Methods and compositions for freeze drying paramagnetic particles

Abstract

Aspects of present disclosure include lyophilized pellets with a longer shelf life, compositions for making lyophilized pellets, and methods for making lyophilized pellets. The lyophilized pellet comprises paramagnetic particles comprising a surface capable of binding to an analyte; a sugar having a glass transition temperature greater than 65° C; or a mixture of two or more sugars, wherein the mixture has a glass transition temperature greater than 65° C; and a water-soluble polysaccharide, wherein the water-soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars.

Description

[0001] PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0002] METHODS AND COMPOSITIONS FOR FREEZE DRYING PARAMAGNETIC PARTICLES

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims benefit under 35 U.S.C. § 1 19(e) of provisional application 63 / 700,184, filed September 27, 2024, which application is hereby incorporated by reference in its entirety.

[0005] INTRODUCTION

[0006] Magnetic separation techniques are widely known to be useful for purification of biologically active substances such as nucleic acids, proteins, viruses, or cells. These techniques avoid the centrifugation and other time-consuming steps of the traditional methods and minimize the risk of cross-contamination. Various types of magnetic particles are commercially available for isolation of a biological molecule of interest from biological samples. Generally, magnetic beads are incubated with the sample in the presence of a suitable lysis buffer. The magnetic beads may be functionalized to bind to nucleic acid or a protein. Following incubation, the magnetic beads are separated from the sample by applying a magnetic field, and unbound material is removed. The beads are optionally washed, and the biological molecule of interest bound to the magnetic beads are eluted.

[0007] Magnetic beads used in these separation techniques can be provided in the form of lyophilized pellets. A mixture of sugar and magnetic particles are lyophilized to form pellets. During storage and manufacturing process, humidity and temperature can negatively impact performance of the magentic particles, resulting in poor stability and shorter shelf life. As such, there is an interest in methods and compositions for improved lyophilized pellets.

[0008] SUMMARY

[0009] Aspects of present disclosure include lyophilized pellets, compositions for making lyophilized pellets, and methods for making lyophilized pellets. The lyophilized pellet comprises paramagnetic particles (PMPs) comprising a surface capable of binding to an analyte; a sugar having a glass transition temperature greater than 65° C; or a mixture of two or more sugars, wherein the mixture of sugars has a glass transition temperature greater than 65° C; and a water-soluble polysaccharide, wherein the water-soluble PCT / US25 / 47509 23 September 2025 (23.09.2025) polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars.

[0010] Aspects of present disclosure also include method of using the lyophilized pellets for analysis of analyte(s) in a sample. In certain embodiments, the sample may be a biological sample.

[0011] In some instances, the sugar is a disaccharide or a trisaccharide or a bioflavonoid, or a mixture thereof. In some instances, the paramagnetic particles comprise a surface capable of binding nucleic acids.

[0012] In some instances, a formulation for making a lyophilized pellet, comprises a sugar or a mixture of two or more sugars in amount of about 5 to 35 weight percentage of total formulation, wherein a glass transition temperature of the sugar or the mixture of sugars is greater than 65° C; a water-soluble polysaccharide in amount of about 1 to 20 weight percentage of total formulation, wherein the water-soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars; and paramagnetic particles in amount of about 45 to 65 weight percentage of total formulation.

[0013] In some instances, a method of making lyophilized pellets, comprises a step of providing the formulation described herein; dispensing droplets of the formulation in liquid nitrogen to generate frozen droplets; and removing water from the frozen droplets to generate lyophilized pellets.

[0014] In some instances, a method of making lyophilized pellets, comprises a step of providing the formulation described herein; and freeze drying the formulation to generate lyophilized powder and compacting the lyophilized powder to generate lyophilized pellets.

[0015] In some instances, a cartridge configured for isolating a target analyte from a sample comprises the lyophilized pellet described herein. The cartridge may include a chamber for incubating a sample with the PMPs. The lyophilized pellet comprising the PMPs may be present in the chamber or may be present adjacent the chamber, e.g., at the inlet to the sample. In instances where the lyophilized pellet is present at the inlet, the lyophilized pellet may be introduced into the chamber by lysis buffer flowing through the inlet into the chamber. The lyophilized pellet may be rehydrated in the lysis buffer, resulting in release of the PMPs.

[0016] In some instances, a method of using the lyophilized pellet for isolating a target analyte from a sample, comprises a step of providing a cartridge comprising a chamber, PCT / US25 / 47509 23 September 2025 (23.09.2025) wherein the chamber is configured for holding a solution; providing the lyophilized pellet in the chamber; wherein the lyophilized pellet is configured for binding to an analyte when the analyte is present in the sample; providing the sample and a lysis buffer in the chamber; mixing the sample and the lysis buffer such that the lyophilized pellet is rehydrated and the paramagnetic particles bind to the analyte; separating the paramagnetic particles from the lysis buffer and optionally washing paramagnetic particles; eluting the target analyte from the paramagnetic particles to provide an elution buffer comprising the isolated analyte.

[0017] BRIEF DESCRIPTION OF THE FIGURES

[0018] Figures 1 , 2 and 3 show diagnostic performances of formulations for making lyophilized pellets after being stored in accelerated temperature conditions.

[0019] DETAILED DESCRIPTION

[0020] Aspects of present disclosure include freeze drying paramagnetic particles, compositions for making freeze drying paramagnetic particles, and methods for making freeze drying paramagnetic particles are provided. Lyophilized pellets, compositions for making lyophilized pellets, and methods for making lyophilized pellets are also provided. Methods of using the lyophilized pellets for analysis analyte(s) in a sample are also provided.

[0021] The lyophilized pellet comprises paramagnetic particles comprising a surface capable of binding to an analyte; a sugar having a glass transition temperature greater than 65° C; or a mixture of two or more sugars, wherein the mixture has a glass transition temperature greater than 65° C; and a water-soluble polysaccharide, wherein the water- soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars.

[0022] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0023] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, PCT / US25 / 47509 23 September 2025 (23.09.2025) between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0024] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0026] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0027] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0028] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0029] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §1 12, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0030] DEFINITIONS

[0031] Before the embodiments of the present disclosure are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0032] “Comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0033] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0034] “Affinity” and “binding affinity” as used interchangeably herein refer to the tendency or strength of binding of the binding member to the analyte. For example, the binding affinity may be represented by the equilibrium dissociation constant (KD), the dissociation rate (kd), or the association rate (ka).

[0035] “Analog” as used herein refers to a molecule that has a similar structure to a molecule of interest (e.g., nucleoside analog, nucleotide analog, sugar phosphate analog, analyte analog, etc.). An analyte analog is a molecule that is structurally similar to an analyte but for which the binding member has a different affinity.

[0036] “Analyte” as used herein refers to a target molecule to be detected in a sample wherein detection of the analyte may be indicative of a biological state of the organism from which the sample was derived. For example, where an analyte is a nucleic acid analyte, detection of the nucleic acid analyte may be indicative of a biological state of the organisms from which the sample was derived including e.g., where detection of viral nucleic acid may indicate infection with a particular pathogen, etc.

