnesium ions. (Item 154) The method according to any one of items 145 to 153, wherein the ionic liquid comprises a positively charged crown ether. (Item 155) The method according to item 154, wherein the crown ether is selected from 12-crown-4, 15-crown-5, and 18-crown-6 functionalized with a cationic group. (Item 156) The method according to any one of items 145 to 153, wherein the metal-bonded compound comprises a positively charged cryptand. (Item 157) The method according to item 156, wherein the positively charged cryptand includes a cryptand functionalized with a cationic group. (Item 158) The aforementioned crown ether has structure

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Claims

1. A method for ejecting fluid droplets containing a target analyte at a selected concentration, (a) Providing a fluid composition in a fluid reservoir, wherein the fluid composition contains the target analyte and comprises an upper region and a lower region below the upper region, wherein the analyte is present at a first concentration in the upper region and at a second concentration in the lower region, the second concentration being different from the first concentration, the upper region being an upper layer containing a first liquid, the lower region being a lower layer containing a second liquid, the fluid composition having a liquid-liquid boundary between the upper and lower layers, and the first and second liquids being selected such that the first liquid has a first affinity for the target analyte and the second liquid has a second affinity for the target analyte, the first and second affinities being different, and the first and second liquids being immiscible in the injection method, (b) A step of applying focused acoustic energy to the fluid reservoir in a manner effective for ejecting fluid droplets from the fluid composition into a droplet receiver, wherein the fluid droplets are ejected from the upper layer of the fluid composition, the ejected droplets contain the target analyte at a selected concentration, and the selected concentration is (i) equivalent to the first concentration when the fluid droplets are ejected from the upper layer by applying the focused acoustic energy at a focal point in the upper layer, and (ii) uniform throughout the droplet. Methods that include...

2. The method according to claim 1, wherein the first liquid and the second liquid are selected such that the target analyte has a first solubility in the first liquid and a second solubility in the second liquid, and the first solubility differs from the second solubility by at least 50%.

3. The method according to claim 1, wherein the first liquid and the second liquid are selected such that the first liquid has a first affinity for the target analyte and the second liquid has a second affinity for the target analyte, and the first affinity and the second affinity are different.

4. The method according to claim 1, wherein one of the first liquid and the second liquid contains an ionic species that ionically bonds to the target analyte.

5. The method according to claim 1, further comprising, prior to step (a), exposing a combination of a sample containing the target analyte and a miscible mixture of the first liquid and the second liquid to conditions that make the first liquid and the second liquid immiscible.

6. The method according to claim 5, wherein the conditions include heating or cooling.

7. The method according to claim 1, further comprising repeating step (b) to inject a plurality of fluid droplets into the droplet receiver.

8. The method according to claim 7, further comprising acoustically detecting the presence of the liquid-liquid boundary before repeating step (b).

9. The method according to claim 1, wherein the fluid reservoir is one of a plurality of fluid reservoirs, each containing a fluid composition containing a target analyte.

10. The method according to claim 9, wherein the fluid reservoirs are arranged in an array.

11. The method according to claim 1, wherein step (b) is performed by acoustically connecting an acoustic droplet injector to the fluid reservoir, activating the injector to generate acoustic energy in the fluid composition toward the reservoir, the acoustic droplet injector comprising an acoustic emission generator and focusing means for focusing the acoustic energy toward a focal point in the reservoir.

12. The method according to claim 11, further comprising the step of (c) positioning the second fluid reservoir and the acoustic droplet dispenser in an acoustic connection relationship after ejecting the fluid droplets, and repeating step (b) for the second fluid reservoir.

13. The method according to claim 1, wherein the fluid composition in the fluid reservoir occupies a volume of 30 μL or less.

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