Acousto-electrophoretic analysis apparatus and method

Abstract

Fluid analysis devices, methods, and systems are disclosed, including an analysis device comprising: a sample vessel having an outer surface, a microchannel within a boundary of the outer surface, a first port extending through the outer surface to the microchannel, and a second port extending through the outer surface to the microchannel; and a piezoelectric transducer coupled to the outer surface of the sample vessel to form an integral structure, the piezoelectric transducer configured to emit ultrasonic sound waves having a first frequency, a second frequency, and a third frequency into and / or into the blood sample within the microchannel, the first frequency is configured to initiate separation of red blood cells and plasma in a blood sample, the second frequency is configured to complete separation of red blood cells and plasma, and the third frequency is configured to rupture cell walls of blood cells, thereby producing a lysed blood sample.

Description

[0001] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 367,347, filed on June 30, 2022. The entire contents of the above-referenced patent applications are hereby expressly incorporated herein by reference. Technical Field

[0002] The present disclosure generally relates to devices, systems and methods for testing blood samples. More specifically, the present disclosure relates to an analytical device as follows: it is configured to separate red blood cells and plasma in a sample vessel by means of ultrasonic sound waves, and the ultrasonic sound waves are generated in the vessel by a piezoelectric transducer driven by one or more first frequencies or frequency ranges. After separating and testing the red blood cells and / or plasma, the analytical device is also configured to lyse the red blood cells in the sample vessel by means of ultrasonic sound waves, shear forces, pressures and / or fluid movement, and the ultrasonic sound waves, shear forces, pressures and / or fluid movement are generated in the vessel by a piezoelectric transducer driven by one or more second frequencies or frequency ranges. In some non-limiting embodiments, the ultrasonic sound waves are generated by a single piezoelectric transducer. The analytical device can be used in conjunction with a blood sample testing analyzer. Background Art

[0003] Point of care testing generally refers to medical testing at or near a patient care location, such as in an emergency room. The desired outcome of such testing is typically a quick and accurate laboratory result to determine the next course of action for patient care. Many such point of care tests involve analyzing a patient's blood sample. Many of these tests use whole blood, plasma, or serum.

[0004] In some tests, the cell walls of red blood cells in a blood sample are broken (lysed) to release hemoglobin. The lysis of red blood cells may be called hemolysis. Typically, hemolysis is accomplished by chemical or mechanical methods.

[0005] Some devices use ultrasound to lyse red blood cells. Some point-of-care testing devices use spectrophotometric optical absorption measurements to determine blood oxygen parameters of whole blood samples in which red blood cells have been lysed. These devices are fluid systems that typically position a patient's blood sample in a special sample chamber to test the blood sample. For example, a system described in U.S. Patent No. 9,097,701 ("Apparatus for Hemolyzing a Blood Sample and for Measuring at Least One Parameter Thereof," published on August 4, 2015) uses two piezoelectric elements, with two balanced resonant elements symmetrically surrounding the sample chamber to lyse red blood cells using acoustophoretic forces. However, these devices are difficult and expensive to manufacture, including the need to achieve highly precise symmetry with the help of specially made resonant elements.

[0006] Once the red blood cells have been lysed, the blood sample can be tested with the aid of a spectrophotometer to analyze the intensity of light of predetermined wavelengths transmitted through the optical window of the cassette. A spectrophotometer is a device used to measure the intensity of light in a portion of the spectrum, especially light transmitted or emitted by a specific substance. A spectrophotometer measures how much light is absorbed by a chemical substance by measuring the intensity of the light beam as it passes through a blood sample or other solution. Each compound in the sample or solution absorbs or transmits light within a specific wavelength range. The absorbance is determined using the Beer-Lambert law. Each compound in the sample or solution absorbs or transmits light at a specific set of wavelengths of interest governed by an extinction coefficient.

[0007] In such tests, critical care hematology parameters can be measured, which may include hematocrit, free and total hemoglobin, bilirubin, lipids, and oxygen saturation (i.e., E hemoglobin fraction). Doctors and clinicians rely on these measurements to make decisions during patient treatment. These measurements are typically performed on large, complex analyzers in central hematology laboratories. However, obtaining fast, accurate, and precise results in a point-of-care environment is in many ways better because it saves time in critical diagnostic situations and avoids sample transportation issues in intensive care units. Some blood gas analyzers offer point-of-care capabilities, but do not offer a single solution that provides the desired time to obtain results, accuracy, precision, and reliability while being simpler and easier to manufacture than existing equipment.

[0008] There is a need for an analytical device that provides improved accuracy and precision of measured parameters of a sample at the patient's point of care within the time it takes to obtain a result and that is easier and less expensive to manufacture. Summary of the invention

[0009] Disclosed are acoustic electrophoresis analysis devices, methods, and systems. The problem of complex, slow, imprecise, and inaccurate blood sample testing for point-of-care use is solved by a device as follows: the device is configured to separate red blood cells and plasma in a whole blood sample in a sample vessel by means of ultrasonic sound waves, which are generated in the sample vessel by a single acoustic transducer (such as a piezoelectric transducer driven with one or more specific first excitation frequencies or excitation frequency ranges). After testing the separated plasma and / or red blood cells, the analysis device can be configured to lyse the red blood cells by means of ultrasonic sound waves, which are generated in the sample vessel by a single piezoelectric transducer driven with one or more specific second excitation frequencies or excitation frequency ranges. Since the analysis device can be configured to lyse the red blood cells after testing the separated plasma and / or red blood cells, the separated plasma and red blood cells cannot be mixed in the analysis device or a separate device to reconstitute whole blood. The analysis device can be configured to directly lyse the red blood cells after testing the separated plasma and / or red blood cells. That is, the same analytical device may be configured to separate red blood cells and plasma in a whole blood sample, and then lyse the red blood cells directly after testing the separated plasma and / or red blood cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments described herein and, together with the specification, explain these embodiments. The drawings are not intended to be drawn to scale, and certain features and certain views of the drawings may be shown exaggerated and not to scale or schematically for the sake of clarity and conciseness. Not every component may be labeled in every drawing. Like reference numerals in the drawings may represent and refer to the same or similar elements or functions. In the drawings:

[0011] Figure 1 is a perspective view of an acoustoelectrophoresis analysis apparatus according to the present disclosure.

[0012] Figure 2 is a top view of an acousto-electrophoresis analysis device according to the present disclosure.

[0013] Figure 3 is a bottom view of the acoustoelectrophoresis analysis device according to the present disclosure.

[0014] Figure 4 is a first end elevation view of an acoustoelectrophoresis analysis apparatus according to the present disclosure.

[0015] Figure 5 is a second end elevation view of an acoustoelectrophoresis analysis device according to the present disclosure.

[0016] Figure 6 is a first side elevation view of an acoustoelectrophoresis analysis apparatus according to the present disclosure.

[0017] Figure 7is a cross-sectional view of an exemplary acoustoelectrophoresis analysis device according to the present disclosure.

[0018] Figure 8 is a cross-sectional view of an exemplary acoustoelectrophoresis analysis device according to the present disclosure.

[0019] Fig. 9 is a perspective view of components of an exemplary sample vessel according to the present disclosure.

[0020] Fig.10 is a graphical representation of the total displacement of an exemplary lysis device according to the present disclosure.

[0021] Fig.11 is a schematic diagram of an exemplary analyzer according to the present disclosure.

[0022] Fig.12 is a schematic diagram of components of an exemplary analyzer according to the present disclosure.

[0023] Fig.13 is a diagrammatic view of an exemplary blood testing method according to one embodiment of the present disclosure.

[0024] Fig.14A is a schematic representation of a whole blood sample inserted into a microchannel according to the present disclosure.

[0025] Fig. 14B is a schematic representation of the separation of red blood cells and plasma fractions in a whole blood sample in a microchannel after having been exposed to ultrasonic sound waves at a first frequency or a first frequency range in accordance with the present disclosure.

[0026] Fig. 14C is a schematic representation of substantially complete separation of red blood cells (dashed area) and plasma (line area) in a whole blood sample in a microchannel after having been exposed to ultrasonic sound waves at a second frequency or second frequency range in accordance with the present disclosure.

[0027] Fig.14D is a schematic representation of a lysed blood sample including plasma and lysed red blood cells in a microchannel after having been exposed to ultrasonic sound waves at a third frequency or a third frequency range in accordance with the present disclosure.

[0028] Fig.15 A method for calculating an absorption spectrum based on known calculations for absorption in liquid media is illustrated.

[0029] Fig.16 The spectral distribution coefficients of hemoglobin forms are shown.

[0030] Fig.17A is a bottom perspective view of an assembly constructed in accordance with the present disclosure.

[0031] Fig. 17B yes Fig.17A A top perspective view of the components of the . DETAILED DESCRIPTION

[0032] The following detailed description refers to the accompanying drawings. The same reference numbers in different drawings may identify the same or similar elements.

[0033] The mechanism proposed in the present disclosure circumvents the above-mentioned problems. The present disclosure describes an acoustic electrophoresis analysis device, an analyzer, and a lysis method, including an acoustic electrophoresis analysis device, which is configured to: first separate red blood cells and plasma in a whole blood sample for testing in a sample vessel by means of ultrasonic sound waves, and the ultrasonic sound waves are generated in the sample vessel by a piezoelectric transducer connected to the sample vessel and driven by one or more first frequencies or a first excitation frequency range; then lyse the red blood cells in the same sample vessel by means of ultrasonic sound waves, shear forces, pressure, cavitation, and / or fluid movement, and the ultrasonic sound waves, shear forces, pressure, cavitation, and / or fluid movement are generated in the sample vessel by a piezoelectric transducer driven by one or more second frequencies or a second excitation frequency range. In a non-limiting embodiment, the acoustic transducer (such as a piezoelectric transducer) is a single piezoelectric transducer. The present disclosure further describes an analyzer configured to receive and interact with an acoustic electrophoresis analysis device to test a sample in a sample vessel, and a method of use.