[0037] “Aptamer” as used herein refers to an oligonucleotide or peptide molecule that can bind to pre-selected targets including small molecules, proteins, and peptides among others with high affinity and specificity. Aptamers may assume a variety of shapes due to their propensity to form helices and single-stranded loops. An oligonucleotide or nucleic acid aptamer can be a single-stranded DNA or RNA (ssDNA or ssRNA) molecule. A peptide aptamer can include a short variable peptide domain, attached at both ends to a protein scaffold.

[0038] “Assessing” includes any form of measurement and includes determining if an element is present or not. The terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably and include quantitative and qualitative determinations. Assessing may be relative or absolute. “Assessing the identity of” includes determining the most likely identity of a particular compound or formulation or substance, and / or determining whether a predicted compound or formulation or substance is present or absent. “Assessing the quality of” includes making a qualitative or quantitative assessment of quality e.g., through the comparisons of a determined value to a reference or standard of known quality.

[0039] “Bead” and “particle” are used herein interchangeably and refer to a substantially spherical solid support. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0040] “Biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. The term “biological sample” includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, and the like.

[0041] “Bodily fluid” as used herein generally refers to fluids derived from a “biological sample” which encompasses a variety of sample types obtained from an individual or a population of individuals and can be used in a diagnostic, monitoring or screening assay. The definition encompasses blood and other liquid samples of biological origin. The definition also includes samples that have been manipulated in any way after their procurement, such as by mixing or pooling of individual samples, treatment with reagents, solubilization, or enrichment for certain components, such as nucleated cells, nonnucleated cells, pathogens, etc.

[0042] “Component,” “components,” or “at least one component,” refer generally to a capture antibody, a detection reagent or conjugate, a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, serum, whole blood, tissue aspirate, or plasma sample, in accordance with the methods described herein and other methods known in the art. Some components can be in solution or lyophilized for reconstitution for use in an assay.

[0043] “Control” as used herein refers to a reference standard for an analyte such as is known or accepted in the art, or determined empirically using acceptable means such as are commonly employed. A “reference standard” is a standardized substance which is used as a measurement base for a similar substance. For example, there are documented reference standards published in the U.S. Pharmacopeial Convention (USP-NF), Food Chemicals Codex, and Dietary Supplements Compendium (all of which are available at http: / / www.usp.org), and other well-known sources. Methods for standardizing references are described in the literature. Also well-known are means for quantifying the amounts of analyte present by use of a calibration curve for analyte or by comparison to an alternate reference standard. A standard curve can be generated using serial dilutions or solutions of known concentrations of analyte, by mass spectroscopy, gravimetric methods, and by other techniques known in the art. Alternate reference PCT / US25 / 47509 23 September 2025 (23.09.2025) standards that have been described in the literature include standard addition (also known as the method of standard addition), or digital polymerase chain reaction.

[0044] “Enzymatic cleavable sequence” as used herein refers to any nucleic acid sequence that can be cleaved by an enzyme. For example, the enzyme may be a protease or an endonuclease, such as a restriction endonuclease (also called restriction enzymes). Restriction endonucleases are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides. Some endonucleases, such as for example Fokl, comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. In some embodiments, the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical.

[0045] “Globular protein” refers to a water-soluble protein that has a roughly spherical shape. Examples of globular proteins include but are not limited to ovalbumin, betaglobulin, C-reactive protein, fibrin, hemoglobin, IgG, IgM, and thrombin.

[0046] “Label” or “detectable label” as used interchangeably herein refers to a tag attached to a specific binding member or analyte by a cleavable linker.

[0047] “Nanoparticle(s)” and “nanobead(s)” are used interchangeably herein and refer to a nanobead or nanoparticle sized to translocate through or across a nanopore used for counting the number of nanobeads / nanoparticles traversing through it.

[0048] "Nucleobase" or "Base" means those naturally occurring and synthetic heterocyclic moieties commonly known in the art of nucleic acid or polynucleotide technology or peptide nucleic acid technology for generating polymers. Non-limiting examples of suitable nucleobases include: adenine, cytosine, guanine, thymine, uracil, 5-propynyl- uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2- thiouracil and 2- thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6- diaminopurine), hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8- aza-adenine). Nucleobases can be linked to other moieties to form nucleosides, nucleotides, and nucleoside / tide analogs.

[0049] “Nucleoside” refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleobase, e.g., adenine, guanine, cytosine, uracil, thymine, 7- deazaadenine, 7-deazaguanosine, that is linked to the anomeric carbon of a pentose sugar at the 1 ' position, such as a ribose, 2'-deoxyribose, or a 2',3'-di-deoxyribose. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0050] “Nucleotide as used herein refers to a phosphate ester of a nucleoside, e.g., a mono-, a di-, or a triphosphate ester, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose.

[0051] “Nucleobase polymer” or “nucleobase oligomer” refers to two or more nucleobases that are connected by linkages to form an oligomer. Nucleobase polymers or oligomers include, but are not limited to, poly- and oligonucleotides (e.g., DNA and RNA polymers and oligomers), poly-and oligo-nucleotide analogs and poly- and oligonucleotide mimics, such as polyamide or peptide nucleic acids. Nucleobase polymers or oligomers can vary in size from a few nucleobases to several hundred nucleobases or to several thousand nucleobases. The nucleobase polymers or oligomers may include from about 2 to 100 nucleobases or from about 8000 to 10000 nucleobases. For example, the nucleobase polymers or oligomers may have at least about 2 nucleobases, at least about 5 nucleobases, at least about 10 nucleobases, at least about 20 nucleobases, at least about 30 nucleobases, at least about 40 nucleobases, at least about 50 nucleobases, at least about 60 nucleobases, at least about 70 nucleobases, at least about 80 nucleobases, at least about 90 nucleobases, at least about 100 nucleobases, at least about 200 nucleobases, at least about 300 nucleobases, at least about 400 nucleobases, at least about 500 nucleobases, at least about 600 nucleobases, at least about 700 nucleobases, at least about 800 nucleobases, at least about 900 nucleobases, at least about 1000 nucleobases, at least about 2000 nucleobases, at least about 3000 nucleobases, at least about 4000 nucleobases, at least about 5000 nucleobases, at least about 6000 nucleobases, at least about 7000 nucleobases, at least about 8000 nucleobases, at least about 9000 nucleobases, or at least about 10000 nucleobases.

[0052] “Polymer brush” refers to a layer of polymers attached with one end to a surface. The polymers are close together and form a layer or coating that forms its own environment. The brushes may be either in a solvent state, when the dangling chains are submerged into a solvent, or in a melt state, when the dangling chains completely fill up the space available. Additionally, there is a separate class of polyelectrolyte brushes, when the polymer chains themselves carry an electrostatic charge. The brushes may be characterized by the high density of grafted chains. The limited space then leads to a strong extension of the chains, and unusual properties of the system. Brushes may be PCT / US25 / 47509 23 September 2025 (23.09.2025) used to stabilize colloids, reduce friction between surfaces, and to provide lubrication in artificial joints

[0053] “Polynucleotides” or “oligonucleotides” refer to nucleobase polymers or oligomers in which the nucleobases are connected by sugar phosphate linkages (sugar- phosphate backbone). Exemplary poly- and oligonucleotides include polymers of 2'- deoxyribonucleotides (DNA) and polymers of ribonucleotides (RNA). A polynucleotide may be composed entirely of ribonucleotides, entirely of 2'-deoxyribonucleotides or combinations thereof. “Nucleic acid” encompasses “polynucleotide” and “oligonucleotides” and includes single stranded and double stranded polymers of nucleotide monomers.

