Point-of-care testing system for antithrombin III

Abstract

Compounds and compositions for determining the level of antithrombin (ATIII) in a sample are described, along with a system for determining the level of ATIII in a point-of-care setting. Methods for forming the compounds and compositions are also described. Methods for using the compounds and compositions to quantify the level of ATIII in a subject are further described. Systems and devices are provided that efficiently determine the level of ATIII in a point-of-care setting to facilitate determining the dosage or heparin or ATIII to administer to a patient.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 302,395, filed January 24, 2022, and U.S. Provisional Application No. 63 / 368,212, filed July 12, 2022, each of which is incorporated by reference herein.

[0002] Sequence Listing The sequence listing set forth in file 573852_SeqListing_ST26.xml is 19 kilobytes in size, was created on December 1, 2022, and is incorporated herein by reference.

[0003] Embodiments of the present disclosure relate generally to testing antithrombin III levels in patients, and more specifically to a point-of-care testing system for testing antithrombin III levels in patients in a care setting with results obtained quickly and efficiently. [Background technology]

[0004] Antithrombin (ATIII) is a circulating serpin (serine protease inhibitor) protein produced by the liver that functions as a regulator / buffer of inflammation and coagulation. ATIII inhibits inflammation and coagulation by binding to and inhibiting thrombin. If humans are born with less than 20% ATIII activity, they exhibit severe hypercoagulability and most die within the first few days of birth. Levels of 30-60% are associated with chronic and sometimes acute life-threatening conditions. ATIII deficiency is one of the most common causes of deep vein thrombosis and spontaneous pulmonary embolism.

[0005] Heparin is the most commonly used intravenous agent for anticoagulation in hospitals today. Heparin acts as an anticoagulant through its interaction with ATIII. The antithrombin activity of ATIII is increased 2000-4000 fold by binding to heparin. Approximately 15% of all hospitalized patients receive heparin at some point during their hospital visit. It is required for use of heart-lung machines. In cardiac and vascular surgery, heparin is administered in moderate to large doses (250-600 u / Kg heparin). Smaller doses are routinely utilized for procedures such as cardiac catheterization, renal dialysis, and many other hemofiltration or radiological procedures.

[0006] Patient response to heparin is highly variable due to polymorphisms within the population, and various diseases, injuries, conditions, or drugs may reduce circulating ATIII. In particular, heparin therapy reduces circulating ATIII. In cardiac surgery, the variability of human response to heparin is a major problem. In patients with low ATIII, heparin does not function well as an anticoagulant. Such patients require administration of exogenous ATIII. Antithrombin supplementation improves the survival rate of cardiac surgery patients. However, excess antithrombin can lead to adverse bleeding events. Ideally, the level of ATIII in a subject would be determined prior to administration of heparin or ATIII. Currently, there is no reliable means to predict or rapidly measure a subject's ATIII level. Existing tests for ATIII indirectly assess anti-Xa activity in the presence or absence of heparin, or utilize complex and / or infrequently performed enzyme or immunoassays in a hospital setting. Summary of the Invention

[0007] Compounds, compositions, and kits useful for determining the level of antithrombin III (ATIII) in a sample are described. The compounds, compositions, and kits include a signal aptamer that binds to thrombin and generates a detectable signal in the presence of ATIII. In some embodiments, the signaling aptamer includes a first thrombin-specific aptamer containing a fluorescent label and a second thrombin-specific aptamer containing a quencher. In some embodiments, the signaling aptamer is provided bound to thrombin in a thrombin signaling aptamer complex. Upon binding to thrombin, hybridizing sequences present in each of the signaling aptamers hybridize to form a duplex region. The formation of the duplex places the fluorescent label in close proximity to the quencher, decreasing the fluorescence of the fluorescent label. In the presence of ATIII, ATIII binds to thrombin and displaces the first signaling aptamer, the second signaling aptamer, or both signaling aptamers. The displacement of the signaling aptamer results in an increase in the fluorescent signal. The increase in fluorescent signal can be measured and used to detect and / or quantitate ATIII in a sample.

[0008] In some embodiments, the kit further contains heparin. Proteins present in a sample, such as a blood sample, can disrupt the aptamer-thrombin complex. ATIII-independent disruption of the aptamer-thrombin complex can lead to an overestimation of the amount of ATIII in the sample. Heparin can be added to the assay to selectively activate ATIII toward the aptamer complex. Heparin enhances the activity of ATIII and accelerates thrombin binding to ATIII. The signal output is higher early when ATIII is activated with heparin than when heparin is not present. The change in the initial rate of signal generation when heparin is added is due to heparin-activated ATIII-dependent signal, not from background protein interactions. Thus, the change in the initial rate of fluorescence increase in the assay when comparing samples tested in the presence of heparin with samples tested in the absence of heparin can be correlated with the ATIII concentration in blood.

[0009] In some embodiments, heparin is bound to a solid support and used to extract ATIII from the sample prior to contact with the thrombin signaling aptamer complex.

[0010] Also described are methods for detecting and / or quantifying the amount of ATIII in a sample using the described compounds, compositions, and kits. In some embodiments, the method includes forming a thrombin signaling aptamer complex comprising thrombin, a first signaling aptamer, and a second signaling aptamer, contacting the thrombin signaling aptamer complex with a sample that contains or is suspected of containing ATIII, and measuring the fluorescence emitted by the fluorescent label. In some embodiments, the method includes contacting the thrombin signaling aptamer complex with a sample that contains or is suspected of containing ATIII, and detecting or measuring an increase in the fluorescent signal emitted by the fluorescent label. In some embodiments, the increase in the fluorescent signal is proportional to the level of ATIII in the sample.

[0011] In some embodiments, the method includes forming a thrombin signaling aptamer complex comprising thrombin, a first signaling aptamer, and a second signaling aptamer; contacting the thrombin signaling aptamer complex with a sample containing or suspected of containing ATIII; and measuring the initial rate of fluorescence increase emitted by the fluorescent label in the presence and absence of heparin. In some embodiments, the method includes contacting the thrombin signaling aptamer complex with a sample containing or suspected of containing ATIII; and detecting or measuring the initial rate of increase of the fluorescent signal emitted by the fluorescent label in the presence and absence of heparin. In some embodiments, the change in the initial rate of fluorescence signal increase in the sample containing heparin compared to the initial rate of fluorescence signal increase in the sample lacking heparin is proportional to the level of ATIII in the sample.

[0012] In some embodiments, the method includes contacting a sample containing or suspected of containing ATIII with heparin linked to a solid support, purifying any ATII in the sample from one or more other components of the sample using the heparin linked to the solid support, forming a thrombin signaling aptamer complex comprising thrombin, a first signaling aptamer, and a second signaling aptamer, contacting the thrombin signaling aptamer complex with the purified sample, and measuring the fluorescence emitted by the fluorescent label. In some embodiments, the method includes contacting a sample containing or suspected of containing ATIII with heparin linked to a solid support, purifying any ATII in the sample from one or more other components of the sample using the heparin linked to the solid support, contacting the thrombin signaling aptamer complex with the purified sample containing or suspected of containing ATIII, and measuring the fluorescence signal emitted by the fluorescent label. In some embodiments, the increase in the fluorescence signal is proportional to the level of ATIII in the sample.

[0013] In some embodiments, the described compounds, compositions, kits, and methods can be used to determine the dose of heparin and / or exogenous ATIII to administer to a subject prior to, concurrently with, or following a medical procedure.

[0014] Embodiments provided herein include a system for determining a level of antithrombin (ATIII) in a subject, the system including: a column defining an inlet and an outlet, where heparin beads are retained within the column; a manifold connected to the inlet of the column, where the manifold includes a plurality of ports, each port configured to receive a fluid, the manifold directing the fluid from each port to the inlet of the column; a first syringe including a wash buffer received at a first port of the plurality of ports; a second syringe including plasma received at a second port of the plurality of ports of the manifold; and a third syringe including plasma received at a third port of the plurality of ports of the manifold. a third syringe comprising a wash buffer received at a fourth port of the plurality of ports of the manifold; and a fourth syringe comprising an aptamer-thrombin complex received at a fourth port of the plurality of ports of the manifold, wherein the aptamer-thrombin complex comprises a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher and thrombin, wherein the first and second hybridization sequences are complementary to one another, and wherein when the first and second thrombin-specific signaling aptamers bind to thrombin, fluorescence of the fluorescent label is quenched.

[0015] According to some embodiments, the system includes a substrate, the substrate includes a waste reservoir separated from the column by a waste valve and a fluorescent reservoir separated from the column by a fluorescent valve. The heparin beads retained in the column are washed in some embodiments in response to a first syringe passing a wash buffer through an inlet of the column. The heparin beads of some embodiments are incubated with plasma in response to a second syringe passing plasma through an inlet of the column, the heparin beads capturing ATIII thereon during incubation with the plasma. The heparin beads with captured ATII are washed in some embodiments with a wash buffer in response to a third syringe passing a wash buffer through an inlet of the column. The heparin beads with captured ATIII are incubated in some embodiments with an aptamer-thrombin complex in response to a fourth syringe passing an aptamer-thrombin complex through an inlet of the column, the ATIII captured on the heparin beads triggering the release of aptamer-F into a solution in the column for signal generation. According to some embodiments, the fluorescence of the solution is read to determine the level of ATIII in the plasma.

[0016] Embodiments provided herein include a method for determining levels of antithrombin (ATIII) in a subject, the method including passing a first wash buffer through a column having heparin beads suspended therein, passing plasma through the column, incubating the plasma with the heparin beads, passing a second wash buffer through the column, passing an aptamer-thrombin complex through the column, incubating the aptamer-thrombin complex with the heparin beads, and generating fluorescence indicative of ATIII levels in the plasma.

[0017] According to some embodiments, a first wash buffer is passed through the column by a first syringe, plasma is passed through the column by a second syringe, a second wash buffer is passed through the column by a third syringe, and the aptamer-thrombin complex is passed through the column by a fourth syringe. The method of some embodiments includes washing the heparin beads with the first wash buffer in response to passing the first wash buffer through the column in which the heparin beads are suspended. The method of some embodiments includes capturing ATIII on the heparin beads in response to incubating the plasma with the heparin beads. The method of some embodiments includes washing the heparin beads with the captured ATIII with the second wash buffer in response to passing the second wash buffer through the column. The method optionally includes triggering the release of aptamer F into a solution in the column for signal generation in response to incubating the heparin beads with the captured ATIII with the aptamer-thrombin complex.