[0034] As used herein, the terms "comprises," "including," "comprising," "including," "having," "having," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. In addition, unless expressly stated otherwise, "or" refers to an inclusive or, not an exclusive or. For example, a condition A or B satisfies any of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0035] In addition, "a" or "an" is used to describe the elements and components of the embodiments herein. This is done for convenience only and to give a general meaning of the inventive concept. The description should be understood to include one or more, and the singular also includes the plural, unless it is obvious that there is another meaning.

[0036] Additionally, use of the term "plurality" is intended to convey "more than one" unless explicitly stated to the contrary.

[0037] As used herein, qualifiers such as "about," "approximately," and combinations and variations thereof are intended to include not only the exact quantities or values ​​specified, but also include some slight deviations that may result, for example, from manufacturing tolerances, measurement errors, wear and tear, stress exerted on various parts, and combinations thereof.

[0038] As used herein, the term "substantially" means that the subsequently described parameter, event, or situation occurs completely, or that the subsequently described parameter, event, or situation occurs to a large extent. For example, the term "substantially" means that the subsequently described parameter, event, or situation occurs at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the time; or means that a size or measurement result is within at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the referenced size or measurement result.

[0039] The use of the term "at least one" or "one or more" will be understood to include one and any number more than one. In addition, the use of the phrase "at least one of X, V, and Z" will be understood to include individual X, individual V, and individual Z, as well as any combination of X, V, and Z.

[0040] The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing between two or more items and is not intended to imply any order or sequence or importance of one item relative to another or any added order unless expressly stated otherwise.

[0041] Finally, as used herein, any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0042] As discussed above, typical previous devices for testing blood samples for point-of-care use are complex, slow, imprecise, and inaccurate. The present disclosure addresses these deficiencies with the aid of devices, systems, and methods for: separating plasma from red blood cells in whole blood in a sample vessel with the aid of ultrasonic sound waves generated by a piezoelectric transducer connected to the sample vessel, the piezoelectric transducer being driven at a first specific excitation frequency having a power sufficient to initiate separation of the red blood cells from the plasma, followed by a second excitation frequency at a power level sufficient to substantially complete the separation of the red blood cells from the plasma, thereby running at least one first test; and then lysing the red blood cells in the sample vessel with the aid of ultrasonic sound waves at a third excitation frequency at a power level sufficient to lyse the red blood cells, thereby producing a lysed blood sample, on which one or more second tests may be run.

[0043] Referring now to the drawings, and in particular to Figure 1-Figure 8 , an acoustophoresis device 10 is shown. Generally speaking, the acoustophoresis device 10 includes a sample vessel 12 and an acoustic transducer 14, which may be a piezoelectric transducer 14. The acoustic transducer 14 will be referred to herein as the piezoelectric transducer 14. The piezoelectric transducer 14 is bonded to the sample vessel 12. In one embodiment, the acoustophoresis device 10 is an integral structure such as formed by the sample vessel 12 and the piezoelectric transducer 14 bonded together using a suitable bonding material (such as epoxy resin). The sample vessel 12 is preferably permanently bonded to the piezoelectric transducer 14 in a non-clamping manner. This non-clamping manner is performed to minimize vibration losses between the sample vessel 12 and the piezoelectric transducer 14.

[0044] The sample vessel 12 is provided with a first substrate 15, which is bonded to a second substrate 16 using a suitable bonding material (such as epoxy). The first substrate 15 has an upper surface 17 and a lower surface 18. The second substrate 16 has an upper surface 20, a lower surface 21, a microchannel 22 within the confines of the upper surface 20, a first port 24 extending from the lower surface 21 and in fluid communication with the microchannel 22, and a second port 26 extending through the lower surface 21 to the microchannel 22 and in fluid communication with the microchannel 22. In one embodiment, the upper surface 17 of the first substrate 15 may have a mounting area for the piezoelectric transducer 14.

[0045] In one embodiment, the sample vessel 12 has a top 40, a bottom 42, a first end 44, a second end 46, a first side 48, and a second side 50, wherein the first side 48 and the second side 50 extend between the first end 44 and the second end 46 and between the top 40 and the bottom 42. In one embodiment, the top 40 and the bottom 42 are flat. In one embodiment, the first side 48 and the second side 50 are flat. In one embodiment, the first end 44 and the second end 46 are flat. In one embodiment, the top 40, the bottom 42, the first end 44, the second end 46, the first side 48 and the second side 50 cooperate to form a three-dimensional cuboid. In some embodiments, the piezoelectric transducer 14 cooperatively engages the outer surface of the sample vessel 212. For example, the piezoelectric transducer 14 and the outer surface can have flat surfaces configured to be positioned together.

[0046] The sample vessel 12 can be partially transparent, substantially transparent, or completely transparent. In one embodiment, the sample vessel 12 is transparent at least above and below the microchannel 22, so that the light beam can pass through the sample vessel 12 through the microchannel 22, interact with any substance in the microchannel 22, and pass out of the sample vessel 12.

[0047] The sample vessel 12 can be constructed of glass. In one embodiment, the sample vessel 12 can be constructed of a material (glass or non-glass) having a Young's modulus in the range of about 50 GPa to about 90 GPa. A material property called Young's modulus or elastic modulus is a measure of the ability of a material to undergo a change in length under longitudinal tension or compression. Young's modulus is equal to longitudinal stress divided by strain. In one embodiment, the sample vessel 12 can be constructed of a plastic having a stiffness and / or Young's modulus similar to that of glass. In one embodiment, the sample vessel 12 can be constructed of alkali borosilicate glass. An example of alkali borosilicate glass is made by Schott Advanced Optics, located at 400 York Avenue, Duryea, zip code 18642, and is commercially available under the name of "D 263T ECO Thin Glass".

[0048] In one embodiment, the sample vessel 12 may have one or more conductive structures 30 that may be metal sputtered or otherwise bonded to the upper surface 17 of the first substrate 15. The one or more conductive structures 30 provide an electrical path to which an element such as a piezoelectric transducer 14 may be connected. The one or more conductive structures 30 may include a first electrode 32 and a second electrode 34 positioned on both sides and extending approximately parallel to the microchannel 22. The first electrode 32 and the second electrode 34 may extend within a parallel range of plus or minus 5 degrees (preferably, extend in parallel). In the illustrated embodiment, both the first electrode 32 and the second electrode 34 are in a linear configuration and are adjacent to, but not covering, the microchannel 22, so as not to block the light beam generated by the spectrophotometer as discussed herein. In some embodiments, the first electrode 32 and the second electrode 34 may be parallel or non-parallel, as long as the first electrode 32 and the second electrode 34 do not block the light beam 116 and allow the capacitance readings from the first electrode 32 and the second electrode 34 to be correlated with the expected channel contents in the microchannel 22 during calibration. In some embodiments, the first electrode 32 and / or the second electrode 34 may have a serpentine configuration in which one or more portions cross over the microchannel 22 , but outside of the intended path of the light beam 116 through the sample vessel 12 .

[0049] The sample vessel 12 has a length from a first end 44 to a second end 46, a width from a first side 48 to a second side 50, a thickness between a top 40 and a bottom 42, and an aspect ratio defining a proportional relationship between the length and the width. The sample vessel 12 has a longitudinal axis along the length and a transverse axis along the width.

[0050] In one embodiment, the aspect ratio of the sample vessel 12 is in the range of from about 0.5 to about 3.0. In one embodiment, the aspect ratio of the sample vessel 12 is in the range of from about 1.4 to about 1.9. In one embodiment, the length can be about 22 millimeters and the width can be about 12 millimeters. In one embodiment, the length can be about 17 millimeters and the width can be about 12 millimeters. In one embodiment, the length can be about 17 millimeters and the width can be about 6 millimeters. In one embodiment, the length can be about 12 millimeters and the width can be about 6 millimeters.

[0051] The microchannel 22 can be configured to receive a fluid sample (including, but not limited to, a blood sample, a "blank" sample, and / or a cleaning solution sample) through the first port 24 and / or the second port 26. The inlet 52 can be connected to the first port 24, and the outlet 54 can be connected to the second port 26 to allow the first tube 56 and the second tube 58 to be fluidically connected to the microchannel 22, respectively. The microchannel 22 has a length, a width, and a height. Typically, the length of the microchannel 22 is oriented along the longitudinal axis of the sample vessel 12, and the width of the microchannel 22 is oriented along the transverse axis of the sample vessel 12. However, it will be understood that the microchannel 22 can be oriented to be angled or offset from the longitudinal axis and / or the transverse axis of the sample vessel 12.

[0052] The microchannel 22 has an aspect ratio that defines a proportional relationship between the width and height of the microchannel 22. In one embodiment, the width to height aspect ratio of the microchannel 22 is in a range from about 0.04 to about 0.175. In one embodiment, the width to height aspect ratio of the microchannel 22 is in a range from about 0.04 to about 0.125. In one embodiment, the width to height aspect ratio of the microchannel 22 is about 0.05.

[0053] In one embodiment, the width of microchannel 22 is about 2 millimeters. In one embodiment, the width of microchannel 222 is about 2.5 millimeters. In one embodiment, the width of microchannel 22 is greater than the illuminated width of the light output region of the absorbance spectrophotometer 102. The illuminated width can be defined as the width of the cross-section of the light output from the absorbance spectrophotometer 102 along the optical path where the optical path intersects the microchannel 22. For example, when the illuminated diameter is between 1 millimeter and 1.5 millimeters, then the width of microchannel 22 can be at least about 1.6 millimeters. The width of microchannel 22 can be determined to achieve sufficient mechanical alignment between microchannel 22 and the optical path. For example, for an illuminated width between 1 millimeter and 1.5 millimeters, the width of microchannel 22 can be about 2 millimeters.

[0054] In one embodiment, the length of microchannel 22 can be between about 10 mm and about 12 mm. In one embodiment, the length of microchannel 22 can be at least about 4 mm. In one embodiment, the length of microchannel 22 can be between about 4 mm and about 20 mm.