[0054] “Polynucleotide analog” or “oligonucleotide analog” refers to nucleobase polymers or oligomers in which the nucleobases are connected by a sugar phosphate backbone comprising one or more sugar phosphate analogs. Typical sugar phosphate analogs include, but are not limited to, sugar alkylphosphonates, sugar phosphoramidites, sugar alkyl- or substituted alkylphosphotriesters, sugar phosphorothioates, sugar phosphorodithioates, sugar phosphates and sugar phosphate analogs in which the sugar is other than 2'-deoxyribose or ribose, nucleobase polymers having positively charged sugar-guanidyl interlinkages such as those described in U.S. Patent No. 6,013,785 and U.S. Patent No. 5,696,253.

[0055] “Receptor” as used herein refers to a protein-molecule that recognizes and responds to endogenous-chemical signals. When such endogenous-chemical signals bind to a receptor, they cause some form of cellular / tissue-response. Examples of receptors include, but are not limited to, neural receptors, hormonal receptors, nutrient receptors, and cell surface receptors.

[0056] As used herein, “spacer” refers to a chemical moiety that extends the cleavable group from the specific binding member, or which provides linkage between the binding member and the support, or which extends the label / tag from the photocleavable moiety. In some embodiments, one or more spacers may be included at the N-terminus or C- terminus of a polypeptide or nucleotide-based tag or label in order to distance optimally the sequences from the specific binding member. Spacers may include but are not limited to 6-aminocaproic acid, 6-aminohexanoic acid; 1 ,3-diamino propane; 1 ,3-diamino ethane; polyethylene glycol (PEG) polymer groups, short amino acid sequences, and such as polyglycine sequences, of 1 to 5 amino acids. In some embodiments, the spacer PCT / US25 / 47509 23 September 2025 (23.09.2025) is a nitrobenzyl group, dithioethylamino, 6 carbon spacer, 12 carbon spacer, or 3-(9-((3- carboxypropyl)(tosyl)carbamoyl)acridin-10-ium-10-yl)propane-1 -sulfonate.

[0057] “Specific binding partner” or “specific binding member” as used interchangeably herein refers to one of two or more different molecules that specifically recognize the other molecule compared to substantially less recognition of other molecules. The one of two different molecules has an area on the surface or in a cavity, which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The molecules may be members of a specific binding pair. For example, a specific binding member may include, but is not limited to, a protein, such as a receptor, an enzyme, and an antibody.

[0058] As used herein, “tag” or “tag molecule” both refer to the molecule (e.g., cleaved from the second binding member or an aptamer dissociated from the target analyte) that is translocated through or across a nanopore and provides an indication of the level of analyte in a sample. These terms refer to a single tag molecule or a plurality of the same tag molecule. Likewise “tags”, unless specified otherwise, refers to one or one or more tags.

[0059] “Threshold” as used herein refers to an empirically determined and subjective cutoff level above which acquired data is considered “signal”, and below which acquired data is considered “noise”. The use of a threshold for digital signal counting is depicted in Fig. 29. A computer program based on CUSUM (Cumulative Sums Algorithm) is employed to process acquired data and detect events based on threshold input from the user. Variation between users is avoided by detection of any many events as possible followed by filtering the data afterwards for specific purposes. For example, as can be seen from this figure, events detected above the set threshold impact the population of events that are counted as signal. With a “loose” threshold a lesser number of events will be counted as signal. With a “tight” threshold a greater number of events will be counted as signal. Setting the threshold as loose or tight is a subjective choice based on the desired sensitivity or specificity for an assay, and whether in a given assessment false positives or false negatives would be preferred. Current blockade signatures from DNA translocations were calculated to be 1.2 nA, which was based on an empirical formula relating current change to the diameter of DNA and the thickness of the nanopore membrane (H. Kwok, et al., PLoS ONE, 9(3), 392880, 2014). PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0060] As used herein, reference to movement (e.g., of a nanoparticle, tag, tag molecule, or other) “through or across” a nanopore means alternately, through, or across, in other words, from one side to another of a nanopore, e.g., from the cis to the trans side, or vice versa.

[0061] “Tracer” as used herein refers to an analyte or analyte fragment conjugated to a tag or label, wherein the analyte conjugated to the tag or label can effectively compete with the analyte for sites on an antibody specific for the analyte. For example, the tracer may be an analyte or analog of the analyte, such as cyclosporine or its analog ISA247, vitamin D and its analogs, sex hormones and their analogs, etc.

[0062] “Translocation event” as used herein refers to an event in which a tag translocates through or across (e.g., from the cis to trans side or vice versa) the layer or nanopore.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety to disclose and describe the methods and / or materials in connection with which the publications are cited. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0064] LYOPHILIZED PELLETS

[0065] Provided herein are lyophilized pellets. The lyophilized pellet may comprise paramagnetic particles comprising a surface capable of binding to an analyte; a sugar having a glass transition temperature greater than 65° C; or a mixture of two or more sugars, wherein the mixture has a glass transition temperature greater than 65° C; and a water-soluble polysaccharide, wherein the water-soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars. The lyophilized pellets of the present invention exhibit better storage stability than the conventional lyophilized pellets.

[0066] As used herein the term “lyophilized pellet” or “freeze drying paramagnetic particles” are used interchangeably to refer to a material that has substantially low water content, e.g., to less than about 5% water. Lyophilization or freeze-drying is an effective PCT / US25 / 47509 23 September 2025 (23.09.2025) process of removing water content by sublimation from a frozen mixture, usually under vacuum. Lyophilized materials typically have a porous structure that arises when bubbles of water vapor expand within the material during the process. Lyophilization has been used to create pellets as small as the constituents of fine powders and larger pellets. Such pellets would be useful in microfluidic systems, where the volumes of reagents are on the scale of a few microliters, and where reaction chambers are only a few millimeters in dimension.

[0067] Typically, the lyophilized pellets of the present invention have an average volume of between about 5 microliters and about 20 microliters (e.g., about 6 microliters, about 9 microliters, about 12 microliters, about 15 microliters, or about 18 microliters). In certain embodiments, the lyophilized pellets have an average volume of between about 8 microliters and about 18 microliters. In certain embodiments, the lyophilized pellets have an average volume of between about 10 microliters and about 15 microliters.

[0068] Typically, the lyophilized pellets or particles can be of any shape or size. In certain embodiments, the pellets are spherical in shape and have an average diameter of between about 0.5 mm and about 5 mm (e.g., about 4 mm, about 3 mm, about 2.5 mm, about 2 mm, or about 1 mm). In certain embodiments, the pellets have an average diameter of between about 1 mm and about 4 mm.