[0018] Embodiments provided herein include an apparatus for determining a level of antithrombin (ATIII) in a subject, the apparatus including a column defining an inlet and an outlet, a manifold defining a plurality of ports and a manifold outlet (the manifold outlet is connected to the inlet of the column), a substrate defining a waste flow path extending between a substrate inlet and a waste reservoir, a waste flow path extending between the substrate inlet and a fluorescent reservoir, a waste valve configured to open and close the waste flow path between the substrate inlet and the waste reservoir, and a fluorescent valve configured to open and close the fluorescent flow path between the substrate inlet and the fluorescent reservoir. The apparatus of some embodiments further includes a first syringe received in a first port of the plurality of ports of the manifold, a second syringe received in a second port of the plurality of ports of the manifold, a third syringe received in a third port of the plurality of ports of the manifold, and a fourth syringe received in a fourth port of the plurality of ports of the manifold. According to certain embodiments, the first syringe holds a first wash buffer, the second syringe holds plasma, the third syringe holds a second wash buffer, and the fourth syringe holds an aptamer-thrombin complex. According to some embodiments, in response to the contents of the first syringe, the second syringe, the third syringe, and the fourth syringe being passed sequentially through the column, the fluorescent reservoir fluoresces based on the level of ATIII in the plasma. [Brief description of the drawings]

[0019] [Figure 1] 1 illustrates a system for determining levels of antithrombin (ATIII) in a subject in a point-of-care setting, according to an exemplary embodiment of the present disclosure. [Diagram 2] 1 shows a column of suspended heparin beads in a system for determining levels of ATIII in a subject in a point-of-care setting, according to an exemplary embodiment of the present disclosure. [Diagram 3]1 illustrates a workflow of a system for determining levels of antithrombin (ATIII) in a subject in a point-of-care setting, according to an exemplary embodiment of the present disclosure. [Figure 4] ATIII (antithrombin) binds to the thrombin signal aptamer complex and displaces both the first (TBA15) and second (TBA29) signaling aptamers. For some signaling aptamer pairs, ATIII binding to thrombin results in dissociation of the first (e.g., TBA15) signaling aptamer. [Diagram 5] 1 is a graph showing the increase in fluorescence with increasing concentrations of ATIII. [Figure 6] 1 is a graph showing fluorescence intensity at various wavelengths for a signaling aptamer concentration of 100 nM. The complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 7] 1 is a graph showing fluorescence intensity at various wavelengths for a signaling aptamer concentration of 200 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 8] 1 is a graph showing fluorescence intensity at various wavelengths for a signaling aptamer concentration of 300 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 9] 1 is a graph showing fluorescence intensity at various wavelengths for a signaling aptamer concentration of 400 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 10] FIG. 1 is a graph showing the fluorescence intensity at various wavelengths for increasing ratios of thrombin to signaling aptamers TBA15-F6 and TBA29-D6. TBA15-F6 and TBA29-D6 were used at a concentration of 200 nM. Thrombin was used at 20 nM to 100 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 11]FIG. 1 is a graph showing fluorescence intensity at various wavelengths for increasing ratios of thrombin to signaling aptamers TBA15-F7 and TBA29-D7. TBA15-D7 and TBA29-D7 were used at a concentration of 200 nM. Thrombin was used at 75 nM to 300 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 12] FIG. 1 is a graph showing the fluorescence intensity at various wavelengths for increasing ratios of thrombin to signaling aptamers TBA15-F8 and TBA29-D8. TBA15-D8 and TBA29-D8 were used at a concentration of 200 nM. Thrombin was used at 75 nM to 300 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 13] FIG. 1 is a graph showing fluorescence intensity at various wavelengths for increasing ratios of thrombin to signaling aptamers TBA15-F9 and TBA29-D9. TBA15-D9 and TBA29-D9 were used at a concentration of 200 nM. Thrombin was used at 75 nM to 300 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 14] FIG. 1 is a graph showing the fluorescence intensity at various wavelengths for increasing ratios of thrombin to signaling aptamers TBA15-F10 and TBA29-D10. TBA15-D10 and TBA29-D10 were used at a concentration of 200 nM. Thrombin was used at 75 nM to 300 nM. Complexes in the figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 15] Graph showing the kinetics of formation of TBA15-F9+TBA29-D9+thrombin (1:1:1) complex at 200 nM of each component. [Figure 16] 1 is a graph showing the kinetics of dissociation of the TBA15-F9+TBA29-D9+thrombin (1:1:1) complex in the presence of 1940 nM ATIII. The complex was present at a concentration of 200 nM. [Figure 17]Figure 1 is a graph showing fluorescence titration of 200 nM conjugates with increasing concentrations of heparin from 0.25 g / L to 2.00 g / L. Conjugates in figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 18] Figure 1 is a graph showing fluorescence titration of 200 nM conjugates with increasing concentrations of HBA from 1 g / L to 4 g / L. Conjugates in figure legend are in order of maximum intensity (highest to lowest maximum intensity). [Figure 19] 1 is a graph showing fluorescence titration of 200 nM thrombin signaling aptamer complex with increasing concentrations of human serum albumin (HSA) and ATIII. [Figure 20] 13 is a graph showing fluorescence titration of 200 nM thrombin signaling aptamer complex with increasing concentrations of heparin and ATIII. [Figure 21] FIG. 13 is a graph showing the effect of heparin on the kinetics of signal generation in an aptamer ATIII assay. [Figure 22] 1 is a graph showing the rate of increase in fluorescent signal for various amounts of ATIII in the absence of added heparin. [Figure 23] 1 is a graph showing the rate of increase in fluorescent signal for various amounts of ATIII in the presence of added heparin. [Figure 24] FIG. 1 is a schematic of heparin-bound Sepharose beads extracting ATIII, where remaining proteins are removed and an aptamer thrombin assay is added to generate a fluorescent signal indicative of ATIII originally present in the plasma sample. [Diagram 25] Graphs showing detection of ATIII: dP+B=deficient plasma+beads (5 μL plasma was diluted in 95 μL PBS); dP+A+B=deficient plasma+spiked ATIII (2 μM)+beads (5 μL plasma was diluted in 95 μL ATIII solution (2 μM)); and dP is detection of deficient plasma without beads, NC is negative control where no ATIII was added to the regular assay. [Figure 26]The top graph shows a graph demonstrating the sensitivity of the heparin bead ATT assay showing RLU for various concentrations of ATII. [Figure 27] 1 is a graph showing the fluorescence of TBA15-F9 (F), TBA15-F9+TBA29-D9 (F+Q), TBA15-F9+TBA29-D9+thrombin (F+Q+T), and TBA15-F9+TBA29-D9+thrombin+ATIII (F+Q+T+AT-III) in various buffers. For each buffer, the bars are in the following order: F, F+Q, F+Q+T, F+Q+T+AT-III. [Figure 28] Graph showing the kinetics of formation of TBA15-F9+TBA29-D9+thrombin complex. [Figure 29] 1 is a graph used in determining the detection limit of ATIII. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] A.Definition Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "oligomer" includes a plurality of oligomers, and so forth. The conjunction "or" should be construed in the inclusive sense, i.e., equivalent to "and / or," unless an inclusive sense is unreasonable in the context.

[0021] In general, the term "about" refers to insubstantial variations in the amounts of components of a composition that do not significantly affect the activity or stability of the composition. When the present specification discloses a particular value of a parameter, the specification should alternatively be understood to disclose the parameter at "about" that value. All ranges should be interpreted to include the endpoint in the absence of an explicit exclusion, such as "not including the endpoint," thus, for example, "within 10 to 15" includes the values ​​10 and 15. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It should be understood that both the summary and detailed description above are merely exemplary and explanatory and are not intended to limit the present teachings. In the event that any material incorporated by reference conflicts with the express content of this disclosure, the express content shall control.

[0022] Unless otherwise stated, embodiments herein that are described as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the recited components. Embodiments herein that recite "consisting essentially of" various components are also contemplated as "consisting of." "Consisting essentially of" means that additional components, compositions, or method steps that do not materially alter the basic and novel characteristics of the compositions and methods described herein may be included in those compositions or methods.

[0023] An "aptamer" is a short (often less than 40 nucleobases in length) single-stranded polynucleotide molecule that selectively binds to a specific molecular target, such as a protein or protein epitope. Aptamers are easily produced by chemical synthesis. Aptamers can have desirable storage properties. In some embodiments, aptamers induce little or no immunogenicity in therapeutic applications.

[0024] A "signaling aptamer" is an aptamer linked to a hybridization sequence and a label. The hybridization sequence can be linked to the 5' or 3' end of the aptamer. In some embodiments, the hybridization sequence is linked to the aptamer via a linker. In some embodiments, a label is linked to the hybridization sequence.

[0025] A "hybridization sequence" is a short (generally 6-12 nucleotides in length) single-stranded polynucleotide that can base pair with a complementary hybridization sequence. In some embodiments, a first signaling aptamer is linked to a first hybridization sequence that is complementary to a second hybridization sequence linked to a second signaling aptamer. The first and second hybridization sequences can base pair (hybridize) to form a double-stranded region.

[0026] A "label" is a detectable molecule and / or a quencher. Detectable molecules include, but are not limited to, fluorescent labels. A fluorescent label (fluorophore) is a fluorescent compound that can re-emit light upon light excitation. A fluorescent label absorbs light energy at a specific wavelength and re-emits light at a longer wavelength. A quencher is a compound that reduces the fluorescence intensity of a fluorescent label. A quencher can absorb the excitation energy released from a fluorescent molecule and dissipate the energy as heat or as light at a longer wavelength. When the fluorescent label and quencher are in sufficient proximity, the emission of the fluorescent label is suppressed.

[0027] The fluorescent label can be, but is not limited to, xanthene, FITC, FAM™, TET™, CAL FLUOR™ (orange or red), ALEXA FLUOR™, QUASAR™, fluorescein, hexachlorofluorescein (HEX), rhodamine, carboxy-X-rhodamine (ROX), tetramethylrhodamine, IAEDANS, EDANS, coumarin, BODIPY FL, Lucifer Yellow, eosin, erythrosine, Texas Red, cyanine, or a CY dye (e.g., Cy3, Cy3.5, Cy5, Cy5.5).

[0028] The quencher can be, but is not limited to, DABCYL, BLACK HOLE QUENCHER™ (BHQ™, e.g., Black Hole Quencher-0, Black Hole Quencher 1, Black Hole Quencher 2, Black Hole Quencher 3, Black Hole Quencher 650), or a TAMRA™ compound.

[0029] Exemplary fluorescent label / quencher pairs include, but are not limited to, fluorescein and dabcyl, fluorescein and black hole quencher, eosin and DABCYL, coumarin / DABCYL, CY5 and black hole quencher 1, CY5 and black hole quencher 2, CY3 and black hole quencher 1, CY3 and black hole quencher 2.

[0030] A "linker" or linking group is a connection between two atoms that links one chemical group or segment of interest (e.g., an aptamer) to another chemical group or segment of interest (e.g., a hybridization sequence) via one or more covalent bonds. In some embodiments, the linker increases the distance between the two atoms. In some embodiments, the linker is a flexible linker that adds flexibility to the connection. Linkers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups (each of which may contain one or more heteroatoms), heterocycles, amino acids, nucleotides, sugars, and polymeric groups. Polymeric groups include, but are not limited to, polyethylene glycol. In some embodiments, the linker is a PEG. n wherein n is an integer from 1 to 50. In some embodiments, the linker is PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, or PEG 10 The linker does not interfere with the binding of the aptamer to its molecular target (e.g., thrombin). The linker can promote duplex formation by complementary hybridization sequences when two aptamers containing complementary hybridization sequences bind to a target molecule.

[0031] A "sample" includes any physiological fluid derived from a subject. Samples include, but are not limited to, blood, serum, plasma, and fractions thereof. Samples can be pre-treated before use, such as preparing plasma from blood. Samples may be used directly as obtained from a subject or after pre-treatment to modify the characteristics of the sample. Pre-treatment includes, but is not limited to, filtration, dilution, and addition of one or more reagents useful for preparing or analyzing the sample. Samples include original samples or original samples that have undergone one or more pre-treatments. Samples can be of any suitable size or volume. In some embodiments, the sample volume is 1 mL or less, 500 μL or less, 250 μL or less, 100 μL or less, 75 μL or less, 50 μL or less, 35 μL or less, 25 μL or less, or 20 μL or less.

[0032] "Thrombin" is a serine protease. In humans, thrombin is encoded by the F2 gene. Prothrombin (clotting factor II) is proteolytically cleaved to form thrombin in the clotting process. Thrombin then acts as a serine protease that converts soluble fibrinogen into insoluble chains of fibrin and catalyzes other coagulation-related reactions. Thrombin is inactivated by antithrombin III.

[0033] Heparin is a naturally occurring polysaccharide (glycosaminoglycan) that inhibits coagulation (i.e., acts as an anticoagulant). Natural heparin consists of molecular chains of various lengths or molecular weights. Heparin can be unfractionated heparin or low molecular weight heparin. LMWH is defined as a heparin salt having an average molecular weight of less than 8000 Da and at least 60% of all chains having a molecular weight of less than 8000 Da.