[0055] In one embodiment, the length of the microchannel 22 can be based at least in part on a predetermined desired number of acoustic nodes to be created in the microchannel 22. For example, for a microchannel 22 having a width of approximately 2 mm and wherein the whole blood wave propagation velocity is approximately 1500 m / s, a single acoustic node is calculated to be at 350 kHz. The acoustic nodes can be distributed in the microchannel 22, evenly spaced along the length of the microchannel 22 (e.g., 2x2 mm=4 mm), wherein the high pressure causes the lysed blood to be evenly distributed. For example, if the predetermined desired number of acoustic nodes is six nodes on each side wall of the microchannel 22, then in the region where the side walls extend approximately parallel (see Fig. 14C , which depicts five antinode regions 67a and six nodes 68, including two end nodes and four nodes between the five antinode regions 67a), the length of the microchannel 22 can then be set to approximately 17 mm, including the tapered inlet and outlet regions.

[0056] The height of the microchannel 22 can vary, as discussed below. The height of the microchannel 22 can be based on the amount of light output from the absorbance spectrophotometer 102 absorbed in the lysed blood and the desired absorption accuracy. For example, the desired absorption can be approximately 1 optical density (OD).

[0057] In one embodiment, the height of microchannel 22 is about 100 microns. In one embodiment, the height of microchannel 22 is about 150 microns. In one embodiment, the height of microchannel 22 is about 250 microns. In one embodiment, the height of microchannel 22 is about 300 microns. In one embodiment, the height of microchannel 22 is between about 80 microns and about 300 microns. In one embodiment, the height of microchannel 22 is between about 80 microns and about 150 microns.

[0058] The first port 24 and the second port 26 are fluidly connected to the microchannel 22 and extend from the microchannel 22 through the lower surface 21 of the second substrate 16. In one embodiment, the first port 24 is fluidly connected to the microchannel 22 and can extend from the microchannel 22 to the top 40, the bottom 42, the first end 44, the second end 46, the first side 48, and / or the second side 50 of the sample vessel 12. In one embodiment, the second port 26 is fluidly connected to the microchannel 22 and can extend from the microchannel 22 to the top 40, the bottom 42, the first end 44, the second end 46, the first side 48, and / or the second side 50 of the sample vessel 12. The first port 24 and the second port 26 can extend to the same or different ones of the top 40, the bottom 42, the first end 44, the second end 46, the first side 48, and / or the second side 50.

[0059] In one embodiment, the first port 24 and the second port 26 each have a diameter between about 0.5 mm (500 microns) and about 1.5 mm (1500 microns). In one embodiment, the first port 24 and the second port 26 each have a diameter of about 0.8 mm (800 microns). The microchannel 22 tapers toward the first port 24 and the second port 26, such as Figure 2 The taper facilitates providing fluid to and / or from the first port 24. The cross-sectional width (e.g., diameter) of the first port 24 and the second port 26 is less than the width of the microchannel 22. For example, the cross-sectional width of the first port 24 and the second port 26 can be 50% to 100% of the width of the microchannel 22.

[0060] The sample vessel 12 may be integrally fabricated, in that the sample vessel 12 is formed from a single piece of material, or the sample vessel 12 may be formed from multiple parts that are interconnected to form a unified entity. Figure 1 As discussed, the sample vessel 212 can be formed from two substrates bonded together. Alternatively, the sample vessel 12 can be formed from three substrates bonded together, such as Fig. 9 as shown in .

[0061] The second substrate 16 can be laminated with the first substrate 15 to form an integral structure. In one embodiment, the first substrate 15 and the second substrate 16 can be annealed to each other. In one embodiment, the first substrate 15 and the second substrate 16 can be thermally plasma bonded to each other. In one embodiment, the first substrate 15 and the second substrate 16 have the same length to width aspect ratio as the sample vessel 12.

[0062] The microchannel 22 may be positioned in the first substrate 15, the second substrate 16, and / or formed partially in the first substrate 15 and partially in the second substrate 16. In one embodiment, the microchannel 22, the first port 24, and the second port 26 are positioned in the first substrate 15. In one embodiment, the microchannel 22 is etched into the first substrate 15 and / or the second substrate 16. In one embodiment, the microchannel 22 is positioned in the first substrate 15, and one or both of the first port 24 and the second port 26 are positioned in the second substrate 16. One or both of the first port 24 and the second port 26 may be positioned in the first substrate 15 and / or the second substrate 16 (and / or extend through the first substrate 15 and / or the second substrate 16).

[0063] like Fig. 9As shown in FIG. 1 , in one embodiment, the sample vessel 12 may include a first substrate 70, a second substrate 72, and a third substrate 80 between the first substrate 70 and the second substrate 72. The first substrate 70, the second substrate 72, and the third substrate 80 may be stacked to form an integral structure. In one embodiment, the first substrate 70, the second substrate 72, and the third substrate 80 may be thermally plasma bonded to each other. In one embodiment, the first substrate 70, the second substrate 72, and the third substrate 80 may be annealed to each other. One or both of the first port 74 and the second port 76 may be positioned in the second substrate 72. In one embodiment, the microchannel 78 is a groove positioned through the third substrate 80. In one embodiment, the thickness of the third substrate 80 may be the same as the height of the microchannel 78. In one embodiment, the third substrate 80 may be 100 microns thick. In one embodiment (not shown), the microchannel 78 may be positioned in the second substrate 72.

[0064] Return to Figure 1 , the piezoelectric transducer 14 is mounted to the sample vessel 12 (such as to a mounting area of ​​the top 40) to form the overall structure of the acoustophoresis device 10. The piezoelectric transducer 14 can have a mounting surface that is mounted to the mounting area of ​​the top 40. In one embodiment, the piezoelectric transducer 14 is at least partially mounted to the top 40 of the sample vessel 12; however, it will be understood that the piezoelectric transducer 14 can be mounted to the top 40, the bottom 42, the first end 44, the second end 46, the first side 48 and / or the second side 50. The piezoelectric transducer 14 is positioned relative to the microchannel 22 so that it does not block light from moving through the microchannel 22 from the top 40 or the bottom 42 of the sample vessel 12. The piezoelectric transducer 14 can be offset from the microchannel 22 so that the piezoelectric transducer 14 allows light to enter the microchannel 22 from outside the sample vessel 12. In one embodiment, piezoelectric transducer 14 has a length and has a longitudinal axis along the length, the longitudinal axis being oriented substantially parallel (e.g., within 5 degrees of parallel) to the longitudinal axis of sample vessel 12. In one embodiment, the width of piezoelectric transducer 14 is less than the length of piezoelectric transducer 14.

[0065] The piezoelectric transducer 14 may be positioned on a side of the sample vessel 12 opposite one or both of the first port 24 and the second port 26 or on the same side as one or more of the first port 24 and the second port 26 .

[0066] The piezoelectric transducer 14 can be bonded to the sample vessel 12. The bond can be thin relative to the thickness of the piezoelectric transducer 14 and the sample vessel 12. The piezoelectric transducer 14 can be bonded to the sample vessel 12 by means of an adhesive. The adhesive can be configured to allow sound waves to propagate and the sound waves to attenuate low. In one embodiment, a liquid adhesive can be applied to the piezoelectric transducer 14, and then the piezoelectric transducer 14 can be attached to the sample vessel 12 via the liquid adhesive. For example, a liquid adhesive with a temperature stability of up to 350°C, excellent adhesion on glass and high hardness (rigidity) can be applied. In one example, the liquid adhesive can be an epoxy resin glue, such as EPO-TEK 353ND (made by Epoxy Technology Co., Ltd., 14 Fortune Drive, Billerica, Massachusetts), which allows ultrasonic waves to propagate and has a Shore D hardness of about 85. In one example, approximately 5 μl of liquid adhesive can be applied. The piezoelectric transducer 14 may be clamped to the sample vessel 12 and the adhesive cured at about 150° C. In one embodiment, the thickness of the adhesive after curing may be about 100 μm. In one embodiment, the thickness of the adhesive after curing may be about 10 μm.

[0067] The piezoelectric transducer 14 can be configured to convert the applied alternating electric field into another form of energy, such as acoustic pressure waves having one or more frequencies and / or frequency ranges. The piezoelectric transducer 14 can be configured to oscillate when the alternating electric field is applied to the piezoelectric transducer 14, thereby generating acoustic pressure waves that are introduced into the sample vessel 12, which can generate one or more standing wave acoustic nodes within the blood sample in the sample vessel 12. Figure 1 As shown in , the piezoelectric transducer 14 may include a first terminal 90 and a second terminal 92, which are configured to be connected to an alternating voltage source. In one embodiment, the first terminal 90 and the second terminal 92 may be electrically connected to the conductive structure 30 to deliver the alternating voltage. In one embodiment, the piezoelectric transducer 14 may be a piezoelectric ultrasonic transducer.

[0068] The piezoelectric transducer 14 may be configured to generate mechanical activity by expanding and contracting when an alternating electric field is applied, thereby producing sound waves having a specific frequency. Fig.10 A graphical representation of one example of the total displacement of piezoelectric transducer 14 in one exemplary operation of piezoelectric transducer 14 is shown.

[0069] In one embodiment, the piezoelectric transducer 14 can be configured to generate an ultrasonic wave having a first frequency, which can be in the range between 950 kHz and 1100 kHz, that begins to cause separation of plasma from other components of the blood sample (such as red blood cells) within a first predetermined time period. Beginning to separate plasma from other components of the blood sample suspended in the plasma can be referred to herein as pre-enrichment, and the first frequency can be referred to herein as the pre-enrichment frequency. The first frequency can be generated with the aid of a first electrical voltage, which can be between 50 volts and 100 volts. The first predetermined time period can be between 5 seconds and 15 seconds. In one embodiment, the first frequency can be in the range between 600 kHz and 630 kHz.

[0070] The piezoelectric transducer 14 may also be configured to transmit ultrasonic waves at a second frequency and / or frequency range and a second electrical voltage within a second predetermined time period, thereby causing complete separation of red blood cells suspended in plasma in the blood sample. The complete separation of plasma from other components of the blood sample suspended in plasma may be referred to herein as enrichment, and the second frequency may be referred to herein as the enrichment frequency. In one embodiment, the second frequency and / or frequency range may be different from (e.g., lower than) the first frequency or range, the second charge may be different from (e.g., higher than) the first charge, and the second predetermined time period may be the same as or different from the first predetermined time period (e.g., greater than the first predetermined time period). For example, the second frequency and / or frequency range may be between 320kHz and 500kHz, and the second charge may be between 70 volts and 100 volts. The second predetermined time period may be between 10 and 25 seconds.