[0069] In certain embodiments, the lyophilized pellets may comprise paramagnetic particles. The paramagnetic particles have an iron oxide core (e.g., singular core or multicore). By paramagnetic particles (PMPs), it is meant magnetic particles capable of having an analyte of interest attached thereon, e.g., capable of having nucleic acids attached thereon. PMPs are magnetically responsive. Magnetically responsive particles include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as FesC . BaFei20i9, CoO, NiO, Mn2Os, CteOs, and CoMnP. PMPs may be comprised of a paramagnetic material enclosed in a non-magnetic polymer, such as, magnetic materials covered with a polymeric material or magnetic material embedded in a polymer matrix. Such particles may be referred to as magnetic or paramagnetic beads.

[0070] The paramagnetic particles and sugar may be present in the polymer matrix and lyophilized. In certain embodiments, the paramagnetic particles have a particle diameter PCT / US25 / 47509 23 September 2025 (23.09.2025) of about 2 nm to about 200 pm (e.g., about 5 nm to about 150 gm, about 10 nm to about 100 gm, about 50 nm to about 100 pm, about 100 nm to about 50 pm, about 500 nm to about 50 pm, about 1 pm to about 10 pm , or about 10 pm to about 50 pm). In certain embodiments, the lyophilized pellets may comprise mixture of paramagnetic particles having particle diameter between about 2 nm to about 200 pm. In certain embodiments, the lyophilized pellets may comprise two or more populations of paramagnetic particles, wherein a first population of paramagnetic particles has a particle diameter between about 2 nm to about 50 pm and a second population of paramagnetic particles have particle diameter between about 50 pm to about 200 pm. In certain embodiments, the paramagnetic particles have a mean particle size of about 1 pm or less.

[0071] In certain embodiments, the paramagnetic particles comprise a silica-coated surface capable of binding nucleic acids. In certain embodiments, the paramagnetic particles comprise a surface capable of binding proteins.

[0072] As used herein "nucleic acid" means either DNA or RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding or electrostatic interaction to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8- position purine modifications, modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, and methylations. Accordingly, the nucleic acids described herein include not only the standard bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) but also non-standard or non-natural nucleotides. Non-standard or non-natural nucleotides also include bases that form non-natural hydrogen- bonding base pairs (e.g., isobases). By "non-standard nucleotide" or "non-natural nucleotide" it is meant a base other than A, G, C, T, or U that is susceptible to incorporation into an oligonucleotide and that is capable of base-pairing by hydrogen bonding, or by hydrophobic, entropic, or van der Waals interactions, with a complementary non-standard or non-natural nucleotide to form a base pair.

[0073] In certain embodiments, the sugar present in the lyophilized pellet is a disaccharide, such as, for example, lactose or cellobiose. Lactose is a disaccharide composed of galactose and glucose. In certain embodiments, the sugar PCT / US25 / 47509 23 September 2025 (23.09.2025) is a tnsacchande, such as, for example, raffinose. Raffinose is a trisaccharide composed of galactose, glucose, and fructose. In some embodiments, the sugar is D-(+)-Raffinose pentahydrate having molecular weight of 591 .51 Daltons. In certain embodiments, the sugar comprises a bioflavonoid, such as, for example, hesperidin or rutin. Rutin is a glycoside combining the flavonol quercetin and the disaccharide rutinose (a-L- rhamnopyranosyl-(1 ^6)-[3-D-glucopyranose). Hesperidin is a flavanone glycoside. In some embodiments, the sugar is hesperidin methyl chaicone having molecular weight of 624.59 Daltons.

[0074] In certain embodiments, sugars may be used in combinations, such as, for example, one or more disaccharides, one or more trisaccharides, one or more bioflavonoids, a disaccharide and a trisaccharide, a disaccharide and a bioflavonoid, or a trisaccharide and bioflavonoid. For example, raffinose and cellobiose, raffinose and hesperidin, rutin and lactose, or hesperidin and lactose. In certain embodiments, the sugar is not trehalose.

[0075] Any sugar or mixture of two or more sugars that provide for a glass transition temperature (Tg) in the lyophilized pellet that is greater than 65° C may be used for generating the lyophilized pellets. For example, a sugar or mixture of sugars may be used for generating the lyophilized pellets and the glass transition temperature of the sugar or mixture of sugars is measured. In certain embodiments, a sugar or mixture of two or more sugars that have a glass transition temperature is greater than 65° C, greater than 70° C or greater than 75° C or greater than 80° C or greater than 85° C may be selected for formulating the lyophilized pellets. In certain embodiments, the glass transition temperature of greater than 65° C and less than 220° C. The glass transition temperature may be measured by differential scanning calorimetry.

[0076] The lyophilized pellets may also comprise a water-soluble polysaccharide. Polysaccharides may act as a bulking agent and help to maintain rigidity of the lyophilized pellets. In certain embodiments, the water-soluble polysaccharide is dextran. Dextran is a complex branched glucan (polysaccharide derived from the condensation of glucose). In certain embodiments, the water-soluble polysaccharide is dextrin. In certain embodiments, the dextrin is derived from maize. In certain embodiments, the water- soluble polysaccharide is not polyvinylpyrrolidone (PVP). In certain embodiments, the water-soluble polysaccharide is not polyvinyl alcohol (PVA). PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0077] In certain embodiments, the water-soluble polysaccharide has a higher molecular weight than the sugar or the mixture of sugars. In certain embodiments, the water-soluble polysaccharide has a molecular weight of about 50,000 Da to 7,000,000 Da (e.g., about 55,000 Da, about 1 ,00,000 Da, about 2,00,000 Da, about 3,00,000 Da, about 4,00,000 Da, about 5,00,000 Da, about 6,00,000 Da, about 7,00,000 Da, about 8,00,000 Da, about 9,00,000 Da, about 1 ,000,000 Da, about 2,000,000 Da, about 3,000,000 Da, about 4,000,000 Da, about 5,000,000 Da, about 6,000,000 Da, or about 6,500,000 Da). In certain embodiments, the water-soluble polysaccharide has a molecular weight of about 60,000 Da to about 7,000,000 Da. In certain embodiments, the water-soluble polysaccharide has a molecular weight of about 1 ,000,000 Da to about 7,000,000 Da. In certain embodiments, the water-soluble polysaccharide has a molecular weight of about 550,000 Da to about 700,000 Da. In certain embodiments, the water-soluble polysaccharide has a molecular weight of about 550,000 Da to about 650,000 Da.

[0078] In certain embodiments, the water-soluble polysaccharide has a higher glass transition temperature than the sugar or the mixture of sugars. In certain embodiments, the water-soluble polysaccharide has a glass transition temperature is greater than about 225° C, or greater than 250° C, or greater than 270° C, or greater than 275° C, or greater than 280° C, or greater than 285° C, may be selected for formulating the lyophilized pellets. In certain embodiments, the glass transition temperature is between about 225° C to about 350° C. The glass transition temperature may be measured by differential scanning calorimetry.

[0079] In certain embodiments, the lyophilized pellet comprises a mixture of two or more sugars, wherein the mixture has a glass transition temperature greater than 65° C and a molecular weight lower than the water-soluble polysaccharide.