[0034] "Heparin beads" include heparin bound (e.g., conjugated or immobilized) to a support or matrix. The support or matrix can be, but is not limited to, sepharose, sepharose beads, agarose beads, gel, acrylic beads, silica. Heparin beads have been widely used for affinity purification of various heparin-binding proteins or ligands, such as antithrombin III.

[0035] "Separation" or "purification" means that one or more components of a sample are removed or separated from other sample components. Sample components include cellular fragments, proteins, carbohydrates, lipids, and nucleic acids. Purification does not imply any degree of purification.

[0036] B. Purpose ATIII is a serine protease inhibitor that, when activated by heparinoid glycosaminoglycans at a specific pentasaccharide sequence on the protein (ATIII), undergoes a conformational change and increases binding affinity for thrombin by 100-1000-fold. Thrombin is a potent inflammation and coagulation amplifier that is released from endothelial cells, platelets, and leukocytes in response to a wide range of proinflammatory stimuli. ATIII is manufactured in the liver as a non-constitutive protein. A normal liver in healthy individuals results in a steady and consistent production of ATIII. In disease states or when certain drugs, such as heparin, are administered, levels of ATIII are reduced. ATIII is carried in the plasma but binds to the endothelial glycocalyx surface (heparin). Thus, the interaction between bound heparin and ATIII forms part of the endothelial brush border barrier against inflammation and thrombosis. ATIII is essential to maintain normal vascular function. Abnormalities in ATIII levels, such as nonproduction or consumption, or the presence of polymorphic, less functional ATIII, can lead to the formation of venous and, more rarely, arterial clots with embolization. ATIII genetic dysfunction is probably the largest cause of spontaneous deep vein thrombosis (DVT) and pulmonary embolism (PE). ATIII not only binds to thrombin, but also inhibits other coagulation cascade proteins in the contact activation cascade. Evidence suggests that the cascade is a direct link from tissue destruction to bradykinin and complement activation. Thus, ATIII is a buffer on that link, creating a system in which small amounts of tissue damage do not cause systemic inflammatory events.

[0037] During some disease states, ATIII levels in plasma are reduced. In sepsis, ATIII levels are reduced, and in severe sepsis, levels are very low. Trauma and vascular catastrophes such as aortic dissection cause an immediate and rapid reduction in circulating ATIII. In pre-eclampsia (toxic pregnancy) and eclampsia, a sudden and catastrophic reduction in ATIII is seen. In trauma, and possibly in toxicity of pregnancy, the reduction in ATIII is due to the production or release of large amounts of tissue factor and thrombin. Liver disease causes a reduction in the production of ATIII. However, the most important cause of the reduction in ATIII is the use of the drug heparin.

[0038] Heparin is an injectable complex of polysaccharides that contains the key pentasaccharide sequence for activating ATIII. High molecular weight (HMW) heparin is isolated from porcine intestine, and the commercially available drug is a mixture of active and inactive (>60% by weight) diffusely distributed molecular weight species of polysaccharides. Low molecular weight (LMW) heparin is more "purified", more "targeted" and specific, but is not as easily controlled without an activity antagonist. HMW heparin, and to some extent LMW heparin at present, are the most widely utilized injectable drugs in medicine worldwide. It is estimated that 30-60% of all patients hospitalized for more than 24 hours are treated with heparin. Heparin consumes ATIII, the body's natural buffering capacity for inflammation. Heparin also displaces or displaces native heparan from the endothelial system, thus making the vascular tree more susceptible to inflammatory attacks. Heparin is also highly antigenic, resulting in heparin PF4 antibodies in most humans exposed to the drug for extended periods of time. Heparin is widely utilized in surgery. Vascular and open-heart surgery would not be possible today without the use of injectable large-dose heparin and its reversal with another protein, protamine. In cardiac surgery, the average decline in ATIII levels is from 80-120% of normal activity toward 40-60% activity. However, a significant outlier group of patients shows a severe and potentially catastrophic decline in ATIII, reaching levels of 15-30% of normal activity. These levels are similar to those seen in non-survivable congenital ATIII deficiency or severe post-traumatic massive coagulopathy of trauma leading to diffuse intravascular coagulation (DIC).

[0039] ATIII levels can be monitored in plasma by central laboratories in most hospitals. A chromogenic assay is performed on isolated spun plasma, which is reported as % of normal activity. It is therefore a functional assay of activity. Such chromogenic assays are available on a STAT basis in some hospitals, but in most hospitals, ATIII testing is available in the standard laboratory testing time frame of 24 hour turnaround. Thus, physicians do not routinely utilize ATIII testing to guide treatment. ATIII levels can change rapidly, predict potential coagulation disorders, and are important in the most widely used drug in medicine today (heparin), but are rarely tested as a protein level. The rapid change and relatively long turnaround time limit the effectiveness of monitoring ATIII levels, as they can change substantially before results are returned.

[0040] Medical protocols have found ways to take advantage of heparin by using surrogate tests for clotting such as the activated partial thromboplastin time (APTT) and the activated clotting time (ACT). Both depend on the number or protein (and cell-ACT) interactions. The tests bear some relationship to ATIII activity, but do not measure it directly, nor do they act in a way that allows for ATIII titration. As a result, monitoring ATIII levels in standard medical practice and disease states lacks precision, accuracy, and speed.

[0041] The embodiments provided herein provide a point-of-care testing system for testing ATIII levels in patients in a care environment with results obtained relatively quickly compared to conventional laboratory-based methods. The embodiments enable real-time point-of-care testing based on the development of a biochemical method for the detection of ATIII. The embodiments include a dual aptamer-based thrombin ATIII binding assay that allows for quantitative detection of pure ATIII spiked in saline and human plasma. The embodiments use two complementary aptamers to bind to thrombin, whose fluorophores are spaced to quench each other (on-state), as shown in FIG. 4. The intensity of the fluorescence is a direct correlation to the concentration of ATIII.

[0042] C. Signal Transduction Aptamers Compounds, compositions, kits, and methods for detecting and / or quantifying levels of ATIII in a sample are described. Embodiments of the present disclosure provide a point-of-care test system for measuring levels of ATIII in patients in a care setting with relatively quickly obtained results.

[0043] In some embodiments, the compounds and compositions include one or more signaling aptamers that bind to thrombin and generate a detectable signal in the presence of ATIII. In some embodiments, the compounds, compositions, and kits include a first signaling aptamer and a second signaling aptamer, each of which binds to thrombin. The first and second signaling aptamers bind to different epitopes on thrombin. In some embodiments, the first aptamer contains a detectable label, such as a fluorescent label, and the second aptamer contains a quencher that reduces or alters the fluorescence of the fluorescent label. In some embodiments, the second aptamer contains a detectable label, such as a fluorescent label, and the first aptamer contains a quencher that reduces or alters the fluorescence of the fluorescent label. The first and second signaling aptamers are configured such that when both signaling aptamers bind to thrombin, the quencher quenches the signal from the fluorescent label. The first and second signaling aptamers are further designed such that ATIII binding to thrombin results in dissociation of at least one of the aptamers from thrombin. Dissociation of the signaling aptamer from thrombin results in dequenching of the fluorescent label and an increase in the detectable signal (i.e., fluorescence).

[0044] The first and second signaling aptamers contain hybridization sequences that can be cleaved from the duplex region when the signaling aptamer is bound to thrombin. In some embodiments, the label on the signaling aptamer is linked to the hybridization sequence. In some embodiments, the hybridization sequence is designed such that the fluorescent label is placed in close proximity to the quencher when the hybridization sequence base pairs to form a duplex. The hybridization sequence forms a sufficiently stable duplex such that the fluorescent label is quenched when the signaling aptamer binds to thrombin in the absence of ATIII. In some embodiments, the hybridization sequence is further designed such that the binding of ATIII to thrombin results in unquenching and increased fluorescence.

[0045] In some embodiments, the signaling aptamer contains a linker between the aptamer sequence and the hybridization sequence. The linker can be used to increase the spacing between the aptamer sequence and the hybridization sequence and / or to add flexibility between the aptamer sequence and the hybridization sequence. The linker is designed to promote duplex formation between the hybridization sequence when the signaling aptamer binds to thrombin.

[0046] In some embodiments, the first signaling aptamer contains the aptamer nucleotide sequence of the thrombin aptamer TBA15. TBA15 is a 15-mer single stranded DNA having the sequence 5'-GGTTGGTGTGGTTGG-3' (SEQ ID NO: 1). The TBA15 aptamer binds to the exosite I epitope of thrombin with a Kd of about 100 nM. Exosite I is the binding site for fibrinogen on thrombin. In other embodiments, the first signaling aptamer comprises the aptamer nucleotide sequence of the thrombin aptamer TBA29 (described in more detail below). In some embodiments, the first signaling aptamer comprises a first hybridization sequence linked to the 3' end of the aptamer. In some embodiments, the first hybridization sequence is linked to the 3' end of the aptamer via a linker. In some embodiments, the linker comprises PEG. In some embodiments, the PEG is PEG. 1~10 (for example, - (CH 2- CH 2- O) 1~10- In some embodiments, the PEG comprises PEG6. In some embodiments, the first signaling aptamer comprises a fluorescent molecule linked to the first hybridization sequence. In some embodiments, the first signaling aptamer comprises a fluorescent molecule linked to the 3' end of the first hybridization sequence.

[0047] In some embodiments, the second signaling aptamer contains the aptamer nucleotide sequence of the thrombin aptamer TBA29. TBA29 is a 29-mer single stranded DNA having the sequence 5'-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' (SEQ ID NO:2). The TBA29 aptamer binds to the exosite II epitope of thrombin with a Kd of about 0.5 nM. The exosite II epitope is involved in the activation of factors V / VIII and mediates heparin binding. In other embodiments, the second signaling aptamer contains the aptamer nucleotide sequence of the thrombin aptamer TBA15. In some embodiments, the second signaling aptamer comprises a second hybridization sequence linked to the 5' end of the aptamer. In some embodiments, the second hybridization sequence is linked to the 5' end of the aptamer via a linker. In some embodiments, the linker comprises PEG. In some embodiments, PEG is 1~10 (for example, - (CH 2- CH 2- O) 1~10- In some embodiments, the PEG comprises PEG6. In some embodiments, the second signaling aptamer comprises a quencher linked to the second hybridization sequence. In some embodiments, the second signaling aptamer comprises a quencher linked to the 5' end of the second hybridization sequence.

[0048] Other thrombin-binding aptamers can be identified using methods known in the art, including, but not limited to, systematic evolution of ligands by exponential enrichment (SELEX) and automated in vitro selection.

[0049] In some embodiments, the hybridization sequence of the signaling aptamer is 5-20 nucleobases in length. In some embodiments, the hybridization sequence of the signaling aptamer is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length. In some embodiments, the hybridization sequence of the signaling aptamer is 6-10 nucleobases in length. In some embodiments, the hybridization sequence of the signaling aptamer is 6, 7, 8, 9, or 10 nucleobases in length. In some embodiments, the hybridization sequence of the signaling aptamer is 9 nucleobases in length.

[0050] In some embodiments, the hybridization sequence of the signaling aptamer hybridizes to form a duplex 6-10 base pairs in length. In some embodiments, the hybridization sequence of the signaling aptamer hybridizes to form a duplex 6, 7, 8, 9, or 10 base pairs in length. In some embodiments, the hybridization sequence of the signaling aptamer hybridizes to form a duplex 9 base pairs in length.

[0051] In some embodiments, the first signaling aptamer is n -3' of a first hybridization sequence, each N is independently any nucleobase, n is an integer between 6 and 10, and the second signaling aptamer comprises a second hybridization sequence that is complementary to the first hybridization sequence. In some embodiments, the hybridized duplex contains four G:C base pairs and five A:T base pairs.

[0052] In some embodiments, the hybridization sequence of the signaling aptamer comprises or consists of the sequence 5'-GTCGTA-3' (SEQ ID NO: 3) or 5'-TACGAC-3' (SEQ ID NO: 4). In some embodiments, the first and second hybridization sequences of the signaling aptamer comprise or consist of 5'-GTCGTA-3' (SEQ ID NO: 3) and 5'-TACGAC-3' (SEQ ID NO: 4).