[0071] In one embodiment, the second frequency can cause one or more acoustic standing waves, one or more acoustic standing waves different from the acoustic standing waves caused by the first frequency. Acoustic standing waves (also called standing waves) are waves that oscillate over time but have a peak amplitude distribution that does not move in space. The acoustic standing waves in the microchannel 22 can form a node region with approximately zero force and approximately no particle movement relative to the rest of the microchannel 22 and an antinode region with the highest force and the most particle movement. When the second frequency is applied, as the red blood cells in the blood sample separate from the plasma, the red blood cells tend to move to the node region in the microchannel 22, and the plasma tends to move to the antinode region. This separation into a known region at a specific and repeatable position within the microchannel 22 allows plasma and / or red blood cell analyte measurement, as will be further described below.

[0072] It should be noted that the first frequency and the second frequency may be collectively referred to herein as the separation frequency, ie, the frequency of the ultrasound or sound waves delivered into the sample vessel is sufficient to separate the red blood cells and plasma of the whole blood sample without rupturing the red blood cells.

[0073] The piezoelectric transducer 14 may also be configured to transmit ultrasonic waves at a third frequency and / or frequency range and a third electrical voltage within a third predetermined time period after the first predetermined time period and the second predetermined time period, thereby causing the red blood cells in the blood sample to lyse. The third frequency and / or frequency range may be referred to as a lysis frequency in this article. The piezoelectric transducer 14 generates an acoustic standing wave in the microchannel 22 when driven by a third charge, and the acoustic standing wave is configured to rupture the cell walls of the cells in the blood sample and is configured to release hemoglobin from the cells mixed with the plasma. In one embodiment, the third frequency and / or frequency range may be different from (e.g., lower than) the first and / or second frequency or range, the third electrical voltage may be different from (e.g., higher than) the first electrical voltage and the same or different from the second charge (e.g., higher than or lower than the second charge), and the third predetermined time period may be the same or different from the first or second predetermined time period (e.g., less than the first or second predetermined time period). For example, the third frequency and / or frequency range may be between 330 kHz and 370 kHz, and the third charge may be between 100 volts and 200 volts. The third predetermined time period may be less than 20 seconds, such as between 0, 1 or 2 and 10 seconds.

[0074] In a non-limiting exemplary embodiment, to cause pre-enrichment, the first frequency may be from 1092 kHz to 1097 kHz, which may be generated during a first predetermined time period of 6 seconds with a first electrical voltage that may be 70 volts. To cause enrichment, the second frequency and / or frequency range may be from 376 kHz to 377 kHz, which may be generated during a second predetermined time period of 12 seconds with a second electrical voltage that may be 80 volts. To cause lysis of red blood cells, the third frequency and / or frequency range may be from 349 kHz to 351 kHz, which may be generated with a third electrical voltage that may be 120 volts.

[0075] In another non-limiting exemplary embodiment, to cause pre-enrichment, the first frequency may be from 1092 kHz to 1097 kHz, which may be generated during a first predetermined time period of 6 seconds with a first electrical voltage that may be 90 volts. To cause enrichment, the second frequency and / or frequency range may be from 376 kHz to 377 kHz, which may be generated during a second predetermined time period of 12 seconds with a second electrical voltage that may be 100 volts. To cause lysis of red blood cells, the third frequency and / or frequency range may be from 349 kHz to 351 kHz, which may be generated with a third electrical voltage that may be 120 volts.

[0076] In a further non-limiting exemplary embodiment, to cause pre-enrichment, the first frequency may be from 600 kHz to 630 kHz, and more specifically from 612 kHz to 613 kHz, which may be generated during a first predetermined time period of 8 seconds with a first charge that may be 5 to 12 volts (e.g., 6 or 10 volts). To cause enrichment, the second frequency and / or frequency range may be from 470 kHz to 500 kHz, and more specifically from 486 kHz to 487 kHz, which may be generated during a second predetermined time period of 15 seconds with a second charge that may be 13 to 20 volts (e.g., 13 or 18 volts). To cause lysis of red blood cells, the third frequency and / or frequency range may be from 343 kHz to 348 kHz, which may be generated with a third charge that may be 13 volts.

[0077] The third frequency may be a resonant frequency that causes resonance in a blood sample introduced into the microchannel 22 of the sample vessel 12, causing the walls of red blood cells in the blood sample to rupture. In one embodiment, the piezoelectric transducer 14 has a first resonant frequency, and the overall structure of the acousto-electrophoresis device 10 has a second resonant frequency that is spectrally spaced from the first resonant frequency, the second resonant frequency being the frequency of the acoustic wave generated by the piezoelectric transducer 14 and introduced into the sample vessel 12, thereby causing cavitation in the blood sample, thereby generating bubbles that rupture in a region of higher pressure and generate shock waves, which then cause the walls of the red blood cells to rupture, thereby releasing hemoglobin from within the red blood cells into the plasma of the blood sample.

[0078] In one example, at the primary resonance of the acoustophoresis device 10 (i.e., the sample vessel 12 coupled to the piezoelectric transducer 14), for example, when the sample vessel 12 is made of glass, the width of the microchannel 22 is about 2 mm, the aspect ratio is 0.05 to 0.125, and the width of the sample vessel 12 is about 12 mm, the aspect ratio is 1.4 to 1.9. The piezoelectric transducer 14 can be configured to generate ultrasonic sound waves in the range of 330 kHz to 370 kHz, the peak pressure in the microchannel 22 is 5 MPa, and the peak velocity is up to 8 m / s.

[0079] However, the ultrasonic sound waves inside the microchannel 22 and the ultrasonic piezoelectric transducer 14 may generate unwanted heat, including unwanted heat in the blood sample in the microchannel 22. To avoid overheating of the blood sample, the piezoelectric transducer 14 can be operated to generate a resonant frequency within a predetermined time period. For example, the piezoelectric transducer 14 can be operated to generate a sound wave with a second resonant frequency between about 1 second and about 2 seconds. In one embodiment, the piezoelectric transducer 14 can be operated to generate a sound wave with a second resonant frequency in less than about one and a half seconds. In one example, the acoustophoresis device 10 can be configured to operate the piezoelectric transducer 14 for equal to or less than 1.5 seconds to result in 99.99% lysis of red blood cells. In one example, the acoustophoresis device 10 can be configured to operate the piezoelectric transducer 14 for about 10 seconds or less.

[0080] In one embodiment, ultrasonic sound waves inside microchannel 22 break blood cells and cell walls into fine particles that produce less light scattering during optical measurements of the blood sample than larger particles.

[0081] In one embodiment, the piezoelectric transducer 14 may be configured to generate ultrasonic sound waves within a frequency range, and the second resonant frequency may be within the frequency range.

[0082] In one embodiment, the piezoelectric transducer 14 may be configured to generate ultrasonic sound waves within a frequency range between 300 kHz - 1.5 MHz.

[0083] The resonant frequency and / or frequency range can be determined based on one or more factors, including: the size, shape, and material of the sample vessel 12; the size and shape of the microchannel of the sample vessel 12; the amount of fluid in the fluid sample; and / or the size, shape, and material of the piezoelectric transducer 14; and / or the size, shape, and material of the adhesive layer between the sample vessel and the piezoelectric transducer.

[0084] For example, when sample vessel 12 is made of glass, the aspect ratio of microchannel 22 is about 0.05 to about 0.125, and the aspect ratio of sample vessel 12 is about 1.4 to about 1.9. Piezoelectric transducer 14 may be configured to generate ultrasonic sound waves in the range of about 330 kHz to about 370 kHz.

[0085] The width of the microchannel 22 may be determined based at least on the speed of acoustic wave propagation inside the blood sample (e.g., about 1500 m / s) and using a predetermined desired number of acoustic nodes as one node in the middle of the microchannel 22, such that the frequency is about 330 kHz to about 370 kHz. The following formula may be used to at least partially determine the first acoustic node inside the microchannel 22 (having an exemplary 2000 μm width and 100 μm depth), without taking into account any minor reflections or other images:

[0086] f=v / λ

[0087] Where f is the frequency, v is the wave speed in the fluid, and λ is the wavelength (where the wavelength is twice the width of the microchannel 22, with a single node across the width of the channel for a half-wavelength resonant mode).

[0088] Because it may be difficult to accurately calculate the resonant frequency of the sample vessel 12 due to manufacturing and / or material differences, in one embodiment, the piezoelectric transducer 14 can be configured to scan the third frequency within a frequency range having a plurality of frequencies, starting from a first scanning frequency, and reaching a third scanning frequency among the plurality of frequencies through one or more second scanning frequencies. In one embodiment, the piezoelectric transducer 14 can be configured to scan the third frequency range in steps (such as steps of 1 kHz frequency). In one embodiment, scanning the third frequency range from the first scanning frequency to the third scanning frequency ensures that the resonant frequency of the acoustophoresis device 10 plus the blood sample is reached, even taking into account differences in the geometry and materials of the acoustophoresis device 10.

[0089] In one embodiment, the piezoelectric transducer 14 may be configured to scan a third frequency range between about 330 kHz and about 370 kHz, such as in steps of about 1 kHz. The piezoelectric transducer 14 may be configured to scan a third frequency range starting from about 330 kHz and reaching about 370 kHz, and / or the piezoelectric transducer 14 may be configured to scan a third frequency range starting from about 370 kHz and reaching about 330 kHz, for example.

[0090] In one embodiment, the piezoelectric transducer 14 can be configured to scan the third frequency range within a time period greater than 0 seconds and less than 5 seconds, less than 4 seconds, less than 3 seconds, less than 2 seconds, and / or less than 1 second. In one embodiment, the piezoelectric transducer 14 can be configured to scan the third frequency range within a time period of about 1 second and about 2 seconds.