[0080] The lyophilized pellets can be stored at ambient or elevated temperature like 50° C for a period of up to 1 month, up to 3 months, up to 6 months, up to 1 year, or up to 2 years without significant loss of functionality of the PMPs, depending on the sugar or mixture of sugars used to make lyophilized pellets. In other words, the lyophilized pellets have a shelf life of up to 1 year, or up to 2 years, or longer in room environments. Shelf life is commonly estimated by two different stability testing procedures: real-time stability tests and accelerated stability tests. In real-time stability testing, the pellets may be stored at recommended storage conditions and monitored until they fail the specification. In accelerated stability tests, the pellets may be stored at elevated stress conditions (such PCT / US25 / 47509 23 September 2025 (23.09.2025) as temperature, humidity, and pH). The lyophilized pellets of the present disclosure have longer shelf life than the conventional lyophilized pellets. In some instances, the lyophilized pellets of the present disclosure have a shelf life that is longer than the shelf life of conventional lyophilized pellets by at least 1 month, at least 2 months, at least 6 months, at least 1 or 2 years at room temperature.

[0081] FORMULATIONS

[0082] Provided herein are formulations for making lyophilized pellets. The formulation comprises a sugar or a mixture of two or more sugars in amount of about 5 to 35 weight percentage of total formulation, wherein a glass transition temperature of the sugar or the mixture of sugars is greater than 65° C; a water-soluble polysaccharide in amount of about 1 to 20 weight percentage of total formulation, wherein the water-soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars; and paramagnetic particles in amount of about 45 to 65 weight percentage of total formulation.

[0083] Typically, the formulation for making lyophilized pellets comprises a sugar or a mixture of two or more sugars, a water-soluble polysaccharide, paramagnetic particles, optionally an IC plasmid and other residues. The sugar(s) act as a spacer among the paramagnetic particles and the polymeric chains of the water-soluble polysaccharide. The water-soluble polysaccharide provides a backbone structure to the lyophilized pellet. The paramagnetic particles enable extraction of target analyte from samples and release later for testing. Other residues are present in an amount less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, less than about 1 % or about 0%). Other residues may be trapped moisture or impurities. In certain embodiments, the formulation may be substantially free of any moisture or impurities. The formulation described herein does not include any biological sample.

[0084] The paramagnetic particles are as described in above paragraphs. In certain embodiments, the paramagnetic particles are in amount of about 45 to 65 weight percentage of total formulation (e.g., about 45 weight percentage, about 50 weight percentage, or about 55 weight percentage). In certain embodiments, the paramagnetic particles are in amount of about 50 to 60 weight percentage of total formulation.

[0085] The sugars are as described in above paragraphs. In certain embodiments, the sugar or the mixture of two or more sugars are in amount of about 5 to 35 weight PCT / US25 / 47509 23 September 2025 (23.09.2025) percentage of total formulation (e.g., about 5 weight percentage, about 10 weight percentage, about 15 weight percentage, about 20 weight percentage, about 25 weight percentage, about 30 weight percentage or about 35 weight percentage). In certain embodiments, the sugar or the mixture of two or more sugars are in amount of about 10 to 20 weight percentage of total formulation. In certain embodiments, the sugar or the mixture of two or more sugars are in amount of about 10 to 15 weight percentage of total formulation.

[0086] The water-soluble polysaccharide is as described in above paragraphs. In certain embodiments, the water-soluble polysaccharide is in amount of about 1 to 20 weight percentage of total formulation (e.g., about 2 weight percentage, about 3 weight percentage, about 4 weight percentage, about 5 weight percentage, about 10 weight percentage, about 15 weight percentage, or about 20 weight percentage). In certain embodiments, the water-soluble polysaccharide is in amount of about 1 to 15 weight percentage of total formulation. In certain embodiments, the water-soluble polysaccharide is in amount of about 1 to 10 weight percentage of total formulation.

[0087] In certain embodiments, the total amount of the sugar and the water-soluble polysaccharide is about 5 to 45 weight percentage of total formulation (e.g., about 10 weight percentage, about 15 weight percentage, about 20 weight percentage, about 25 weight percentage, about 30 weight percentage, about 35 weight percentage, about 40 weight percentage, or about 45 weight percentage). In certain embodiments, the total amount of the sugar and the water-soluble polysaccharide is about 5 to 20 weight percentage of total formulation.

[0088] In certain embodiments, the lyophilized pellet comprises a mixture of two or more sugars, wherein the mixture has a glass transition temperature greater than 65° C and a molecular weight lower than the water-soluble polysaccharide.

[0089] The formulations may extend the shelf life of the pellets up to 2 years period based on accelerated stability tests and real-time stability tests. In some instances, the formulations may extend the shelf life of the pellets up to about 18 months, about 12 months or about 6 months.

[0090] METHODS

[0091] Provided herein are methods of making lyophilized pellets. The method comprises a step of providing the formulation described herein; dispensing droplets of the PCT / US25 / 47509 23 September 2025 (23.09.2025) formulation in liquid nitrogen to generate frozen droplets; and removing water from the frozen droplets to generate lyophilized pellets.

[0092] Typically, one or more sugars, a water-soluble polysaccharide and paramagnetic particles are combined with a solvent (e.g., water) to make a solution. The solution is then placed by a dispensing method, (such as by a pipette), into liquid nitrogen. For example, the solution is introduced into a pipette, and the tip is positioned above the liquid nitrogen, and a droplet of solution is dispensed into the liquid. Preferably the solution is vortexed during at least the period of time it is being introduced into the pipette, the time that the tip is being positioned, and the time that the solution is being dispensed. The tip of pipette is preferably kept far (at least 3 inches away) from the liquid nitrogen to avoid sample congealing in the pipette tip. The droplets rapidly freeze from bottom up within seconds of contact with the liquid nitrogen so that there is no significant loss due to evaporation and no significant change in the physical shape of the dispensed pellet. In this way, a frozen droplet that is almost spherical is created. The frozen droplets are introduced into a lyophilization room and subjected to a vacuum while still frozen for pressure and time sufficient to remove the solvent (e.g., by sublimation) from the droplets, thereby forming lyophilized pellets. The lyophilized pellets are packed and sealed from the environment. The period of residency in the lyophilizer sufficient to produce lyophilized pellets will vary according to pellet size and composition but is typically about 20-40 hours.

[0093] Also provided herein is another method of making lyophilized pellets. The method comprises a step of providing the formulation described herein; and freeze drying the formulation to generate lyophilized powder and compacting the lyophilized powder to generate lyophilized pellets.

[0094] Also provided herein are methods of using the lyophilized pellet described herein, for isolating a target analyte from a sample. The method comprises a step of providing a cartridge comprising a chamber, wherein the chamber is configured for holding a solution; providing the lyophilized pellet in the chamber; wherein the lyophilized pellet is configured for binding to an analyte when the analyte is present in the sample; providing the sample and a lysis buffer in the chamber; mixing the sample and the lysis buffer such that the lyophilized pellet is rehydrated and the paramagnetic particles bind to the analyte; separating the paramagnetic particles from the lysis buffer and optionally washing PCT / US25 / 47509 23 September 2025 (23.09.2025) paramagnetic particles; eluting the target analyte from the paramagnetic particles to provide an elution buffer comprising the isolated analyte.

[0095] Also provided herein are cartridges configured for isolating a target analyte from a sample. The cartridge comprises the lyophilized pellet described herein.