[0053] In some embodiments, the hybridization sequence of the signaling aptamer comprises or consists of the sequence 5'-GTCGTAT-3' (SEQ ID NO:5) or 5'-ATACGAC-3' (SEQ ID NO:6). In some embodiments, the first and second hybridization sequences of the signaling aptamer comprise or consist of the sequences 5'-GTCGTAT-3' (SEQ ID NO:5) and 5'-ATACGAC-3' (SEQ ID NO:6).

[0054] In some embodiments, the hybridization sequence of the signaling aptamer comprises or consists of the sequence 5'-GTCGTAGT-3' (SEQ ID NO: 7) or 5'-ACTACGAC-3' (SEQ ID NO: 8). In some embodiments, the first and second hybridization sequences of the signaling aptamer comprise or consist of 5'-GTCGTAGT-3' (SEQ ID NO: 7) and 5'-ACTACGAC-3' (SEQ ID NO: 8).

[0055] In some embodiments, the hybridization sequence of the signaling aptamer comprises or consists of the sequence 5'-GTCGTAAGT-3' (SEQ ID NO: 9) or 5'-ACTTACGAC-3' (SEQ ID NO: 10). In some embodiments, the first and second hybridization sequences of the signaling aptamer comprise or consist of 5'-GTCGTAAGT-3' (SEQ ID NO: 9) and 5'-ACTTACGAC-3' (SEQ ID NO: 10).

[0056] In some embodiments, the hybridization sequence of the signaling aptamer comprises or consists of the sequence 5'-GTCGTAAGCT-3' (SEQ ID NO:11) or 5'-AGCTTACGAC-3' (SEQ ID NO:12). In some embodiments, the first and second hybridization sequences of the signaling aptamer comprise or consist of 5'-GTCGTAAGCT-3' (SEQ ID NO:11) and 5'-AGCTTACGAC-3' (SEQ ID NO:12).

[0057] In some embodiments, the hybridization sequence of the signaling aptamer comprises or consists of the sequence 5'-GTCGTAAGT-3' (SEQ ID NO: 9) or 5'-ACTTACGAC-3' (SEQ ID NO: 10). In some embodiments, the TBA15 signaling aptamer comprises a 3' hybridization sequence consisting of 5'-GTCGTAAGT-3' (SEQ ID NO: 9). In some embodiments, the TBA15 signaling aptamer comprises a 3' hybridization sequence consisting of 5'-ACTTACGAC-3' (SEQ ID NO: 10). In some embodiments, the TBA29 signaling aptamer comprises a 5' hybridization sequence consisting of 5'-GTCGTAAGT-3' (SEQ ID NO: 9). In some embodiments, the TBA29 signaling aptamer comprises a 5' hybridization sequence consisting of 5'-ACTTACGAC-3' (SEQ ID NO: 10). In some embodiments, the TBA15 signaling aptamer comprises a 3' hybridization sequence consisting of 5'-GTCGTAAGT-3' (SEQ ID NO: 9) and the TBA29 signaling aptamer comprises a 5' hybridization sequence consisting of 5'-ACTTACGAC-3' (SEQ ID NO: 10). In some embodiments, the TBA15 signaling aptamer comprises a 3' hybridization sequence consisting of 5'-ACTTACGAC-3' (SEQ ID NO: 10) and the TBA29 signaling aptamer comprises a 5' hybridization sequence consisting of 5'-GTCGTAAGT-3' (SEQ ID NO: 9).

[0058] In some embodiments, the signaling aptamer comprises a linker between the protein binding region (aptamer) and the hybridization sequence. In some embodiments, the linker is a flexible linker. The linker can be a nucleobase linker or a non-nucleobase linker. The non-nucleobase linker can be, but is not limited to, a PEG or an aliphatic chain. In some embodiments, the linker is a PEG group. The PEG group can be (PEG) n where n is an integer from 1 to 20. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is 6.

[0059] In some embodiments, a first signaling aptamer containing a first hybridization sequence and a fluorescent label is paired with a second signaling aptamer containing a second hybridization sequence complementary to the first hybridization sequence, and a quencher that reduces or alters the fluorescence emitted by the fluorescent label.

[0060] In some embodiments, the kit further contains heparin. The heparin can be naturally occurring or synthetic. The heparin can be unfractionated or low molecular weight heparin.

[0061] In some embodiments, the kit further comprises heparin linked to a support. The heparin bound to the support can be, but is not limited to, heparin beads. The heparin linked to a solid support can be used to purify any ATIII present in the sample from one or more other components in the sample.

[0062] In some embodiments, the kit includes suitable assay containers (e.g., sample tubes or wells) for determining ATIII in the presence and absence of heparin. In some embodiments, the kit includes suitable assay containers for determining the initial rate of signal generation in the presence and absence of heparin. Measurement of the fluorescent signal in the presence and absence of heparin can be used to correct for proteins other than ATII in the sample that may disrupt the aptamer-thrombin complex. Proteins in the sample other than ATIII that disrupt the aptamer-thrombin complex may lead to the assay overestimating the amount or concentration of ATIII in the sample. Because heparin selectively activates ATIII toward the aptamer complex, the signal is higher at early time points when ATIII is activated with heparin than when heparin is absent. By adding heparin to the aptamer assay and comparing the initial rate in the presence of heparin to the initial rate in the absence of heparin, the change in the initial rate of signal generation is due to heparin-activated ATIII. The change in initial rate between the signal measured in the presence and absence of heparin correlates with the concentration of ATIII in the blood.

[0063] D. Composition In some embodiments, a thrombin signal aptamer complex is described. The thrombin signal aptamer complex comprises thrombin bound by the first and second signal aptamers described. In some embodiments, the thrombin signal aptamer complex is in a composition. The composition may contain one or more components that facilitate detection or measurement of fluorescence.

[0064] Thrombin can be prepared by a variety of methods known in the art, and the term "thrombin" is not intended to imply a particular method of production. Thrombin can be purified from naturally occurring sources, can be recombinant, can be synthetic, or can be synthesized from cells such as bacterial, insect, yeast or mammalian cells in culture. Thrombin can be purified from plasma. Both human and non-human thrombin can be used within the present invention. In some embodiments, the thrombin is human thrombin.

[0065] The thrombin signaling aptamer complex can be provided in a solution or a lyophilized powder or cake. The thrombin signaling aptamer complex can also be provided on a solid support, such as a test strip. In some embodiments, the signaling aptamer can be provided in a solution at a concentration of about 100 nM to about 400 nM. In some embodiments, the signaling aptamer can be provided in a solution at a concentration of about 100 nm, about 200 nM, about 300 nM, or about 400 nM. In some embodiments, the signaling aptamer can be provided in a solution at a concentration of about 200 nM. In some embodiments, the thrombin signaling aptamer complex can be provided in a solution at a concentration of about 100 nM to about 400 nM. In some embodiments, the thrombin signaling aptamer complex can be provided in a solution at a concentration of about 100 nM, about 200 nM, about 300 nM, or about 400 nM. In some embodiments, the thrombin signaling aptamer complex may be provided in solution at a concentration of about 200 nM.

[0066] In some embodiments, the molar ratio of the first signaling aptamer to the second signaling aptamer is about 1:1. In some embodiments, the molar ratio of the first and second signaling aptamers to thrombin is about 1:1:2 to 2:2:1 (first signaling aptamer:second signaling aptamer:thrombin). In some embodiments, the molar ratio of the first and second signaling aptamers to thrombin is about 1:1:1 (first signaling aptamer:second signaling aptamer:thrombin). In some embodiments, the molar ratio of the TBA15 signaling aptamer to the TBA29 signaling aptamer is about 1:1. In some embodiments, the molar ratio of the TBA15 signaling aptamer and the TBA29 signaling aptamer to thrombin is about 1:1:2 to 2:2:1 (TBA15 signaling aptamer:TBA29 signaling aptamer:thrombin). In some embodiments, the molar ratio of the TBA15 signaling aptamer and the TBA29 signaling aptamer to thrombin is about 1:1:1 to 1:1:1.25 (TBA15 signaling aptamer:TBA29 signaling aptamer:thrombin). In some embodiments, the molar ratio of the TBA15 signaling aptamer and the TBA29 signaling aptamer to thrombin is about 1:1:1 (TBA15 signaling aptamer:TBA29 signaling aptamer:thrombin).

[0067] When a sample containing ATIII is added to the thrombin signal aptamer complex or the composition containing the thrombin signal aptamer complex, ATIII binds to thrombin, resulting in dissociation of the thrombin signal aptamer complex and an increase in fluorescence from the fluorescent label present on one of the signal aptamers. The increase in fluorescence is proportional to the amount of ATIII in the sample. Dissociation of the thrombin signal aptamer complex includes dissociation of the first signal aptamer, the second signal aptamer, or both signal aptamers from thrombin. In some embodiments, ATIII binding to thrombin dissociates the first signal aptamer.

[0068] When a sample containing ATIII is added to the thrombin signal aptamer complex or the composition containing the thrombin signal aptamer complex in the presence of heparin, heparin activates ATIII to bind to thrombin, resulting in dissociation of the thrombin signal aptamer complex and an increase in fluorescence from the fluorescent label present on one of the signal aptamers. The initial rate of increase in fluorescence is proportional to the amount of ATIII in the sample. Dissociation of the thrombin signal aptamer complex includes dissociation of the first signal aptamer, the second signal aptamer, or both signal aptamers from thrombin. In some embodiments, ATIII binding to thrombin dissociates the first signal aptamer.

[0069] Heparin can be prepared by a variety of methods known in the art, and the term heparin is not intended to imply a particular method of production or a particular molecular weight of heparin. Thrombin can be purified from naturally occurring sources, synthetic, or synthesized from cells such as bacterial, insect, yeast, or mammalian cells in culture. In some embodiments, heparin can be provided as a solution. In some embodiments, heparin is provided coated onto an assay vessel. The assay vessel can be a tube, vial, multi-well plate, or other microfluidic device.

[0070] In some embodiments, heparin is linked to a solid support. The solid support can be, but is not limited to, beads, magnetically attractable particles, nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, slides, and multi-well plates. In some embodiments, the sample is purified to remove one or more components of the sample from the ATIII in the sample before contacting the sample with the thrombin signal aptamer complex. Purifying the sample can include isolating the ATIII bound to heparin from the sample using a solid support, or washing the components of the sample that are not bound to heparin from the solid support.

[0071] In some embodiments, the level of ATIII in the sample is determined by comparing the increase in fluorescence with a standard curve. The standard curve can be obtained by determining the increase in fluorescence of various known concentrations of ATIII when measured under the same conditions.

[0072] In some embodiments, the level of ATIII in the sample is determined by comparing the initial increase rate of fluorescence in the presence of heparin with the initial increase rate in the absence of heparin and comparing with a standard curve.The standard curve can be obtained by determining the increase level of fluorescence of various known concentrations of ATIII when measured under the same conditions.

[0073] E.Kit Also provided is a kit that includes one or more reagents that are used in carrying out the methods disclosed herein, or a kit that includes one or more compounds or compositions disclosed herein.Such a kit can be diagnostic in nature.The kit of the exemplary embodiment provides a point-of-care test system that obtains relatively rapid results for the level of ATIII in patients.

[0074] The kit generally includes a pair of signal transduction aptamers or thrombin signal transduction aptamer complexes described in a suitable container or receptacle. The kit may also include additional reagents or devices useful in determining the level of ATIII in a sample. Such additional reagents or devices may be, but are not limited to, one or more of a buffer, ATIII, a syringe, and a hypodermic needle. The kit can be used to quantify the level of ATIII in one or more samples. The container may be formed from a variety of materials, including, but not limited to, glass and pharma- ceutical compatible plastics.