[0091] In one embodiment, additionally or alternatively, the acousto-electrophoresis device 10 can lyse blood cells in a blood sample by inducing shear and vibration modes in the microchannel 22 of the sample vessel 12. The displacement (possibly primarily lateral displacement) of the rigidly coupled ultrasonic piezoelectric transducer 14 causes the sample vessel 12 coupled to the piezoelectric transducer 14 to vibrate and move. When activated, the ultrasonic piezoelectric transducer 14 changes shape, thereby contracting and extending (lateral displacement) as shown in FIG. Fig.12 The movement of the ultrasonic piezoelectric transducer 14 is transferred to the sample vessel 12 , thereby changing the geometry and / or volume of the microchannel 22 , which induces shear and vibration in the microchannel 22 of the sample vessel 12 . Fig.12 A graphical representation of one example of the total displacement of piezoelectric transducer 14 in one exemplary operation of piezoelectric transducer 14 is shown.

[0092] The displacement of the piezoelectric transducer 14 may cause vibration of the sample vessel 12 and generate shear forces within the sample vessel 12, which may then cause and / or contribute to the lysis of the blood sample in the microchannel 22 of the sample vessel 12 due to a combination of high pressure, shear forces, and / or fluid movement inside the microchannel 22. Therefore, in some embodiments, the lysis of the blood sample in the microchannel 22 may be caused by a combination of acoustic standing waves, pressure, cavitation, shear forces, and / or fluid movement within the blood sample.

[0093] When the piezoelectric transducer 14 is activated, shear stress may be generated at the junction between the piezoelectric transducer 14 and the sample vessel 12. The shear stress may result in a high pressure inside the microchannel 22. For example, in one embodiment, the preferred high pressure may be about 5 MPa. In one embodiment, the pressure may be in the range of about 3 MPa to about 7 MPa. The pressure level may be controlled by the contraction / extension level of the piezoelectric transducer 14, which may depend on the electric field strength of the piezoelectric transducer 14.

[0094] The combination of acoustic standing waves inside the microchannel 22 and shear and / or vibration of the sample vessel 12 induces significant cavitation and associated shock waves in the whole blood sample in the microchannel 22, which causes the cell walls of red blood cells to rupture, thereby releasing hemoglobin from the red blood cells.

[0095] exist Figure 2-Figure 8In one embodiment illustrated in the figure, the acoustophoresis device 10 can be combined with or otherwise connected to a printed circuit board (PCB) 94. The acoustophoresis device 10 can be constructed in the same manner as the acoustophoresis device 204 described in U.S. Serial No. 63 / 366,552, the entire contents of which are hereby incorporated by reference herein. The PCB 94 can be provided with a conductive structure (not shown) on an outer surface, or the conductive structure can be embedded in the interior of the PCB 94 as known in the art. The conductive structure of the PCB 94 can electrically connect the conductive structure 30 of the acoustophoresis device 10 to one or more electrical connectors 96 (only one of which is numbered). One or more electrical connectors 96 can be configured to connect the acoustophoresis device 10 to an analyzer, such as the analyzer 100 further described herein. The PCB 94 can be constructed in the same manner as the support substrate 202 described in U.S. Serial No. 63 / 366,552. Additionally, the acoustophoresis device 10 may be attached to the PCB 94 by soldering mounting pads on the sample vessel 12 to mounting pads on the PCB 94 in the manner described in US Serial No. 63 / 366,552.

[0096] Reference now Fig.11 and Fig.12 In some embodiments, the acoustophoresis device 10 can be a component of an analyzer 100. The analyzer 100 can include the acoustophoresis device 10, an absorbance spectrophotometer 102, a fluid distribution system 104 (e.g., including a peristaltic pump), and / or a controller 106. In one embodiment, the acoustophoresis device 10 can be removed and / or replaced from the other components of the analyzer 100. In one embodiment, the analyzer 100 can also include a support 108, which is configured to receive a PCB 94 that supports the other components of the acoustophoresis device 10. In one embodiment, the acoustophoresis device 10 can be held by the support 108 so that the acoustophoresis device 10 can vibrate and / or move within a range of vibration and / or movement.

[0097] In one embodiment, the analyzer 100 may also include one or more processors 140 coupled to one or more non-transitory computer-readable media 142 storing instructions that are provided to the one or more processors 140 and that, when executed by the one or more processors 140, cause the one or more processors 140 to perform the functions described below. In one embodiment, the one or more processors 140 and the one or more non-transitory computer-readable media 142 may be part of the controller 106. However, it will be understood that one or more of the processors 140 and / or the non-transitory computer-readable media 142 may be external to the controller 106 and / or external to other components of the analyzer 100. In one embodiment, the analyzer 100 may include and / or be connectable to one or more sensor cartridges 143 ( Fig.12 ), and / or one or more test kits 145.

[0098] In one embodiment, the analyzer 100 may include an optical input 112 and an optical receiver 114 positioned adjacent to the sample vessel 12, the optical input 112 being positioned to emit a light beam 116 through the top 40, the bottom 42, and the microchannel 22, and the optical receiver 114 being positioned to receive the portion of the light beam 116 after at least a portion of the light beam 116 has passed through the top 40, the bottom 42, and the microchannel 22. In one embodiment, the optical input 112 may be connected to a first fiber optic bundle 118, which connects a light source 120 to the optical input 112. The optical receiver 114 may be connected to a second fiber optic bundle 119, which connects the optical receiver 114 to the absorbance spectrophotometer 102. The light source 120 may be, for example, one or more light emitting diodes or a tungsten halogen lamp. In one embodiment, the light may be white light having a wavelength in the range of from about 450-700 nanometers.

[0099] The absorbance spectrophotometer 102 can be configured to measure the intensity of light in a portion of the spectrum, particularly light transmitted or emitted by a specific substance in the fluid sample in the microchannel 22 of the sample vessel 12. The absorbance spectrophotometer 102 can be configured to measure how much light is absorbed by a chemical substance by measuring the intensity of the light as the light beam passes through a blood sample or other fluid sample. Each compound in a sample or solution absorbs or transmits light in a specific wavelength range.

[0100] The fluid dispensing system 104 can have an inlet 130 that can be fluidically connected to the first port 24, and an outlet 132 that can be fluidically connected to the second port 26 of the sample vessel 12 of the acousto-electrophoresis device 10. The fluid dispensing system 104 can move one or more fluid samples (such as a blank sample or a blood sample or a cleaning solution) through the inlet 130, through the first port 24 into the microchannel 22 of the sample vessel 12. In one embodiment, the fluid dispensing system 104 can flush the microchannel 22, thereby discharging the material in the microchannel 22 from the outlet 132 through the second port 26 of the sample vessel 12. The fluid dispensing system 104 can be, for example, a peristaltic pump that can be operated automatically, manually, or a combination of automatically and manually. The fluid dispensing system 104 can be operated by a controller 106. In one embodiment, the inlet 130 can be a first tube 56, and the outlet 132 can be a second tube 58, and the inlet 52 can connect the first tube 56 to the first port 24, and the outlet 54 can connect the second tube 58 to the second port 26.

[0101] The controller 106 may be electrically connected to the piezoelectric transducer 14 of the acoustophoresis device 10. The controller 106 may be configured to provide an electrical signal to the piezoelectric transducer 14 that, when received by the piezoelectric transducer 14, causes the piezoelectric transducer 14 to emit ultrasonic sound waves at one or more frequencies and / or frequency ranges, including at the resonant frequency of the overall structure of the acoustophoresis device 10 plus the fluid sample.

[0102] like Fig.11 As shown in FIG. 1 , in one embodiment, the controller 106 may have a first electrical contact 134 and a second electrical contact 136. The first electrical contact 134 and the second electrical contact 136 may be electrically connected to the first terminal 90 and the second terminal 92 of the piezoelectric transducer 14 of the acousto-electrophoresis device 10, respectively, so that an electric potential may be provided to the piezoelectric transducer 14.

[0103] The support 108 can hold the acoustophoresis device 10 in place between the optical input 112 and the optical receiver 114, and can position the acoustophoresis device 10 to be operably connected to the fluid distribution system 104 and the controller 106. The support 108 can be configured to stabilize the acoustophoresis device 10 in place without applying a force that would significantly change the acoustic impedance of the overall structure of the acoustophoresis device 10. For example, the support 108 can include one or more clamps that apply a clamping force equal to or less than about 20 Newtons (N).

[0104] In one embodiment, the analyzer 100 may also include a neon calibrator 122, an optical feedback detector, one or more digital temperature sensors (not shown), and / or one or more thermal control elements, such as a Peltier element (not shown).

[0105] Reference now Figure 13-14D In one embodiment, a method 200 for analyzing blood may include a step 202 of obtaining or receiving a whole blood sample 60 comprising red blood cells 62 and plasma 64 for analysis.

[0106] In step 204, the whole blood sample 60 is positioned in the microchannel 22 of the acousto-electrophoresis device 10 by pumping the whole blood sample 60 into the microchannel 22 through the inlet 130 and the first port 24 by means of the fluid distribution system 104. The controller 106 can sense when the whole blood sample 60 is positioned in the microchannel 22 using the first electrode 32 and the second electrode 34 positioned on both sides of the microchannel 22 to measure, for example, the admittance magnitude and phase.

[0107] In step 206, the whole blood sample 60 in the microchannel 22 is pressurized. To pressurize the whole blood sample 60 in the microchannel 22, the controller 106 can be programmed to clamp or pinch the inlet 130 after the blood sample has been positioned in the microchannel 22, and reverse the fluid distribution system 104 to pressurize the whole blood sample 60 in the microchannel 22. In one embodiment, the whole blood sample 60 can be pressurized to about 5 psi. In other embodiments, the whole blood sample 60 can be pressurized to a pressure between about 1 psi and about 10 psi.

[0108] In step 208, the controller 106 provides a first electrical signal to the piezoelectric transducer 14, which causes the piezoelectric transducer 14 to emit ultrasonic waves at a first frequency and / or frequency range to cause pre-enrichment of the red blood cells 62 and the plasma 64. In particular, the first frequency can be generated within a first predetermined time period by means of a first electrical voltage, thereby causing the red blood cells 62 and the plasma 64 in the whole blood sample 60 to begin to separate. For example, the first frequency and / or frequency range can be between 950 kHz and 1100 kHz, and the first electrical voltage can be between 50 volts and 100 volts. The first predetermined time period can be between 5 seconds and 15 seconds.