[0096] Exemplary Target Analytes

[0097] As will be appreciated by those in the art, any analyte that can be specifically bound by a first binding member and a second binding member may be detected and, optionally, quantified using methods and devices of the present disclosure.

[0098] In some embodiments, the analyte may be a biomolecule. Non-limiting examples of biomolecules include macromolecules such as, proteins, lipids, and carbohydrates. In certain instances, the analyte may be hormones, antibodies, growth factors, cytokines, enzymes, receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, creatine kinase, and the like), toxins, drugs (e.g., drugs of addiction), metabolic agents (e.g., including vitamins), and the like. Non-limiting embodiments of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, or the like.

[0099] In certain embodiments, the analyte may be a post-translational ly modified protein (e.g., phosphorylated, methylated, glycosylated protein) and the first or the second binding member may be an antibody specific to a post-translational modification. A modified protein may be bound to a first binding member immobilized on a solid support where the first binding member binds to the modified protein but not the unmodified protein. In other embodiments, the first binding member may bind to both the unmodified and the modified protein, and the second binding member may be specific to the post- translationally modified protein.

[0100] In some embodiments, the analyte may be a cell, such as, circulating tumor cell, pathogenic bacteria, viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, Filoviruses (ebola), hepatitis viruses (e.g., A, B, C, D, and E); HPV, etc.; spores, etc.

[0101] A non-limiting list of analytes that may be analyzed by the methods presented herein include A 42 amyloid beta-protein, fetuin-A, tau, secretogranin II , prion protein, Alpha-synuclein, tau protein, neurofilament light chain, parkin, PTEN induced putative PCT / US25 / 47509 23 September 2025 (23.09.2025) kinase 1 , DJ-1 , leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin, Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 a), transthyretin, Vitamin D-binding Protein, proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (semen), p14 endocan fragment, Serum, ACE2, autoantibody to CD25, hTERT, CAI25 (MUC 16), VEGF, slL-2, Osteopontin, Human epididymis protein 4 (HE4), Alpha-Fetoprotein , Albumin, albuminuria, microalbuminuria, neutrophil gelatinase-associated lipocalin (NGAL) , interleukin 18 (IL-18) , Kidney Injury Molecule -1 (KIM-1 ) , Liver Fatty Acid Binding Protein (L-FABP) , LMP1 , BARF1 , IL-8, carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB1 , and LZTS1 , alpha-amylase, carcinoembryonic antigen, GA 125, IL8 , thioredoxin, beta-2 microglobulin levels - monitor activity of the virus, tumor necrosis factor-alpha receptors - monitor activity of the virus, CA15-3, follicle- stimulating hormone (FSH), leutinizing hormone (LH), T-cell lymphoma invasion and metastasis 1 (TIAM1 ), N-cadherin, EC39, amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostate specific antigen), thymosin fBI5, insulin, plasma C-peptide, glycosylated hemoglobin (HBA1c), C-Reactive Protein (CRP), lnterleukin-6 (IL-6), ARHGDIB (Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin-1 ), PFN1 (profilin- 1 ), GSTP1 (Glutathione S-transferase P), S100A1 1 (Protein S100- A1 1 ), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein, mitochondrial), LYZ (Lysozyme C precursor), GPI (Glucose-6-phosphate isomerase), HIST2H2AA (Histone H2A type 2-A), GAPDH (Glyceraldehyde-3- phosphate dehydrogenase), HSPG2 (Basement membrane-specific heparan sulfate proteoglycan core protein precursor), LGALS3BP (Galectin-3-binding protein precursor), CTSD (Cathepsin D precursor), APOE (Apolipoprotein E precursor), IQGAP1 (Ras GTPase-activating-like protein IQGAP1 ), CP (Ceruloplasmin precursor), and IGLC2 (IGLC1 protein), PCDGF / GP88, EGFR, HER2, MUC4, IGF-IR, p27(kip1 ), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1 (3-phosphoinositide dependent protein kinase-1 ), TSC1 , TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1 ), S6K, src, KRAS, MEK mitogen-activated protein kinase 1 , cMYC, TOPO II topoisomerase (DNA) II alpha 170 kDa, FRAP1 , NRG1 , ESR1 , ESR2, PGR, CDKN1 B, MAP2K1 , NEDD4-1 , FOXO3A, PPP1 R1 B, PXN, ELA2, CTNNB1 , AR, EPHB2, KLF6, ANXA7, NKX3-1 , PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA), leptin (LEP), advanced glycosylation end product-specific receptor (AGER aka RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14 molecule (CD14), PCT / US25 / 47509 23 September 2025 (23.09.2025) ferritin (FTH1 ), insulin-like growth factor binding protein 1 (IGFBP1 ), interleukin 2 receptor, alpha (IL2RA), vascular cell adhesion molecule 1 (VCAM1 ) and Von Willebrand factor (VWF), myeloperoxidase (MPO), IL1 a, TNFa, perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm’s Tumor-1 protein, Aquaporin-1 , MLL3, AMBP, VDAC1 , E. coll enterotoxins (heat-labile exotoxin, heatstable enterotoxin), influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum toxins, Shiga toxin, Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxins A and B, etc.

[0102] Exemplary targets of nucleic acid aptamers that may be measured in a sample such as an environmental sample, a biological sample obtained from a patient or subject in need using the subject methods and devices include: drugs of abuse (e.g. cocaine), protein biomarkers (including, but not limited to, Nucleolin, nuclear factor-kB essential modulator (NEMO), CD-30, protein tyrosine kinase 7 (PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform, immunoglobulin p Heavy Chains (IGHM), Immunoglobulin E, av[33 integrin, a-thrombin, HIV gp120, NF-KB, E2F transcription factor, HER3, Plasminogen activator inhibitor , Tenascin C,CXCL12 / SDF-1 , prostate specific membrane antigen (PSMA), gastric cancer cells, HGC-27); cells (including, but not limited to, non-small cell lung cancer (NSCLC), colorectal cancer cells, (DLD-1 ), H23 lung adenocarcinoma cells, Ramos cells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM, acute myeloid leukemia (AML) cells (HL60), small-cell lung cancer (SOLO) cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells, HER-2- overexpressing human breast cancer cells, SK-BR-3, pancreatic cancer cell line (Mia- PaCa-2)); and infectious agents (including, but not limited to, Mycobacterium tuberculosis, Staphylococcus aureus, Shigella dysenteriae, Escherichia coll O157:H7, Campylobacter jejuni, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella 08, Salmonella enteritidis).

[0103] Exemplary targets of protein or peptide aptamers that may be measured in a sample obtained from a patient or subject in need using the subject methods and devices include, but are not limited to: HBV core capsid protein, CDK2, E2F transcription factor, Thymidylate synthase, Ras, EB1 , and Receptor for Advanced Glycated End products (RAGE). Aptamers, and use and methods of production thereof are reviewed in e.g., Shum et al., J Cancer Ther. 2013 4:872; Zhang et al., Curr Med Chem. 2011 ;18:4185; PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0104] Zhu et al., Chem Commun (Camb). 2012 48:10472; Crawford et al., Brief Funct Genomic Proteomic. 2003 2:72; Reverdatto et al., PLoS One. 2013 8:e65180.