[0075] In some embodiments, the kit comprises one or more containers containing any one or more of the described signal transduction aptamers or complexes. In some embodiments, the kit comprises a plurality of test strips containing any one or more of the described signal transduction aptamers or complexes. In some embodiments, the kit comprises a unit dose, which refers to a composition comprising a predetermined amount of any of the described signal transduction aptamers or complexes suitable for determining the level of ATIII in a sample, for example, optionally with one or more additional reagents. In some embodiments, the kit contains one or more control samples.

[0076] In some embodiments, the kit can further contain heparin. In some embodiments, the heparin is linked to a support. The heparin bound to the support can be, but is not limited to, heparin beads. In some embodiments, the kit includes a multi-well plate in which at least some of the wells include heparin. In some embodiments, the kit is provided with a multi-well plate in which some wells are coated with heparin and some wells are not coated with heparin. In some embodiments, the kit includes heparin-coated wells and non-heparin-coated wells suitable for analyzing samples in the presence and absence of heparin.

[0077] In some embodiments, the signaling aptamer, thrombin signaling aptamer complex, and / or heparin are provided in a liquid. The liquid may be sterile or non-sterile. In some embodiments, the signaling aptamer, thrombin signaling aptamer complex, and / or heparin are provided in a lyophilized form that can be reconstituted upon addition of a suitable solvent. The solvent used for reconstitution may be provided in a separate container.

[0078] In some embodiments, the signaling aptamer, thrombin signaling aptamer complex, and / or heparin are in solution, lyophilized powder or cake, or as a coating in a sample container, which may be a tube, a microplate well, or other container suitable for performing the assay.

[0079] In some embodiments, the kit includes a label, marker, insert, bar code, and / or reader indicating that the contents of the kit may be used to determine the level of ATIII in a sample. In some embodiments, the kit includes instructional materials that explain the use of the kit to measure the level of ATIII in a sample.

[0080] 1 shows an exemplary system that may be part of a kit that includes a heparin-loaded bead assay. The system shown is an exemplary embodiment that may be structurally modified while still producing similar results. As shown, the system includes a plurality of syringes configured to deliver fluid through a column 100 to a system board 110. The system board 110 includes a waste reservoir 120 and a fluorescence reservoir 130. A waste valve 122 and a fluorescence valve 132 control the flow of fluid from the column 100 to the waste reservoir 120 and the fluorescence reservoir 130, respectively.

[0081] The column-based method of the illustrated embodiment includes syringes loaded into a manifold 140. The syringes include a phosphate buffered saline (PBS) wash buffer syringe 142, a plasma syringe 144, a PBS syringe 146, and an aptamer assay syringe 148. Fluid from the syringes can be pumped from the syringes into the manifold 140 and into the column 100 manually or using a mechanical device to drive the respective plungers. A mechanical device to pump fluid from the syringes can be beneficial to maintain a consistent flow rate and pressure from the syringes through the column 100. As further illustrated in FIG. 2, the column 100 includes an inlet 102, a volume of compact Sepharose beads 104 held within a chamber of the column, a membrane 106 to maintain the compact Sepharose beads within the column, and an outlet 108. Fluid from the syringes is pumped through the column to complete the wash, ATIII capture, wash, and assay steps, in that order.

[0082] The PBS wash buffer is first pumped from a syringe 142 through the inlet 102 of the cylinder 100, across the compact Sepharose beads 104, and out the outlet 108 of the column. The membrane 106 holds the compact Sepharose beads 104 within the column. The PBS wash buffer is received in the waste reservoir 120 in response to the waste valve 122 being in the open position and the fluorescent valve 132 being in the closed position. The diluted plasma in the syringe 144 is then pumped through the column, across the Sepharose beads, and is likewise received in the waste reservoir 120. The PBS wash buffer in the syringe 146 is then pumped across the compact Sepharose beads 106 and is received in the waste reservoir 120. Once the second PBS wash buffer has been passed through the column 100, the waste valve 122 is closed and the fluorescent valve 132 is opened. An aptamer assay syringe 148 is used to pass the incubated aptamer assay through column 100 for fluorescence production, the fluid is received in fluorescence reservoir 130, and the fluorescence is read to determine ATIII levels.

[0083] FIG. 3 illustrates the workflow of heparin beads 202 in heparin bead-based detection of ATIII 204 in plasma 206. Heparin beads 202 are shown at 200 as heparin sepharose beads. Heparin beads 202 are confined to a column at 210 (column 100 in FIG. 1 ) and the heparin beads 202 are washed with PBS wash buffer in the direction of the flow arrow. Washing and incubation of the heparin beads 202 with diluted plasma 206 isolates ATIII 204 and removes interfering molecules 208 in the diluted plasma 206. After incubation with plasma, ATIII 204 is captured on the surface of the heparin beads 202 through the interaction between ATIII and heparin, as shown at 220. Heparin beads 202 are washed again with PBS wash buffer at 230 to remove off-target proteins and subsequently incubated with the aptamer assay. ATIII captured on the beads competitively induces the release of aptamer F into solution, contributing to signal generation at 240. Determination of fluorescence intensity reflects ATIII concentration. Fluorescence intensity is read through the fluorescence well 130, but all reactions occur within the column. Aptamer F provides high intensity fluorescence, allowing the determination of ATIII levels in plasma.

[0084] Using the system described above with respect to Figures 1-3 provides a method of testing a patient's ATIII levels. The process can be performed in a point-of-care environment with results obtained relatively quickly compared to traditional lab-based methods. The process developed by the applicant can rapidly identify a patient's ATIII levels, so that any ATIII deficiencies can be rapidly identified and addressed. The system of Figures X-Z provides a unique process by which ATIII can be measured by fluorescence using a specific array of compounds passed through a column holding heparin beads, as detailed above.

[0085] F. Method Methods are described for detecting and / or quantifying the amount of ATIII in a sample using the described compounds and compositions. In some embodiments, the method includes forming a thrombin signaling aptamer complex comprising thrombin, a first signaling aptamer, and a second signaling aptamer, contacting the thrombin signaling aptamer complex with a sample that contains or is suspected of containing ATIII, and measuring the fluorescence emitted by the fluorescent label. In some embodiments, the method includes contacting the thrombin signaling aptamer complex with a sample that contains or is suspected of containing ATIII, and detecting or measuring an increase in the fluorescent signal emitted by the fluorescent label. In some embodiments, the increase in the fluorescent signal is proportional to the level of ATIII in the sample.

[0086] Methods are described for detecting and / or quantifying the amount of ATIII in a sample using the described compounds and compositions. In some embodiments, the method includes forming a thrombin signaling aptamer complex comprising thrombin, a first signaling aptamer, and a second signaling aptamer; contacting the thrombin signaling aptamer complex with a sample containing or suspected of containing ATIII in both the presence and absence of heparin; and measuring the fluorescence emitted by the fluorescent label in the presence and absence of heparin. In some embodiments, the method includes contacting the thrombin signaling aptamer complex with a sample containing or suspected of containing ATIII in the presence and absence of heparin; and detecting or measuring the increase in the fluorescent signal emitted by the fluorescent label in the presence and absence of heparin. In some embodiments, the initial rate of increase in fluorescence is determined, and the initial rate of increase in fluorescence in the presence or absence of heparin relative to the initial rate of increase in fluorescence in the absence of heparin is proportional to the level of ATIII in the sample.

[0087] Methods are described for detecting and / or quantifying the amount of ATIII in a sample using the described compounds and compositions. In some embodiments, the method includes contacting a sample containing or suspected to contain ATIII with heparin linked to a solid support, purifying any ATIII in the sample from one or more other components of the sample using the heparin linked to a solid support, forming a thrombin signaling aptamer complex comprising thrombin, a first signaling aptamer, and a second signaling aptamer, contacting the thrombin signaling aptamer complex with the purified sample, and measuring the fluorescence emitted by the fluorescent label. In some embodiments, the method includes contacting the thrombin signaling aptamer complex with a purified sample containing or suspected to contain ATIII, and detecting or measuring the fluorescent signal emitted by the fluorescent label, where the purified sample is formed by incubating the sample with heparin linked to a solid support, and any ATIII in the sample is bound by the heparin.

[0088] The described method for determining the level of ATIII in a sample is fast, simple and accurate.The method can be performed before, at the same time and / or after medical procedures.Medical procedures include, but are not limited to, cardiac surgery, vascular surgery, heart-lung machine bypass, cardiac catheterization, dialysis, extracorporeal membrane oxygenation, filtration procedures and radiation procedures.

[0089] In some embodiments, the compounds, compositions, and methods described can be used to determine the level of ATIII in a subject within about 10%, within about 9%, within about 8%, within about 7%, within about 6%, within about 5%, within about 4%, within about 3%, within about 2%, or within about 1%. In some embodiments, the compounds, compositions, and methods described can be used to determine the level of ATIII in a subject within about 5%. In some embodiments, the methods can be used to measure the level of ATIII in a sample in 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less.

[0090] In some embodiments, the methods described are used to determine whether a subject is ATIII deficient. In some embodiments, the methods described are used to determine the dosage of heparin and / or exogenous ATIII to administer to a patient.

[0091] In some embodiments, the level of ATIII measured in the sample is compared to a predetermined control or level. The predetermined control or level may be derived from a population of subjects known to respond to heparin and / or known to be ATIII deficient. In some embodiments, a value below the predetermined control indicates that the subject is ATIII deficient and would benefit from administration of exogenous ATIII. In some embodiments, if the level ATII in the sample is lower than the predetermined level, exogenous ATIII is administered to the subject. In some embodiments, a value higher than the predetermined control indicates that the subject is not ATIII deficient and is likely to respond to heparin treatment. In some embodiments, if the level ATII in the sample is higher than the predetermined level, heparin is administered to the subject.

[0092] A method of treating a patient with an anticoagulant is described that includes obtaining a serum, plasma, or blood sample from a patient, contacting the sample with a thrombin signal aptamer complex described, measuring an increase in fluorescence, determining a level of ATIII in the sample based on the increase in fluorescence, and administering heparin and / or exogenous ATIII to the patient based on the determined level of ATIII in the sample.

[0093] G. Enumeration of Embodiments 1. A kit for determining the level of antithrombin (ATIII) in a subject, comprising: (a) a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label; (b) a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher; and (c) heparin, The kit, wherein the first and second hybridization sequences are complementary to one another and when the first and second thrombin-specific signaling aptamers bind to thrombin, fluorescence of the fluorescent label is quenched.

[0094] 2. The kit of embodiment 1, wherein the first thrombin-specific aptamer comprises TBA15.

[0095] 3. The kit of embodiment 1 or 2, wherein the second thrombin-specific aptamer comprises TBA29.

[0096] 4. A kit described in any one of embodiments 1 to 3, wherein the first and second hybridization sequences are 6 to 10 nucleic acid bases in length.

[0097] 5. The kit of embodiment 4, wherein the first and second hybridization sequences are 9 nucleobases in length.

[0098] 6. A kit according to any one of embodiments 1 to 5, wherein the first hybridization sequence comprises the sequence 5'-GTCGTA-3' and the second hybridization sequence comprises 5'-TACGAC-3', or the first hybridization sequence comprises the sequence 5'-TACGAC-3' and the second hybridization sequence comprises 5'-GTCGTA-3'.

[0099] 7. The kit of embodiment 6, wherein the first hybridization sequence comprises the sequence 5'-GTCGTAAGT-3' and the second hybridization sequence comprises the sequence 5'-ACTTACGAC-3', or the first hybridization sequence comprises the sequence 5'-ACTTACGAC-3' and the second hybridization sequence comprises the sequence 5'-GTCGTAAGT-3'.

[0100] 8. A kit described in any one of embodiments 1 to 7, wherein the first and / or second signaling aptamer comprises a linker, which connects the thrombin-specific aptamer to the hybridization sequence.

[0101] 9. The kit of embodiment 8, wherein the linker comprises polyethylene glycol (PEG).