[0109] In step 210 (e.g., after step 208), the controller 106 provides a second electrical signal to the piezoelectric transducer 14, which causes the piezoelectric transducer 14 to emit ultrasonic waves at a second frequency and / or frequency range and a second electrical voltage for a second predetermined time period, thereby causing the red blood cells 62 and plasma 64 in the whole blood sample 60 to be completely separated. For example, the second frequency and / or frequency range can be between 320 kHz and 500 kHz, and the second electrical voltage can be between 70 volts and 100 volts. The second predetermined time period can be between 10 seconds and 25 seconds. In an exemplary embodiment, at least a portion of the plasma 64 is separated / moved or enriched to the surface or outer edge of the microchannel 22, so that at least a portion of the plasma 64 (separated from the red blood cells 62) is located at the antinode area near the surface of the microchannel 22.

[0110] In step 212 (e.g., after steps 208 and 210), the controller 106 activates the light source 120 during a second predetermined time period (when the red blood cells 62 and plasma 64 are completely separated) to transmit the light beam 116 to the optical input 112 through the first fiber optic bundle 118, which directs at least a portion of the light beam 116 through the microchannel 22 at a predetermined location, the predetermined location being aligned with an antinode region within the microchannel 22, the antinode region being the plasma 64 and being substantially free of red blood cells 62. The optical receiver 114 receives the portion of the directed light beam 116 directed through the microchannel at the predetermined location, and transmits the received portion of the light beam 116 to the absorbance spectrophotometer 102 through the second fiber optic bundle 119 to obtain a plasma spectral reading. In one exemplary embodiment, the predetermined location of the microchannel 22 where the light beam 116 is directed may be an antinode region located along the outer edge of the microchannel 22, the antinode region having plasma 64 and being substantially free of red blood cells 62, such that a plasma spectral reading is performed or implemented on the plasma 64 located at the antinode region along the outer edge of the microchannel 22. For example, a portion of the light beam 116 passing through the plasma 64 located at the antinode region along the outer edge / surface of the microchannel 22 is measured by an optical receiver (optical receiver of an absorbance spectrophotometer) to determine the optical absorbance of the plasma 64 (at the antinode region near the outer edge / surface of the microchannel 22), such as by comparing the intensity of the light beam 116 received by the plasma 64 with the intensity of the light beam 116 output by the plasma 64 within a desired frequency range. The determined optical absorbance may then be used to determine the amount or concentration of a plasma component of particular interest (such as bilirubin or free hemoglobin).

[0111] In step 214, the microchannel is pressurized again.

[0112] In step 216, after the absorbance spectrophotometer 102 has acquired the plasma spectrum reading, the controller 106 provides a third electrical signal to the piezoelectric transducer 14, the third electrical signal causing the piezoelectric transducer 14 to emit ultrasonic waves at a third frequency and / or frequency range (including at the resonant frequency of the overall structure of the acoustophoresis device 10 plus the whole blood sample 60), and / or causing the piezoelectric transducer 14 to stretch and contract, thereby generating shear force in the whole blood sample 60 in the microchannel 22, thereby causing the walls of the red blood cells 62 of the whole blood sample 60 to rupture or lyse, thereby producing a lysed blood sample 66.

[0113] In step 218, the controller 106 activates the light source 120 to transmit the medium 116 to the optical input 112, and the optical input 112 transmits at least a portion of the light beam 116 through the lysed blood sample 66 and to the optical receiver 114, and the optical receiver 114 transmits the portion of the light beam 116 that passes through the lysed blood sample 66 to the absorbance spectrophotometer 102 to obtain a measurement result of the lysed blood sample 66.

[0114] The method 200 may also include determining one or more blood oxygenation parameters of the lysed blood sample based at least in part on the signal indicative of the portion of the light beam 116 received by the absorbance spectrophotometer 102 .

[0115] It should be noted that the method 200 can be implemented without pressurizing the whole blood sample 60 in steps 206 and 214. For example, in one embodiment, lysing the red blood cells 62 in step 216 can be performed directly after taking a plasma spectral reading of the separated plasma 64 in step 212. It should be further noted that the method 200 can be implemented such that whole blood reconstitution does not occur, or in other words, the separated red blood cells 62 and plasma 64 in steps 208 or 210 cannot be mixed to reconstitute into whole blood to produce a reconstituted whole blood sample. Therefore, instead of producing a reconstituted whole blood sample, the method 200 produces a lysed blood sample 66 in step 216, and as such, the method 200 may not include reversibility of the plasma separation steps 208 or 210. In addition, it should be noted that method 200 is implemented within a microchannel 22, so that the steps of pre-enriching red blood cells 62 and plasma 64 (step 208), completely separating and measuring the red blood cells 62 and plasma 64 in the whole blood sample 60 (steps 210, 212), and lysing the red blood cells 62 of the whole blood sample 60 and measuring them (steps 216, 218) are all advantageously implemented within the same single (e.g., linear) microchannel 22 of the same sample vessel 12, thereby advantageously avoiding the need for additional downstream channels or chambers in the sample vessel 12 or the need for additional equipment to transfer or collect the separated plasma or red blood cells.

[0116] like Fig.15As shown in , the absorption spectrum can be calculated based on known calculations for absorption in liquid media. Fig.16 As shown in , determining one or more blood oxygen parameters may also include analyzing spectral distribution coefficients of hemoglobin forms, such as one or more of the following: oxygenated hemoglobin (O2HB), deoxyhemoglobin (HHB), carboxyhemoglobin (COHB), methemoglobin (METHB) and plasma bilirubin (NBILI) as well as interfering substances cyanomethemoglobin (CN_MET_B), sulfhemoglobin (SULF_HIGH) and methylene blue (METH_BLUE_A).

[0117] Determining one or more blood oxygenation parameters may be based on measuring spectrophotometric optical absorption, ie, the absorption of light by components in the lysed blood sample 66 .

[0118] Determining one or more blood oxygen parameters may include measuring at least total hemoglobin (THB) and one or more hemoglobin fractions, such as the following: oxyhemoglobin (O2HB), deoxyhemoglobin (HHB), carboxyhemoglobin (COHB), methemoglobin (METHB).

[0119] In one embodiment, the method 200 may include inputting and discharging a cleaning solution into the microchannel 22 of the sample vessel 12 before and / or after introducing the whole blood sample 60 into the microchannel 22. The method 200 may also include activating the piezoelectric transducer 14 to generate sound waves and / or shear forces to agitate the cleaning solution in the microchannel 22. In one embodiment, the sample vessel 12 may be used, cleaned, and reused. In one embodiment, the acoustophoresis device 10 may not be reusable and may be replaced for each new whole blood sample 60. In such an embodiment, the acoustophoresis device 10 may be discarded after a single use.

[0120] The method 200 may also include calibrating the analyzer 100 with the fluid sample. In one embodiment, the fluid sample may be a test sample called a "blank sample" that may be used to calibrate the analyzer 100. The blank sample may contain a dye solution that may be used to measure the transmission scatter of the medium 116.

[0121] In one embodiment, the volume of the whole blood sample 60 may be approximately 12 microliters. The whole blood sample 60 typically includes plasma 64 and red blood cells 62 (which may comprise 45%-60% of the blood sample), and may include lipids.

[0122] In one embodiment, the whole blood sample 60 is maintained at a consistent temperature. In one embodiment, the temperature of the whole blood sample 60 is thirty-seven degrees Celsius (37° C.) plus or minus approximately 0.3 degrees. In one embodiment, the temperature of the whole blood sample 60 is less than forty degrees Celsius (40° C.) to avoid damaging the whole blood sample 60. In one embodiment, the whole blood sample 60 is maintained at a substantially consistent temperature using one or more temperature sensors (not shown) and / or one or more thermal control elements (not shown).

[0123] An example of the analyzer 100 and the acoustic electrophoresis device 10 in use will now be described. In one example, the sample vessel 12 can be made of glass and can have a length to width aspect ratio in the range of about 1.4 to about 1.9, and the microchannel 22 can have a height to width aspect ratio of about 0.05 (e.g., having a height of about 100 microns and a width of about 2 millimeters). The sample vessel 12 can be inserted into the path in which the medium 116 will travel between the optical input 112 and the optical receiver 114 of the absorbance spectrophotometer 102. It should be understood that the analyzer 100 can be provided with various instruments (including mirrors and / or waveguides) to guide the medium through the path. The fluid distribution system 104 can insert the whole blood sample 60 into the microchannel 22 of the sample vessel 12.

[0124] The controller 106 can be electrically connected to the piezoelectric transducer 14 of the sample vessel 12 and can provide an electrical signal to the piezoelectric transducer 14 to cause the piezoelectric transducer 14 to emit a first ultrasonic sound wave in a frequency range of about 950 kHz to about 1100 kHz in steps of about 1 kHz. The frequency range can be transmitted over a time period of about 5 seconds to about 15 seconds. This causes the red blood cells 62 to begin to separate from the plasma 64 in the whole blood sample 60, as shown in FIG. Fig. 14B For clarity, Figures 14A-14D In FIG. 6 , red blood cells 62 are shown as circles and plasma 64 is represented by lines.

[0125] Then, the controller 106 can provide an electrical signal to the piezoelectric transducer 14 to cause the piezoelectric transducer 14 to transmit a second ultrasonic wave in a frequency range of about 320 kHz to about 500 kHz in steps of about 1 kHz. The frequency range can be transmitted within a time period of about 10 seconds to about 25 seconds. The second ultrasonic wave is transmitted through the whole blood sample 60 to complete the separation of the red blood cells 62 and the plasma 64, as shown in FIG. Fig. 14C as shown in .