[0105] As used herein, “analyte”, “target analyte”, “analyte of interest” may be derived from any suitable source. In some cases, the analyte may comprise a liquid, fluent particulate solid, or fluid suspension of solid particles. In some cases, the analyte may be processed prior to the analysis described herein. For example, the analyte may be separated or purified from its source prior to analysis; however, in certain embodiments, an unprocessed analyte may be assayed directly. The source of the analyte molecule may be synthetic (e.g., produced in a laboratory), the environment (e.g., air, soil, fluid samples, e.g., water supplies, etc.), an animal, e.g., a mammal, a plant, or any combination thereof. In a particular example, the source of an analyte is a human bodily substance (e.g., bodily fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, tissue, organ, or the like). Tissues may include, but are not limited to skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervix tissue, skin, etc. The analyte may be a liquid analyte or a liquid extract of a solid analyte. In certain cases, the source of the analyte may be an organ or tissue, such as a biopsy sample, which may be solubilized by tissue disintegration / cell lysis.

[0106] A wide range of volumes of the fluid analyte may be analyzed. In a few exemplary embodiments, the analyte volume may be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 pL, about 0.1 pL, about 1 pL, about 5 pL, about 10 pL, about 100 pL, about 1 mL, about 5 mL, about 10 mL, or the like. In some cases, the volume of the fluid analyte is between about 0.01 pL and about 10 mL, between about 0.01 pL and about 1 mL, between about 0.01 pL and about 100 pL, or between about 0.1 pL and about 10 pL.

[0107] In some cases, the fluid analyte may be diluted prior to use in an assay. For example, in embodiments where the source of an analyte molecule is a human body fluid (e.g., blood, serum), the fluid may be diluted with an appropriate solvent (e.g., a buffer such as PBS buffer). A fluid sample may be diluted about 1 -fold, about 2-fold, about 3- fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.

[0108] In some cases, the analyte may undergo pre-analytical processing. Pre-analytical processing may offer additional functionality such as nonspecific protein removal and / or PCT / US25 / 47509 23 September 2025 (23.09.2025) effective yet cheaply implementable mixing functionality. General methods of pre- analytical processing may include the use of electrokinetic trapping, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art. In some cases, the fluid analyte may be

[0109] 5 concentrated prior to use in an assay. For example, in embodiments where the source of an analyte molecule is a human body fluid (e.g., blood, serum), the fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. A fluid sample may be concentrated about 1 -fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater, prior to use.

[0110] In certain embodiments, the analyte is not amplified (i.e., the copy number of the analyte is not increased) prior to the measurement of the analyte. For example, in cases where the analyte is DNA or RNA, the analyte is not replicated to increase copy numbers of the analyte. In certain cases, the analyte is a protein or a small molecule.

[0111] 15 EXAMPLES

[0112] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

[0113] Raw Materials

[0114] Tris(hydroxymethyl)aminomethane (Tris), Ethylenediaminetetraacetic acid (EDTA), water, paramagnetic particles, Ng (Neisseria gonorrhoeae), Ct (Chlamydia

[0115] 25 trachomatis), Dextran, D-(+)-Raffinose pentahydrate, Hesperidin methyl chaicone, Trehalose, Lactose, Cellubiose, Mannitol, Stachyose, Rutin, Naringin, maltodextrin, Cyclodextrin, Polyvinylpyrrolidone (PVP), and Polyvinyl alcohol (PVA). PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0116] Example 1 - Preparation of lyophilized pellets

[0117] 1 . A 2x sugar solution (20 g) as per the table below was prepared by weighing 2 g Raffinose, 2 g Hesperidin and 8 mL Dextran 500k (20%) into a 50 mL tube. Then

[0118] 5 8 mL water was added to quantity sufficient (Q.S.) to target weight. The solution was vortexed on a Vortexer for 1 minute to completely mix the solution. The solution was then heated up in 60°C water bath for 1 hour to dissolve floating particles, then it was cooled down to room temperature. The solution should be clear without floating particles. The prepared 2x sugar solution can be used

[0119] 10 immediately or stored at 4°C for no more than 1 month.

[0120] 2. A formulation was prepared according to the table below, by first mixing the calculated volumes of 2x Sugar Solution, Tris, EDTA, and water in a container,

[0121] 15 then the formulation was vortexed on a Vortexer for 15 - 30 seconds to mix. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0122] 3. The tube of pre-aliquoted IC at 1.00E7 cp / pL was thawed on ice. The PMP suspension was attached in container on a rotator mixer and was set at 35 rpm to thoroughly homogenize the particles for at least 5 minutes.

[0123] 4. Corresponding volume of homogenized PMP suspension was added into the formulation from the above steps, the tube was put on the rotator mixer and mixed at 35 rpm for at least 5 minutes. Corresponding volume of IC was added to the formulation, and the tube was on the rotator mixer and mixed at 35 rpm for at least 5 minutes.

[0124] 5. The tube was transferred to lyophilization room. In the lyophilization room, a lyophilization program “Liquid Nitrogen” was first loaded to cool down chamber (chamber temperature was set at -35°C), and waited until the chamber temperature was lower than 0°C.

[0125] 6. Labeled aluminum pans were prechilled on lyophilizer shelf, and an aluminum bucket was put inside a foam box. Liquid nitrogen was poured inside the aluminum bucket to at least 1 centimeter depth such that the dispensed lyophilized pellets were submerged at all times. A pipette was used for liquid dispense by cutting 2- 3 mm end of the pipette tip.

[0126] 7. The formulation was vortexed for at least 15 - 30 seconds, the tube was briefly flicked down, and then 15 pL of the formulation was gently dispensed into liquid nitrogen, with at least 5 seconds of interval between dispenses to avoid pellet merging. The formulation was re-vortexed and inverted for 15 - 30 seconds every 20 - 30 dispenses.

[0127] 8. The filter was prechilled in liquid nitrogen in another foam box. Once all pellets of the same formulation were dispensed, the prechilled aluminum pan was taken out of the lyophilizer shelf. Immediately (within 5 seconds), the pellets submerged in liquid nitrogen were poured onto the prechilled filter to drain the liquid nitrogen. Immediately (within 5 seconds), the drained frozen pellets were poured onto the PCT / US25 / 47509 23 September 2025 (23.09.2025) prechilled aluminum pan. The pan was transferred back to the lyophilizer shelf (chamber temperature < 0°C) within 15 seconds.

[0128] 9. The liquid nitrogen was poured in the aluminum bucket again and the dispensing process was repeated for other formulations. Once all the pellets are transferred

[0129] 5 into the lyophilizer shelves, the lyophilization cycle was started.

[0130] 10. After the lyophilization cycle was stopped, the aluminum pans were transferred to a dry room and covered with tissue paper to avoid mixing pellets between conditions due to static charge.

[0131] 1 1 . Maximum 10 lyophilized pellets each (2 - 3 mm in diameter and spherical shape)

[0132] 10 were placed in a 2mL tube. Such tubes were then put in the aluminum pouch with desiccant packs, sealed and stored.