[0102] 10. The kit of embodiment 9, wherein the PEG is PEG6.

[0103] 11. The kit of any one of embodiments 1 to 10, wherein the fluorescent label comprises FITC, fluorescein, hexachlorofluorescein, rhodamine, carboxy-X-rhodamine, tetramethylrhodamine, IAEDANS, EDANS, coumarin, BODIPY FL, Lucifer Yellow, Eosin, Erythrosine, Texas Red, or Cyanine.

[0104] 12. The kit of any one of embodiments 1-11, wherein the quencher comprises a DABCYL, BLACK HOLE QUENCHER, or TAMRA compound.

[0105] 13. A kit described in any one of embodiments 1 to 12, further comprising thrombin.

[0106] 14. The kit of embodiment 13, wherein the thrombin, the first signaling aptamer, and the second signaling aptamer form a complex.

[0107] 15. A method for determining ATIII levels in a sample from a subject, comprising: (a) contacting a first composition with a first portion of a sample from a subject, the first composition comprising heparin; thrombin; a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher, wherein the first and second hybridization sequences are complementary to each other and form a duplex, and fluorescence of the fluorescent label is quenched; (b) contacting a second composition with a second portion of the sample, the second composition comprising thrombin; a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher, wherein the first and second hybridization sequences are complementary to each other and form a duplex, and the fluorescence of the fluorescent label is quenched, and the second composition does not contain heparin; (c) measuring an initial rate of increase in fluorescence of the first composition and an initial rate of increase in fluorescence of the second composition; A method wherein the rate of initial increase in fluorescence of the first composition relative to the rate of initial increase in fluorescence of the second composition is proportional to the level of ATIII in the sample.

[0108] 16. The method of embodiment 17, wherein the sample comprises a serum sample, a blood sample, or a plasma sample.

[0109] 17. A method of treating a patient with an anticoagulant, comprising: (a) determining the level of ATIII in a patient according to claim 15; (b) administering heparin and / or exogenous ATIII to the patient based on the level of ATIII in the sample determined in step (a).

[0110] 18. The kit of embodiment 1, wherein the kit further comprises a microfluidic device, at least one of the wells of the microfluidic device being coated with heparin and at least one of the wells of the microfluidic device being not coated with heparin.

[0111] 19. A method for determining a level of ATIII in a subject, comprising: (a) contacting a sample from a subject with heparin linked to a solid support, wherein any ATIII in the sample binds to the heparin; (b) purifying the sample to remove one or more components of the sample that do not bind to heparin to form a purified sample; (c) contacting the purified sample with a thrombin signaling aptamer complex; (d) measuring fluorescence, wherein the level of fluorescence is proportional to the level of ATIII in the sample. EXAMPLES

[0112] Example 1. Exemplary signaling aptamers. TBA15-F6(5'-GGTTGGTGTGGTTGG-(PEG) 6- GTCGTA-FITC-3') (SEQ ID NO: 13) TBA29-D6(5'-DABCYL-TACGAC-(PEG) 6- AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' (SEQ ID NO: 14), TBA15-F7(5'-GGTTGGTGTGGTTGG-(PEG) 6- GTCGTAT-FITC-3') (SEQ ID NO: 15) TBA29-D7 (5'-Dabcyl-ATACGAC-(PEG)6-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3') (SEQ ID NO: 16) TBA15-F8(5'-GGTTGGTGTGGTTGG-(PEG) 6- GTCGTAGT-FITC-3') (SEQ ID NO: 17) TBA29-D8(5'-Dabcyl-ACTACGAC-(PEG) 6- AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' (SEQ ID NO: 18) TBA15-F9(5'-GGTTGGTGTGGTTGG-(PEG) 6- GTCGTAAGT-FITC-3') (SEQ ID NO: 19) TBA29-D9 (5'-Dabcyl-ACTTACGAC-(PEG)6-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3') (SEQ ID NO: 20) TBA15-F10(5'-GGTTGGTGTGGTTGG-(PEG) 6- GTCGTAAGCT-FITC-3') (SEQ ID NO: 21) TBA29-D10(5'-Dabcyl-AGCTTACGAC-(PEG) 6- AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' (SEQ ID NO: 22)

[0113] Example 2. Reagents. All DNA (Integrated DNA Technologies) was stored at −20° C. with Tris-HCl buffer (Tris-acetate 20 mM, KCl 5 mM, NaCl 140 mM, MgCl 2 1 mM, CaCl 2 1 mM, pH=7.5).

[0114] Human α-thrombin (Haematologic Technologies, Inc.) was dissolved in 50% glycerol / water (v / v) and stored at -20°C.

[0115] Antithrombin (Grifols Therapeutics Inc.) was purchased as a powder, mixed with 10 mL of deionized water, and divided into 10 vials, all stored at 4° C. The concentration was more than 50-fold higher than the normal human antithrombin concentration. The normal concentration of antithrombin III in plasma is 2.3 μM, with a half-life of 3 days.

[0116] Heparin sodium salt (Santa Cruz biotechnology) was stored at 4°C.

[0117] Albumin from human serum (Sigma) was stored at 4°C.

[0118] Example 3. Formation of thrombin signal aptamer complex. Thrombin was prepared at a concentration of 200 nM. The ratio between TBA15 signaling aptamer and TBA29 signaling aptamer was 1:1. Signaling aptamer was prepared at concentrations of 100 nM, 200 nM, 300 nM and 400 nM. Thrombin signaling aptamer complexes were formed by combining thrombin with different concentrations of signaling aptamer. For each, thrombin and signaling aptamer solutions were incubated together for 30 minutes at room temperature in a total volume of 100 μL Tris-HCl buffer (Tris-acetate 20 mM, KCl 5 mM, NaCl 140 mM, MgCl2 1 mM, CaCl2 1 mM, pH 7.5). For controls, TBA15 signaling aptamer alone (control 1) or TBA15 signaling aptamer + TBA29 signaling aptamer (control 2) were used. Fluorescence spectroscopy was used for detection.

[0119] As shown in FIG. 6, at a concentration of 100 nM, the peak fluorescence intensity was 3.80×10 for the thrombin signaling aptamer complex. 5 and I FDTHR The peak fluorescence intensity of the TBA15 signaling aptamer alone was set at 1.029 × 10 6and was set as I0. The peak fluorescence intensity of the TBA15-F9+TAB29-D9 signaling aptamer was 1.011×10 6 and I FD The fluorescence intensity of the TBA15 signaling aptamer alone was normalized as 100%. FD The thrombin signaling aptamer complex (I FDTHR The relative fluorescence intensity of the fluorescein-containing solution (pH 7.0 / I0) was 36.92%.

[0120] As shown in Figure 7, at a concentration of 200 nM, the peak fluorescence intensity of TBA15-F9 alone was 1.31 × 10 6 The peak fluorescence intensity of TBA15-F9+TBA29-D9 was 1.23H10 6 is 2.66 × 10 5 The fluorescence intensity of TBA15-F9 alone was normalized as 100% and set as I0. The fluorescence intensity of TBA15-F9+TBA29-D9 was normalized as I FD Then, I FD The / I0 was 93.89%. The thrombin signaling aptamer complex (I FDTHR The relative fluorescence intensity of the fluorescein-containing solution (pH 7.0 / I0) was 20.23%.

[0121] As shown in Figure 8, at a concentration of 300 nM, the peak fluorescence intensity of TBA15-F9 alone was 2.36 × 10 6 The peak fluorescence intensity of TBA15-F9+TBA29-D9 was 2.06×10 6 The peak fluorescence intensity of the thrombin signal aptamer complex was 3.11 × 10 5 The fluorescence intensity of TBA15-F9 alone was normalized as 100% and set as I0. The fluorescence intensity of TBA15-F9+TBA29-D9 was normalized as I FD It was set as TBA15-F9+TBA29-D(I FD The relative fluorescence intensity of the thrombin signaling aptamer complex (I FDTHR The relative fluorescence intensity of the fluorescein-containing solution (I0) was 13.17%.

[0122] As shown in FIG. 9, at a concentration of 400 nM, the peak fluorescence intensity of TBA15-F9 alone was 2.90×10 6 The peak fluorescence intensity of TBA15-F9+TBA29-D9 was 2.06×10 6 The peak fluorescence intensity of the thrombin signal aptamer complex was 3.36 × 10 5 The fluorescence intensity of TBA15-F9 alone was normalized as 100% and set as I0. The fluorescence intensity of TBA15-F9+TBA29-D9 was normalized as I FD It was set as TBA15-F9+TBA29-D(I FD The relative fluorescence intensity of the thrombin signaling aptamer complex (I FDTHR The relative fluorescence intensity of the fluorescein at 100 nm / I0 was 11.58%.

[0123] Based on these experiments, a 1:1:1 ratio of thrombin to TBA15 signaling aptamer to TBA29 signaling aptamer was selected for additional experiments (Figures 6-9).

[0124] Example 4. Hybridization sequences. Five hybridization sequences (with complementary hybridization sequences) were tested for use with the signaling aptamers. The hybridization sequences were as described for TBA15-F6+TBA29-D6, TBA15-F7+TBA29-D7, TBA15-F8+TBA29-D8, TBA15-F9+TBA29-D9, and TBA15-F10+TBA29-D10. Controls were as described above.

[0125] The TBA15 signaling aptamer and the TBA29 signaling aptamer were prepared at a concentration of 200 nM and combined in a 1:1 ratio. Thrombin signaling aptamer complexes were formed by combining the signaling aptamer with various concentrations of thrombin. The ratio of aptamer to thrombin was (1:1:0.1 to 1:1:1.5). For each, the thrombin and signaling aptamer solutions were incubated together for 30 min in a total volume of 100 μL of Tris-HCl buffer at room temperature. For controls, the TBA15 signaling aptamer alone or the TBA15 signaling aptamer + TBA29 signaling aptamer were used. Fluorescence spectroscopy was used for detection.

[0126] For aptamers TBA15-F6 and TBA29-D6, no specific trends were observed with different concentrations of thrombin (Figure 10). No specific trends were observed with different concentrations of thrombin. The data suggest that the two aptamers did not bind to thrombin and / or the hybridization sequences of the two signaling aptamers did not form a duplex.

[0127] For aptamers TBA15-F7 and TBA29-D7, the fluorescence intensity decreased with increasing thrombin concentration (Figure 11). The peak fluorescence intensity of TBA15-D7 alone was 2.806 x 10 6 , which is normalized to 100%, and I 70 The peak fluorescence intensity of TBA15-F7+TBA29-D7 (1:1) was 2.768 × 10 6 and I FD7 The peak fluorescence intensity of TBA15-F7 + TBA29-D7 + thrombin (1:1:1) was 1.95 × 10 6 and I FDTHR7 It was set as I FD7 / I 70 The result was 98.64%. FDTHR7 / I 70was 69.49%. The data showed the two signaling aptamers bound to thrombin and the hybridization sequence formed a duplex. FITC fluorescence was quenched by dabcyl. For TBA15-F7+TBA29-D7 in solution, the fluorescence did not appear to be quenched, indicating that binding to thrombin was required for duplex formation and quenching of fluorescence by the hybridization sequence.

[0128] For aptamers TBA15-F8 and TBA29-D8, the fluorescence intensity decreased with increasing thrombin concentration (Figure 12). The peak fluorescence intensity of TBA15-D8 alone was 2.76 x 10 6 , which is normalized to 100%, and I 80 The peak fluorescence intensity of TBA15-F8+TBA29-D8 (1:1) was 2.54 × 10 6 and I FD8 The peak fluorescence intensity of TBA15-F8 + TBA29-D8 + thrombin (1:1:1) was 1.79 × 10 6 and I FDTHR8 It was set as I FD8 / I 80 The result was 92.03%. FDTHR8 / I 80 was 64.86%. The data showed two signaling aptamers bound to thrombin, and the hybridization sequences formed a duplex. FITC fluorescence was quenched by dabcyl. Compared to TBA15-F6 and TBA15-F7, improved quenching was observed for TBA15-F8 and TBA29-F8 in the presence of thrombin. FD8 / I 80 The slight decrease in β-thrombin indicates that TBA15-F8 and TBA29-F8 may interact weakly in the absence of thrombin.