[0126] The standing waves generated by the second ultrasonic sound wave cause the red blood cells 62 and plasma 64 to move to known portions of the microchannel 22, such as Fig. 14CThe antinode regions 67 (only the antinode regions 67a and 67b are numbered in the figure) and the node regions 68 (only one of which is numbered in the figure) between the antinode regions 67 form a known portion of the microchannel 22. The separated plasma 64 is enriched in (e.g., separated / moved to) the antinode regions 67 in the microchannel 22 where there are substantially no red blood cells 62, while the red blood cells 62 move to the node regions 68. Fig. 14C As illustrated in , a plurality of staggered node regions 68 and antinode regions 67 (such as exemplary antinode region 67a) may be formed along the outer edge of the microchannel 22, and a plurality of antinode regions 67 (such as exemplary antinode region 67b) may be formed along the central axis of the microchannel 22. The outer edge of the microchannel 22 may also be referred to as the side, wall, or surface of the microchannel 22, such that the antinode region 67a is formed near the side, wall, or surface of the microchannel 22, and the antinode region 67b is formed at the center of the microchannel 22. The node region 68 may be formed in the remaining space of the microchannel 22. The desired pattern of antinode regions 67 and node regions 68 may be calculated using the techniques described above.

[0127] Then, the controller 106 can cause the light source 120 to emit a light beam 116, which is directed by the optical input 112 through a predetermined portion (i.e., one antinode region 67) of the plasma 64 of the microchannel 22 that has been separated from the red blood cells 62 of the whole blood sample 60. At least a portion of the light beam 116 passes through the plasma 64 and is received by the optical receiver 114, and is directed to the absorbance spectrophotometer 102 to obtain a plasma measurement result. Measurements of plasma can also be made using absorbance spectroscopy. Patient blood samples (i.e., whole blood samples) include plasma and red blood cells. As discussed above, red blood cells (RBCs) are separated from plasma to physically separate plasma to measure plasma components of particular interest, such as bilirubin or free hemoglobin. RBCs contain hemoglobin in the following forms: oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin. In some cases, free hemoglobin may be found in plasma. Free hemoglobin may result from improper phlebotomy (drawing of a blood sample) or from a patient's physiological condition, such as hemolytic anemia.

[0128] In one exemplary embodiment, the predetermined portion of the microchannel 22 through which the light beam 116 passes is an antinode region 67a located along the outer edge of the microchannel 22. In this embodiment, the controller 106 is configured to cause the light source 120 to emit the light beam 116 to pass through the antinode region 67a at the side of the microchannel 22 where the plasma has been separated. Therefore, at least a portion of the light beam 116 passes through the plasma 64 at the antinode region 67a formed near the surface of the microchannel 22 and is received by the optical receiver 114 of the absorbance spectrophotometer 102 to obtain a plasma measurement result of the plasma component at the antinode region 67a (i.e., at the side of the microchannel 22). The optical receiver 114 and / or the absorbance spectrophotometer 102 can be aligned with the side of the microchannel 22 to receive the portion of the light beam 116 that passes through the plasma 64 at the antinode region 67a, and perform optical detection on the plasma formed along the side of the microchannel 22 (i.e., the antinode region 67a) to detect the component or analyte by measuring the plasma at the antinode region 67a. Since there is less optical interference at the side of the microchannel 22 (compared to the center of the microchannel 22), the measurement or detection of the plasma component at the antinode region 67a along the side of the microchannel 22 advantageously results in improved plasma measurement accuracy compared to the measurement at the antinode region 67b along the central axis of the microchannel 22.

[0129] Once the plasma measurement has been obtained, the controller 106 can provide an electrical signal to the piezoelectric transducer 14 to cause the piezoelectric transducer 14 to transmit a third ultrasonic sound wave in a frequency range from about 330 kHz to about 370 kHz in steps of about 1 kHz. The frequency range can be transmitted within a time period of about 2 seconds.

[0130] The frequency, intensity and duration of the third ultrasonic sound wave lyse the red blood cells 62 in the whole blood sample 60, thereby causing the red blood cells 62 to release hemoglobin from the red blood cells 62, such as Fig.14D . The frequency range of the third ultrasonic sound wave includes the resonant frequency of the overall structure of the acousto-electrophoresis device 10 and the whole blood sample 60, thereby causing cavitation in the whole blood sample 60, which ruptures the cell walls of the red blood cells 62 in the whole blood sample 60. Additionally or alternatively, the controller 106 may cause one or more processors 140 to transmit a signal to the piezoelectric transducer 14, which causes the piezoelectric transducer 14 to extend and contract, thereby generating a shear force in the whole blood sample 60 in the microchannel 22, which ruptures the cell walls of the red blood cells 62 in the whole blood sample 60, thereby generating a lysed blood sample 66. In the lysed blood sample 66, the lysed red blood cells are shown as dots.

[0131] The cell walls of a majority (more than 50%) of the red blood cells 62 may be ruptured.

[0132] After the lysed blood sample 66 has been produced, the controller 106 may activate the light source 120 of the analyzer 100 to transmit a medium 116, such as light, through the sample vessel 12 into the lysed blood sample 66. The optical receiver 114 may receive at least a portion of the medium 116 that exits the lysed blood sample 66 and the sample vessel 12. The optical receiver 114 may include one or more photodiodes, for example, for generating an electrical signal as a result of receiving the medium 116.

[0133] The analyzer 100 or one or more computer processors 140 may determine one or more analytes present in the lysed blood sample based at least in part on a signal indicative of light received by the optical receiver 114 of the absorbance spectrophotometer 102. The analyzer 100 or one or more computer processors may also analyze spectral distribution coefficients of hemoglobin forms, such as one or more of the following: carboxyhemoglobin (COHB), oxyhemoglobin (O2HB), methemoglobin (METHB), deoxyhemoglobin (HHB), neonatal bilirubin (NBILI), cyanomethemoglobin (CN_MET_B), sulfhemoglobin (SULF_HIGH), methylene blue dye (METH_BLUE_A).

[0134] The analyzer 100 or one or more computer processors 140 may measure total hemoglobin (THB) and / or one or more hemoglobin fractions, such as the following: oxyhemoglobin (O2HB), methemoglobin (METHB), deoxyhemoglobin (HHB), carboxyhemoglobin (COHB).

[0135] The analyzer 100 or one or more computer processors 140 may output the analysis results. The output may be displayed on one or more displays. The output may be used to determine treatment for the patient.

[0136] Fig.17A and Fig. 17B, an assembly 300 for use in an analyzer 100 constructed in accordance with the present invention is shown. The assembly 300 is provided with an acoustophoresis device 10, a PCB 94, and a flow cell holder 301. The flow cell holder 301 is configured to be mounted to the PCB 94, thereby defining a cavity 302 that is sized and dimensioned to receive the acoustophoresis device 10. When the acoustophoresis device 10 is positioned within the cavity 302, the flow cell holder 301 and the PCB 94 press against the acoustophoresis device 10 to securely hold the acoustophoresis device 10 within the cavity 302, thereby preventing movement of the acoustophoresis device 10 relative to the flow cell holder 301 and the PCB 94. In this regard, the flow cell holder 301 is provided with a body 308 having a first surface 310 configured to cooperatively engage at least a portion of the acoustophoresis device 10 and a second surface 312 opposite to the first surface 310. The body 308 also includes an inner wall 314 extending between the first surface 310 and the second surface 312, thereby defining an opening 316 extending through the body 308. The opening 316 is positioned within the body 308 so that when the acousto-electrophoresis device 10 is placed within the cavity 302, the opening 316 is aligned with the microchannel 22. Aligning the opening 316 with the microchannel 22 permits a light beam to pass through the microchannel 22 and the opening 316, so that the contents (e.g., sample) within the microchannel 22 can be measured (e.g., absorbance measurement). The body 308 of the flow cell holder 301 also includes an inlet orifice 320 and an outlet orifice 322 extending from the first surface 310 to the second surface 312. The inlet orifice 320 securely receives the first tube 56, and the outlet orifice 322 securely receives the second tube 58. For example, the inner diameter of the inlet orifice 320 can be smaller than the outer diameter of the first tube 56, thereby securely receiving the first tube 56. The configuration of the outlet orifice 312 and the second tube 58 can be similar to the inlet orifice 320 and the first tube 56. In this embodiment, the inlet and outlet tube fittings 52 and 54 are optional. In some embodiments, the body 308 of the flow cell holder 301 presses the first tube 56 and the second tube 58 against the sample vessel 12 adjacent to the first port 24 and the second port 26, thereby forming and maintaining a fluid connection between the first tube 56 and the first port 24 and between the second tube 58 and the second port 26.

[0137] The flow cell holder 301 can be fixed to the PCB 94 so as to hold the acousto-electrophoresis device 10 on the PCB 94 without soldering. For example, the PCB 94 and the body 308 can be provided with a series of aligned holes 340, which are suitable for receiving fasteners 342 that connect the flow cell holder 301 to the PCB 94. In the example shown, the holes in the body 308 that receive the fasteners 342 are not shown. In the example shown, the PCB 94 and the body 308 are provided with four aligned holes 340 that are suitable for receiving and / or receiving the fasteners 342. The fasteners 342 can be, for example, screws.

[0138] In this example, mounting pads on sample vessel 12 are pressed against mounting pads on PCB 94 to form an electrical connection therebetween. Assembly 300 may be a component of analyzer 100 and used to perform absorbance measurements on blood samples as described above.

[0139] The following is a numbered list of non-limiting illustrative embodiments of the inventive concepts disclosed herein:

[0140] 1. A fluid analysis device, comprising:

[0141] a sample vessel having an outer surface, a microchannel within the confines of the outer surface, a first port extending through the outer surface to the microchannel, and a second port extending through the outer surface to the microchannel such that a blood sample can be inserted into the microchannel through the first port; and

[0142] a piezoelectric transducer bonded to an outer surface of the sample vessel to form an integral structure,

[0143] A controller configured to send signals to the piezoelectric transducer at a separation frequency and a lysis frequency, wherein the separation frequency is configured to cause the piezoelectric transducer to transmit separation sound waves into the sample vessel sufficient to separate red blood cells and plasma of a blood sample without rupturing the red blood cells, and the lysis frequency is configured to cause the piezoelectric transducer to transmit lysis sound waves into the sample vessel sufficient to induce shear forces in the microchannel, wherein the shear forces are configured to induce cavitation in the blood sample, thereby rupturing the cell walls of the red blood cells in the blood sample and releasing hemoglobin from the red blood cells.