[0133] Lyophilization Program “Liquid Nitrogen” PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0134] Example 2

[0135] Formulations for making lyophilized pellets are described in Table 1 , Table 2 and

[0136] 5 Table 3 below:

[0137] Table 1

[0138] The above formulations were lyophilized to generate pellets. The dry lyophilized 10 pellets were stored at 50°C for 4-months and tested at certain time points like time zero, 1 month, 2 months, 3 months, 4 months or more. Ng (N. gonorrhoeae) and Ct (C. trachomatis) assays were used as an indicator for the stability performance. Figure 1 shows that the lyophilized pellets of formulations TH40, TH45 and TH48 exhibit a better diagnostic performance than TH38 (control) after being stored in accelerated 15 temperature conditions. For Ng assay, the TH38 (control) sample achieves 4 / 9 (4 positives over 9 measurements) positive rate, while TH40 having lactose, TH45 having 11% raffinose, and TH48 having 12% raffinose achieve almost 10 / 10 positive rate. PCT / US25 / 47509 23 September 2025 (23.O9.2O25)

[0139] Table 2

[0140] The above formulations were lyophilized to generate pellets. The dry lyophilized pellets were stored at 50°C for 4.5-months and tested at certain time points like time zero, 5 1 month, 2 months, 3 months, 4 months or more. Ng (N. gonorrhoeas) and Ct (C. trachomatis) assays were used as an indicator for the stability performance. Figure 2 shows that the lyophilized pellets of formulation TH27 exhibit a better diagnostic performance than TH23 (control). After the dry lyophilized pellets were stored at 50°C for 4.5 months, the formulation TH27 containing cellobiose showed 10 / 10 Ng positive, while 10 the TH23 (control) sample containing trehalose showed only 8 / 10 Ng positive rate. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0141] Table 3

[0142] The above formulations were lyophilized to generate pellets. The dry lyophilized pellets were stored at 50°C for 4-months and tested at certain time points like time zero, 5 1 month, 2 months, 3 months, 4 months or more. Ng (A / , gonorrhoeae) and Ct (C. trachomatis) assays were used as an indicator for the stability performance. Figure 3 shows that the lyophilized pellets of formulations TH16 and TH19 exhibit a better diagnostic performance than TH 12 (control) after being stored in accelerated temperature conditions.

[0143] 10 Table 4 below demonstrates performance of different formulations for making lyophilized pellets.

[0144] PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0145] Table 4:

[0146] The formulations of present disclosure can offer good stability and therefore longer shelf life.

[0147] 5 In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject

[0148] 10 matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims. PCT / US25 / 47509 23 September 2025 (23.09.2025)

[0149] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or

[0150] 5 scope of the appended claims.

[0151] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0152] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §1 12(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim

[0153] 25 only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 1 12 (f) or 35 U.S.C. §1 12(6) is not invoked.

Claims

PCT / US25 / 47509 23 September 2025 (23.09.2025)CLAIMSWhat is claimed is:1 . A lyophilized pellet, comprising: paramagnetic particles comprising a surface capable of binding a target analyte; a sugar having a glass transition temperature greater than 65° C; or a mixture of two or more sugars, wherein the mixture has a glass transition temperature greater than 65° C; and a water-soluble polysaccharide, wherein the water-soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars.

2. The lyophilized pellet of claim 1 , wherein the sugar is a disaccharide.

3. The lyophilized pellet of claim 1 , wherein the sugar is a trisaccharide.

4. The lyophilized pellet of any one of claims 1 -3, wherein the sugar comprises a bioflavonoid.

5. The lyophilized pellet of any one of claims 1 -4, wherein the sugar is raffinose, cellobiose, hesperidin, rutin, or lactose.

6. The lyophilized pellet of any one of claims 1 -5, wherein the water-soluble polysaccharide has molecular weight of about 50,000 Da to 7,000,000 Da.

7. The lyophilized pellet of any one of claims 1 -6, wherein the water-soluble polysaccharide is dextran.

8. The lyophilized pellet of any one of claims 1 -7, wherein the paramagnetic particles have a particle diameter of about 2 nm to about 200 pm.

9. The lyophilized pellet of any one of claims 1 -8, wherein the paramagnetic particles comprise a surface capable of binding nucleic acids.

10. The lyophilized pellet of any one of claims 1 -9, wherein the paramagnetic particles comprise a surface capable of binding proteins.

11. A formulation for making a lyophilized pellet, comprising: a sugar or a mixture of two or more sugars in amount of about 5 to 35 weight percentage of total formulation, wherein a glass transition temperature of the sugar or the mixture of sugars is greater than 65°C;PCT / US25 / 47509 23 September 2025 (23.09.2025) a water-soluble polysaccharide in amount of about 1 to 20 weight percentage of total formulation, wherein the water-soluble polysaccharide has a higher molecular weight and a higher glass transition temperature than the sugar or the mixture of sugars; paramagnetic particles in amount of about 45 to 65 weight percentage of total formulation.

12. The formulation of claim 11 , wherein the total amount of the sugar and the water- soluble polysaccharide is about 5 to 45 weight percentage of total formulation.

13. The formulation of any one of claims 11 -12, wherein the sugar is a disaccharide.

14. The formulation of any one of claims 11 -12, wherein the sugar is a trisaccharide.

15. The formulation of any one of claims 11 -12, wherein the sugar comprises a bioflavonoid.

16. The formulation of any one of claims 11 -15, wherein the sugar is raffinose, cellobiose, hesperidin, rutin, or lactose.

17. The formulation of any one of claims 11 -16, wherein the water-soluble polysaccharide is dextran.

18. The formulation of any one of claims 11 -17, wherein the water-soluble polysaccharide has molecular weight of about 50,000 Da to 7,000,000 Da.

19. The formulation of any one of claims 11 -18, wherein the paramagnetic particle has a particle diameter of about 2 nm to about 200 pm.

20. The formulation of any one of claims 11 -19, wherein the paramagnetic particles are functionalized to bind nucleic acids.21 . The formulation of any one of claims 11 -20, wherein the paramagnetic particles are functionalized to bind proteins.

22. A method of making lyophilized pellets, comprising providing the formulation of any one of claims 11 -21 ; and freeze drying the formulation to generate lyophilized powder and compacting the lyophilized powder to generate lyophilized pellets.

23. A method of making lyophilized pellets, comprising providing the formulation of any one of claims 11 -21 ; dispensing droplets of the formulation in liquid nitrogen to generate frozen droplets; and removing water from the frozen droplets to generate lyophilized pellets.PCT / US25 / 47509 23 September 2025 (23.09.2025)24. A cartridge configured for isolating a target analyte from a sample, the cartridge comprising the lyophilized pellet of any one of claims 1 -10.

25. A method of using the lyophilized pellet of any one of claims 1-10, for isolating a target analyte from a sample, comprising: providing a cartridge comprising a chamber, wherein the chamber is configured for holding a solution; providing the lyophilized pellet in the chamber; wherein the lyophilized pellet is configured for binding to an analyte when the analyte is present in the sample; providing the sample and a lysis buffer in the chamber; mixing the sample and the lysis buffer such that the lyophilized pellet is rehydrated and the paramagnetic particles bind to the analyte; separating the paramagnetic particles from the lysis buffer and optionally washing paramagnetic particles; eluting the target analyte from the paramagnetic particles to provide an elution buffer comprising the isolated analyte.

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