[0129] For aptamers TBA15-F9 and TBA29-D9, the fluorescence intensity decreased with increasing thrombin concentration (Figure 13). The peak fluorescence intensity of TBA15-D9 alone was 2.81 x 10 6, which is normalized to 100%, and I 90 The peak fluorescence intensity of TBA15-F9+TBA29-D9 (1:1) was 2.35 × 10 6 and I FD9 The peak fluorescence intensity of TBA15-F9 + TBA29-D9 + thrombin (1:1:1) was set at 0.85 × 10 6 and I FDTHR9 It was set as I FD9 / I 90 The result was 83.63%. FDTHR9 / I 90 was 30.25%. The data showed two signaling aptamers bound to thrombin and the hybridization sequences formed a duplex. FITC fluorescence was quenched by dabcyl. TBA15-F9 and TBA29-F9 showed improved quenching compared to the signaling aptamer with an 8-base hybridization sequence. Similar to TBA15-F8 and TBA29-F8, TBA15-F9 and TBA29-F9 showed some quenching in the absence of thrombin.

[0130] For aptamers TBA15-F10 and TBA29-D10, the fluorescence intensity decreased with increasing thrombin concentration (Figure 14). The peak fluorescence intensity of TBA15-D10 alone was 2.30 x 10 6 , which is normalized to 100%, and I 100 The peak fluorescence intensity of TBA15-F10 + TBA29-D10 (1:1) was set at 0.775 × 10 6 and I FD10 The peak fluorescence intensity of TBA15-F10 + TBA29-D10 + thrombin (1:1:1) was set at 0.16 × 10 6 and I FDTHR10 It was set as I FD10 / I 100 The percentage was 33.69%. FDTHR10 / I 100was 6.95%. The data showed the two signaling aptamers bound to thrombin, and the hybridization sequences formed a duplex. FITC fluorescence was quenched by dabcyl. TBA15-F10 and TBA29-F10 showed superior quenching compared to the signaling aptamer with a 9-base hybridization sequence. TBA15-F10 and TBA29-F10 also showed significant quenching in the absence of thrombin.

[0131] Based on the results, TBA15-F9+TBA29-D9 were used for further experiments.

[0132] Example 5. Heparin selectivity. The ability of the thrombin signaling aptamer complex to accurately detect ATIII in the presence of heparin and human serum proteins was tested. 200 nM TBA15-F9 alone or 200 nM TBA15-F9 + TBA29-D9 + thrombin (1:1:1) were used as controls. After complex formation, increasing concentrations of heparin (0.25 g / L to 2.00 g / L, Figure 17; or 0.0 g / L to 1.6 g / L, Figure 20) were added and incubated at room temperature for 30 min. The normal concentration of heparin in human plasma is 1.5 to 3.0 g / L. 1940 nM ATIII was then added and fluorescence spectroscopy was used for detection. The fluorescence intensity at 1940 nM ATIII alone was normalized to 100%. The fluorescence intensity was not affected by the presence of heparin (Figures 17 and 20). The data show that the thrombin signal aptamer complex did not react with heparin and the presence of heparin did not adversely affect ATIII detection.

[0133] Example 6. Heparin-binding antigen (HBA) selectivity. Similar to heparin, the ATIII assay was also analyzed for HBA sensitivity. HBA is a protein produced by certain bacteria that can interfere with heparin activity and the heparin assay. 200 nM of TBA15-F9, TBA29-D9 and thrombin (1:1:1) were incubated at room temperature for 30 min to form the thrombin signaling aptamer complex. Various concentrations of HBA (1 g / L-4 g / L) were then added to the complex and incubated at room temperature for 30 min. ATIII was then added to the reaction and detected by fluorescence spectroscopy. The fluorescence intensity was not affected by the presence of heparin-binding antigen, indicating that the thrombin signaling aptamer complex does not react with heparin and the presence of HBA does not adversely affect ATIII detection (Figure 18).

[0134] Similarly, the ATIII assay was also analyzed for human serum albumin sensitivity. Albumin is the most ubiquitous protein circulating in plasma and affects the surface activity of the glycocalyx on endothelial cells. Albumin interacts with glucose-aminoglycans, especially heparin. Sensitivity to HSA was tested in the same way as sensitivity to HBA. After complex formation, 1940 nM ATIII and increasing concentrations of HSA were added. The fluorescence intensity at 1940 nM ATIII alone was normalized to 100%. The presence of HSA did not affect the fluorescence intensity, indicating that HBA does not adversely affect the reaction and that the presence of HSA does not adversely affect ATIII detection (Figure 19).

[0135] Example 7. Antithrombin response curves. To investigate the utility of the thrombin signaling aptamer complex in quantifying ATIII, various concentrations of ATIII were used. Increasing concentrations of ATIII were added to the thrombin signaling aptamer complex and fluorescence detected by fluorescence spectroscopy. There are three replicates for each sample (Figure 5).

[0136] Antithrombin was titrated into the TBA15-F9-TBA29-D9-thrombin complex. The complex concentration was 200 nM. The ratio of TBA15-F9:TBA29-D9:thrombin was 1:1:1. Antithrombin concentration was increased from 10 nM to 1960 nM. The fluorescence intensity of the TBA15-F9 alone control was normalized as 100% and set as I0. I / I0 set on the y-axis of FIG. 20 means the intensity of each sample compared to the intensity of FITC in solution alone. Each concentration was repeated three times. The data shows that the signaling aptamer complex can be used to quantify ATIII in samples.

[0137] Example 8. Combination of antithrombin (ATIII) and heparin. The ability of heparin to promote the reaction between antithrombin and heparin was analyzed. 200 nM TBA15-F9, TBA29-D9, and thrombin (1:1:1) were incubated at room temperature for 30 minutes to form the thrombin signal aptamer complex. Increasing concentrations of antithrombin were added to the complex, incubated at room temperature for 30 minutes, and fluorescence was detected by fluorescence spectroscopy. There were three replicates for each sample.

[0138] Example 9. Kinetic studies. A. TBA15-F9+TBA29-D9+Thrombin Kinetics. 10 μL of 2 μM TBA15-F9 and 10 μL of 2 μM thrombin were prepared and combined in 78 μL of Tris-HCl. 10 μL of 2 μM TBA29-D9 was added to the solution while monitoring the fluorescence. The reaction was complete in about 160 seconds (FIG. 15).

[0139] B. Complex + Antithrombin Kinetics. 12 μL of 2 μM TBA15-F9, 12 μL of 2 μM TBA29-D9 and 12 μL of 2 μM thrombin (1:1:1 ratio) were combined in 76 μL of Tris-HCl for 30 minutes at room temperature. Then, 8 μL of 28.47 μM (1940 nM) ATIII was titrated into the solution while monitoring the fluorescence. The reaction was complete in about 600 seconds (FIG. 16).

[0140] Example 10. Kinetic response of aptamer assays in the presence of heparin. Antithrombin III was prepared at a concentration of 500 nM from a 100 μM stock.

[0141] Heparin was used at a concentration of 1920 μM and various dilutions were made as shown below. 5x dilution = 384 µM heparin: 20 µL of 1920 µM heparin + 80 µL of water 10x dilution = 192 µM heparin: 50 µL of 384 µM heparin + 50 µL of water. 20x dilution = 96 µM heparin: 50 µL of 192 µM heparin + 50 µL of water. 50x dilution = 38.4 μM heparin: 40 μL of 96 μM heparin + 60 μL of water. 100x dilution = 19.2 μM heparin: 50 μL of 38.4 μM heparin + 50 μL of water. 1000x dilution = 1.92 μM heparin: 10 μL of 19.2 μM heparin + 90 μL of water. 2000x dilution = 0.96 μM heparin: 50 μL of 1.92 μM heparin + 50 μL of water. 4000x dilution = 0.48 μM heparin: 50 μL of 0.96 μM heparin + 50 μL of water.

[0142] Aptamer solution: [Table 1]

[0143] procedure: 1. 5 μL (500 nM) ATIII was mixed with 5 μL water in the first and second reaction wells to serve as a control (no heparin). 2. In separate wells, 5 μL (500 nM) ATIII was mixed with 5 μL each of 384 μM heparin, 192 μM heparin, 96 μM heparin, 38.4 μM heparin, 19.2 μM heparin, 1.92 μM heparin, 0.96 μM heparin, and 0.48 μM heparin. 3. 40 μL of the aptamer solution was then added to each reaction well. 4. Fluorescence was measured over time as described above.

[0144] As shown in Figure 21, increasing concentrations of heparin in the assay resulted in stronger activation of ATIII and faster reaction rates when the aptamer solution was added. The control (no heparin) sample showed faster kinetics than samples made using 1.92 μM, 0.96 μM, and 0.45 μM heparin. In duplicate experiments, the control sample showed faster kinetics than samples made using 96 μM heparin solution, indicating that heparin did not activate ATIII fast enough to react with the aptamer solution.

[0145] Example 11. Effect of heparin at different ATIII concentrations. Heparin was prepared at a concentration of 192 μM.

[0146] ATIII was used at a concentration of 100 μM and various dilutions were made as shown below. 20μM: 200μL 100μM ATIII + 800μL water (final assay concentration = 2μM ATIII) 10μM: 100μL 100μM ATIII + 900μL water (final assay concentration = 1μM ATIII) 5μM: 500μL 10μM ATIII + 500μL water (final assay concentration = 500nM ATIII) 2μM: 400μL 5μM ATIII + 600μL water (final assay concentration = 200nM ATIII) 1 μM: 200 μL 5 μM ATIII + 800 μL water (final assay concentration = 100 nM ATIII)

[0147] Aptamer solution: [Table 2]

[0148] procedure: 1. 5 μL (192 μM) heparin was mixed with 10 μL water in the first and second reaction wells to serve as a control (no ATIII). 2. In separate wells, 5 μL (192 μM) heparin was mixed with 5 μL each of 20 μM, 10 μM, 5 μM, 2 μM, and 1 μM ATIII. In separate wells, 5 μL of water was mixed with 5 μL each of 20 μM, 10 μM, 5 μM, 2 μM, and 1 μM ATIII. 3. 40 μL of the aptamer solution was then added to each reaction well. 4. Fluorescence was measured over time as described above.

[0149] The data was normalized and graphed as shown in Figures 22 and 23. The fluorescent signal was higher in the presence of heparin (compare Figure 22 (without heparin) with Figure 23 (with heparin), confirming the heparin activity of ATIII. The detection limit of ATIII in the absence of heparin was 1 μM. The detection limit of ATLLL in the absence of heparin was 500 nM (0.5 μM).

[0150] Example 12. Heparin-containing ATIII assay device. A. A microplate is prepared with various sample wells coated with or without heparin. Optionally, positive control wells are coated with or without heparin and with or without various predetermined amounts of ATIII. Optionally, positive control samples containing various predetermined amounts of ATIII are provided. Samples from one or more subjects are added to the wells, and the amount of ATIII in the samples is determined as described. The difference in the initial rate of signal output observed between wells correlates with ATIII concentration.

[0151] B. The above approach is incorporated into various other kits and prepackaged assays. The kits and assays can include, but are not limited to, tubes (e.g., microcentrifuge tubes), vials, microtiter plates, microfluidic devices, and other high-throughput devices. The kits can contain various predefined amounts of ATIII or tubes or wells coated with predefined amounts of ATIII, heparin or tubes or wells coated with heparin, the described aptamers, and optionally a control sample containing thrombin (e.g., alpha thrombin). The kits and assays can further include instructions for use.

[0152] Example 13. Purification of ATII from biological samples - Heparin bead assay. Samples are collected from subjects and incubated with heparin linked to solid support.In some methods, the samples are then washed to remove the components of the samples that are not bound to heparin.Then, ATIII assay is carried out in the presence of solid support.Figure 24 is a schematic diagram of heparin-bound sepharose beads that extract ATIII, where remaining proteins are removed and aptamer thrombin assay is added to generate a fluorescent signal that indicates the ATIII that originally exists in plasma samples.

[0153] As above, the following samples were analyzed: (a) 10 μM: TBA15-F9 + TBA29-D9 + thrombin complex + heparin beads + 10 μM ATIII (b) 5 μM: TBA15-F9 + TBA29-D9 + thrombin complex + heparin beads + 5 μM ATIII (c) 2 μM: TBA15-F9 + TBA29-D9 + thrombin complex + heparin beads + 2 μM ATIII (d) 1 μM: TBA15-F9 + TBA29-D9 + thrombin complex + heparin beads + 1 μM ATIII (e) 500nM: TBA15-F9 + TBA29-D9 + thrombin complex + heparin beads + 500nM ATIII (f) Beads-NC: TBA15-F9 + TBA29-D9 + thrombin complex + heparin beads + 0 nM ATIII (g) dP+B: TBA15-F9+TBA29-D9+thrombin complex+heparin beads+ATIII-deficient plasma (5 μL of plasma was diluted in 95 μL of PBS). (h) dP+A+B: TBA15-F9+TBA29-D9+thrombin complex+heparin beads+ATIII-deficient plasma (5 μL of plasma was diluted in 95 μL of 2 μM ATIII). (i) dP: TBA15-F9 + TBA29-D9 + thrombin complex + heparin + deficient plasma (j) NC: TBA15-F9+TBA29-D9+thrombin complex+heparin.

[0154] FIG. 25 shows that ATIII from plasma bound to heparin is detected in the assay. The signal of dP+B (deleted plasmid+heparin beads) is similar to the negative control beads, indicating that non-specific molecules in the sample that can induce ATIII-independent signals were effectively eliminated after extraction, and is compared to the signal generated for the deleted plasmid (dP) that was not purified with heparin beads. dP+A+B simulated a plasma sample from a subject and generated a high signal. Adding ATIII to the sample (dP+A+B) resulted in a highly detectable signal. Thus, the use of heparin beads increased the specificity of the assay in detecting ATIII in plasma.

[0155] FIG. 26 shows that the described method can detect as little as 100 nM ATIII, which is more sensitive than ATIII assays performed in the absence of heparin (detection limit of 1 μM) or in the presence of added heparin but without sample purification (detection limit of 500 nM).

[0156] Example 14. Buffer Analysis The performance of the assay was evaluated using Tris-HCl buffer, PBS buffer, and Mg 2+ and Ca 2+ The intensities were normalized to 200 nM TBA15-F9. 2+ and Ca 2+ Compared with Tris HCl and PBS buffer with Mg2+, PBS buffer provided higher signals in TBA15-F9+TBA29-D9 and TBA15-F9+TBA29-D9+thrombin+ATIII. Self-hybridization of TBA15-F9+TBA29-D9 in PBS without Mg2+ and Ca2+ was weak, and the TBA15-F9+TBA29-D9+thrombin complex was stable (Figure 27).

[0157] Example 15. Formation kinetics The kinetics of the formation of TBA15-F9+TBA29-D9+thrombin complex was analyzed. As shown in Figure 28, 96.82% of stable TBA15-F9+TBA29-D9+thrombin complex was formed in 60 minutes compared to 90 minutes. Therefore, 60 minutes is sufficient time for the formation of TBA15-F9+TBA29-D9+thrombin complex for use in the assay.

[0158] Example 16. Limitations of detection analysis The LoD of the described assay was calculated to be 55.16 nM with an assay time of 30 minutes using 3 standard deviations. The results shown in Figure 29 indicate that the assay is sensitive enough to detect ATIII at levels below those normally present in plasma, i.e., about 2 μM.

Claims

1. 1. A system for determining a level of antithrombin (ATIII) in a subject, comprising: a column defining an inlet and an outlet, the heparin beads being retained within the column; a manifold connected to the inlet of the column, the manifold including a plurality of ports, each port configured to receive a fluid, the manifold directing the fluid from each port to the inlet of the column; and a first syringe containing a first wash buffer received at a first port of the plurality of ports of the manifold; a second syringe containing plasma received at a second port of the plurality of ports of the manifold; a third syringe containing a second wash buffer received at a third port of the plurality of ports of the manifold; and a fourth syringe containing an aptamer-thrombin complex received at a fourth port of the plurality of ports of the manifold, wherein the aptamer-thrombin complex comprises a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher and thrombin, wherein the first and second hybridization sequences are complementary to each other, and when the first and second thrombin-specific signaling aptamers bind to the thrombin, fluorescence of the fluorescent label is quenched.

2. 10. The system of claim 1, further comprising a substrate, the substrate comprising: a waste reservoir separated from the column by a waste valve; and a fluorescence reservoir separated from the column by a fluorescence valve.

3. 2. The system of claim 1, wherein the heparin beads retained within the column are washed in response to the first syringe passing the first wash buffer through the inlet of the column.

4. 4. The system of claim 3, wherein the heparin beads are incubated with the plasma in response to the second syringe passing the plasma through the inlet of the column, and the heparin beads capture ATIII thereon during incubation with the plasma.

5. 5. The system of claim 4, wherein the heparin beads with the captured ATIII are washed with the second wash buffer in response to the third syringe passing the second wash buffer through the inlet of the column.

6. The system of claim 5, wherein the heparin beads with the captured ATIII are incubated with the aptamer-thrombin complex in response to the fourth syringe passing the aptamer-thrombin complex through the inlet of the column, and the ATIII captured on the heparin beads causes the release of aptamer-F into solution within the column for signal generation.

7. The system of claim 6 , wherein the fluorescence of the solution is read to determine the level of ATIII in the plasma.

8. 1. A method for determining a level of antithrombin (ATIII) in a subject, comprising: passing a first wash buffer through the column containing the suspended heparin beads; passing a sample through the column, the sample comprising plasma from the subject; passing a second wash buffer through the column; passing a solution comprising an aptamer-thrombin complex through the column, the aptamer-thrombin complex comprising a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher and thrombin, the first and second hybridization sequences being complementary to each other, such that when the first and second thrombin-specific signaling aptamers bind to the thrombin, fluorescence of the fluorescent label is quenched; collecting an eluate from the column, the eluate comprising the aptamer-thrombin complex and ATIII from the sample; and measuring fluorescence in the solution, the level of the fluorescence being indicative of the level of ATIII in the plasma.

9. 9. The method of claim 8, wherein the first wash buffer is passed through the column by a first syringe, the plasma is passed through the column by a second syringe, the second wash buffer is passed through the column by a third syringe, and the aptamer-thrombin complex is passed through the column by a fourth syringe.

10. 9. The method of claim 8, wherein the heparin beads are washed by passing the first wash buffer through the column in which the heparin beads are suspended.

11. The method of claim 10, wherein ATIII present in the sample is captured onto the heparin beads by passing the sample through the column.

12. 12. The method of claim 11, wherein passing the second wash buffer through the column purifies the ATIII present in the sample from one or more components present in the sample.

13. 13. The method of claim 12, wherein ATIII present in the sample disrupts binding of the first and second signaling aptamers from the thrombin, resulting in an increase in fluorescence of the fluorescent label.

14. 1. An apparatus for determining a level of antithrombin (ATIII) in a subject, comprising: a column defining an inlet and an outlet; a manifold defining a plurality of ports and a manifold outlet, the manifold outlet being connected to the inlet of the column; a substrate defining a waste flow path extending between a substrate inlet and a waste reservoir, and a fluorescence flow path extending between the substrate inlet and a fluorescence reservoir; a waste valve configured to open and close the waste flow path between the substrate inlet and the waste reservoir; a fluorescence valve configured to open and close the fluorescence flow path between the substrate inlet and the fluorescence reservoir.

15. a first syringe received in a first port of the plurality of ports of the manifold; a second syringe received in a second port of the plurality of ports of the manifold; and a third syringe received in a third port of the plurality of ports of the manifold; and 15. The apparatus of claim 14, further comprising: a fourth syringe received in a fourth port of the plurality of ports of the manifold.

16. 16. The device of claim 15, wherein the first syringe holds a first wash buffer, the second syringe holds plasma, the third syringe holds a second wash buffer, and the fourth syringe holds an aptamer-thrombin complex, the aptamer-thrombin complex comprising a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher and thrombin, wherein the first and second hybridization sequences are complementary to each other, and wherein when the first and second thrombin-specific signaling aptamers bind to the thrombin, fluorescence of the fluorescent label is quenched.

17. 17. The device of claim 16, wherein in response to the contents of the first syringe, the second syringe, the third syringe, and the fourth syringe being passed sequentially through the column, the fluorescent reservoir fluoresces based on the level of ATIII in the plasma.

18. 1. A kit for determining the level of antithrombin (ATIII) in a subject, comprising: (a) a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label; (b) a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher; and (c) thrombin; (d) heparin beads, The kit, wherein the first and second hybridization sequences are complementary to one another and when the first and second thrombin-specific signaling aptamers bind to thrombin, fluorescence of the fluorescent label is quenched.

19. 20. The kit of claim 18, wherein the first signaling aptamer, the second signaling aptamer, and the thrombin form a complex.

20. 20. The kit of claim 18, wherein the first signaling aptamer, the second signaling aptamer, and the thrombin are provided in a ratio of about 11:1:1 to about 1:1:1.

25.

21. 20. The kit of claim 18, wherein the first signaling aptamer, the second signaling aptamer, and / or the thrombin are provided in a buffer comprising Tris-HCl buffer, phosphate buffered saline (PBS) buffer, or PBS buffer without magnesium or calcium.

22. 20. The kit of claim 19, wherein the thrombin, the first signaling aptamer, and the second signaling aptamer are provided in a complex at a concentration of about 200 nM.

23. 1. A method for determining a level of ATIII in a subject, comprising: (a) contacting a sample from the subject with heparin linked to a support, wherein any ATIII in the sample binds to the heparin; (b) purifying the sample to remove one or more components of the sample that do not bind to the heparin to form a purified sample; (c) contacting the purified sample with an aptamer-thrombin complex to form an assay solution, the aptamer-thrombin complex comprising a first signaling aptamer comprising a first thrombin-specific aptamer, a first hybridization sequence, and a fluorescent label, and a second signaling aptamer comprising a second thrombin-specific aptamer, a second hybridization sequence, and a quencher and thrombin, the first and second hybridization sequences being complementary to one another, and wherein when the first and second thrombin-specific signaling aptamers bind to the thrombin, fluorescence of the fluorescent label is quenched; (d) measuring fluorescence in the assay solution, the level of fluorescence being proportional to the level of ATIII in the sample.

24. The system of any one of claims 1 to 7, the method of any one of claims 8 to 13 and 22, the device of claim 16 or 17, or the kit of any one of claims 18 to 22, wherein the first thrombin-specific aptamer comprises TBA15 and the second thrombin-specific aptamer comprises TBA29.

25. 25. The system, method, apparatus, or kit of claim 24, wherein the first hybridization sequence comprises the sequence 5'-GTCGTAAGT-3' and the second hybridization sequence comprises the sequence 5'-ACTTACGAC-3', or the first hybridization sequence comprises the sequence 5'-ACTTACGAC-3' and the second hybridization sequence comprises the sequence 5'-GTCGTAAGT-3'.

26. 26. The system, method, device, or kit of claim 24 or 25, wherein the first and / or second signaling aptamer comprises a linker, the linker connecting the thrombin-specific aptamer to the hybridization sequence.

27. The linker is polyethylene glycol (PEG) or ６ 27. The system, method, apparatus, or kit of claim 26, comprising:

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