[0144] 2. The fluid analysis apparatus of illustrative embodiment 1, wherein the sample vessel is constructed of glass.

[0145] 3. A fluid analysis device according to any one of the foregoing illustrative embodiments, wherein the separation frequency includes a pre-enrichment frequency and an enrichment frequency, wherein the controller is configured to send a signal to the piezoelectric transducer at a pre-enrichment frequency between approximately 950 kHz and approximately 1100 kHz, and wherein the controller is also configured to send a signal to the piezoelectric transducer at an enrichment frequency sufficient to cause the piezoelectric transducer to transmit enrichment sound waves into the sample vessel without rupturing the red blood cells.

[0146] 4. The fluid analysis apparatus of any one of the preceding illustrative embodiments, wherein the enrichment frequency is a frequency between approximately 320 kHz and approximately 500 kHz.

[0147] 5. The fluid analysis apparatus of any one of the preceding illustrative embodiments, wherein the lysis frequency is a frequency between approximately 300 kHz and approximately 370 kHz.

[0148] 6. A fluid analysis device according to any of the preceding illustrative embodiments, wherein the lysis frequency is a frequency range between approximately 300 kHz and approximately 370 kHz, and the piezoelectric transducer is configured to scan the frequency range between approximately 300 kHz and approximately 370 kHz.

[0149] 7. A fluid analysis device according to any one of the aforementioned illustrative embodiments, wherein the outer surface is a first outer surface having a mounting area, the mounting area having a first shape, and wherein the piezoelectric transducer has a second outer surface, the second outer surface having a second shape corresponding to the first shape, and the second outer surface of the piezoelectric transducer is bonded to the mounting area.

[0150] 8. The fluid analysis apparatus of any one of the preceding illustrative embodiments, wherein the piezoelectric transducer matingly engages an outer surface of a sample vessel.

[0151] 9. A fluid analysis device according to any one of the foregoing illustrative embodiments, wherein the acoustic waves are configured to cause separation of plasma into an antinode region located near the outer surface of the microchannel, and wherein the optical detector is positioned to receive light passing through the plasma located at the antinode region and is configured to detect plasma components based on measurements of the plasma located at the antinode region located near the outer surface.

[0152] 10. A fluid analyzer, comprising:

[0153] A fluid analysis device, comprising:

[0154] a sample vessel having an outer surface, a microchannel within the confines of the outer surface, a first port extending through the outer surface to the microchannel, and a second port extending through the outer surface to the microchannel such that a blood sample can be inserted into the microchannel through the first port; and

[0155] a piezoelectric transducer coupled to an outer surface of the sample vessel to form an integral structure, the piezoelectric transducer being configured to generate a first ultrasonic sound wave having a first frequency, a second ultrasonic sound wave having a second frequency, and a third ultrasonic sound wave having a third frequency, the first ultrasonic sound wave being configured to initiate separation of red blood cells and plasma in a blood sample in a microchannel, the second ultrasonic sound wave being configured to substantially complete separation of red blood cells and plasma in a blood sample in a microchannel, and the third ultrasonic sound wave being configured to vibrate the sample vessel so as to induce shear force in the microchannel, the third ultrasonic sound wave and the shear force being configured to induce cavitation in the blood sample in the microchannel so as to rupture cell walls of red blood cells in the blood sample and release hemoglobin from the red blood cells;

[0156] an absorbance spectrophotometer comprising an optical transmitter and an optical receiver positioned adjacent to the sample vessel, the optical transmitter positioned to transmit the optical medium through the microchannel, the optical receiver positioned to receive at least a portion of the optical medium after the portion has passed through the microchannel;

[0157] a fluid distribution system having an outlet connected to the first port and an inlet connected to the second port; and

[0158] A controller is electrically connected to the piezoelectric transducer, the controller being configured to send an electrical signal to the piezoelectric transducer, the electrical signal causing the piezoelectric transducer to emit the first ultrasonic sound wave, the second ultrasonic sound wave, and the third ultrasonic sound wave when received by the piezoelectric transducer.

[0159] 11. The fluid analyzer of illustrative embodiment 10, wherein the sample vessel is constructed of glass.

[0160] 12. The fluid analyzer of any of illustrative embodiments 10-11, wherein the first frequency is a frequency between approximately 950 kHz and approximately 1100 kHz.

[0161] 13. The fluid analyzer of any of illustrative embodiments 10-12, wherein the second frequency is a frequency between approximately 320 kHz and approximately 500 kHz.

[0162] 14. The fluid analyzer of any of illustrative embodiments 10-13, wherein the third frequency is a frequency between approximately 300 kHz and approximately 370 kHz.

[0163] 15. The fluid analyzer of one of illustrative embodiments 10-14, wherein the third frequency is a frequency range between approximately 300 kHz and approximately 370 kHz, and the piezoelectric transducer is configured to scan the frequency range between approximately 300 kHz and approximately 370 kHz.

[0164] 16. A fluid analyzer according to any one of illustrative embodiments 10-15, wherein the outer surface of the sample vessel is a first outer surface having a mounting area, the mounting area having a first shape, and wherein the piezoelectric transducer has a second outer surface, the second outer surface having a second shape corresponding to the first shape, and the second outer surface of the piezoelectric transducer is bonded to the mounting area.

[0165] 17. The fluid analyzer of any of illustrative embodiments 10-16, wherein the piezoelectric transducer matingly engages an outer surface of a sample vessel.

[0166] 18. A fluid analyzer according to any one of the foregoing illustrative embodiments, wherein the second ultrasonic sound wave is configured to cause plasma to separate into an antinode region located near the outer surface of the microchannel, and wherein the absorbance spectrophotometer is configured to perform measurements on the plasma at the antinode region located near the outer surface to determine the plasma analyte.

[0167] 19. A blood analysis method comprising the following steps:

[0168] transferring a whole blood sample having red blood cells and plasma into a microchannel of a sample vessel;

[0169] Separating red blood cells from plasma in a microchannel;

[0170] obtaining a first absorbance spectrum reading of the plasma separated from the red blood cells;

[0171] lysing red blood cells within the microchannel to provide a lysed blood sample; and

[0172] A second absorbance spectrum reading is obtained of the lysed blood sample within the microchannel.

[0173] 20. A method according to any of the preceding illustrative embodiments, wherein separating red blood cells from plasma within a microchannel is further defined as inducing a first acoustic wave within the microchannel at a sufficient frequency and duration to initiate separation of red blood cells from plasma, and inducing a second acoustic wave within the microchannel at a sufficient frequency and duration to substantially complete separation of red blood cells from plasma.

[0174] 21. The method of any one of the preceding illustrative embodiments, wherein the sufficient frequency of the first sound wave is a frequency between approximately 950 kHz and approximately 1100 kHz and the duration is a time period between approximately 5 seconds and approximately 15 seconds.

[0175] 22. The method of any one of the preceding illustrative embodiments, wherein the sufficient frequency of the second sound wave is a frequency between approximately 320 kHz and approximately 500 kHz and the duration is a time period between approximately 10 seconds and approximately 25 seconds.

[0176] 23. A method according to any one of the preceding illustrative embodiments, wherein lysing red blood cells in a microchannel to provide a lysed blood sample is further limited to inducing a third acoustic wave in the microchannel with a sufficient frequency and duration to induce a shear force in the microchannel, wherein the shear force is configured to induce cavitation in the blood sample, causing the cell walls of the red blood cells to be lysed and hemoglobin to be released from the red blood cells.

[0177] 24. The method of any one of the preceding illustrative embodiments, wherein the sufficient frequency of the third sound wave is a frequency between approximately 320 kHz and approximately 370 kHz and the duration is a time period between approximately 2 seconds and approximately 20 seconds.

[0178] 25. A method according to any one of the preceding illustrative embodiments, wherein the separation step causes the plasma to be enriched in an antinode region near the surface of the microchannel, and wherein the step of obtaining a first absorbance spectrum reading is performed on the plasma located at the antinode region near the surface of the microchannel.

[0179] in conclusion

[0180] Conventionally, blood analysis cannot be performed at the patient's point of care, or is time consuming and expensive. According to the present disclosure, an analyzer 100 is disclosed that provides improved accuracy and precision of measured parameters of a blood sample at the patient's point of care within the time required to obtain results, and is easier to manufacture and less expensive. The controller 106 of the analyzer 100 sends a predetermined signal to the piezoelectric transducer 14 to transmit sound waves into the sample vessel 12 to first separate the plasma 64 from other components of the whole blood (such as red blood cells 62) to obtain plasma measurements, and then lyse the red blood cells 62 in the sample vessel 12 with the aid of ultrasonic sound waves, pressure, cavitation, fluid movement and / or shear forces, which are generated in the sample vessel 12 by a single piezoelectric transducer 14 driven at one or more specific excitation frequencies or frequency ranges.

[0181] The foregoing description provides illustration and description, but is not intended to be exhaustive or to limit the inventive concepts to the precise forms disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the methods set forth in the present disclosure.

[0182] Even though particular combinations of features and steps are recited in the claims and / or disclosed in the specification, these combinations are not intended to limit the present disclosure. In fact, many of these features and steps may be combined in ways not explicitly recited in the claims and / or disclosed in the specification. Although each dependent claim listed below may be directly dependent on only one other claim, the present disclosure includes each dependent claim in combination with all other claims in the claim group.

[0183] Unless explicitly described outside of the preferred embodiment, any element, act or instruction used in this application should not be construed as essential or necessary to the present invention.In addition, the phrase "based on" is intended to mean "based at least in part on", unless explicitly stated otherwise.

Claims

1. A fluid analysis device, comprising: a sample vessel having an outer surface, a microchannel within the confines of the outer surface, a first port extending through the outer surface to the microchannel, and a second port extending through the outer surface to the microchannel such that a blood sample can be inserted into the microchannel through the first port; as well as a piezoelectric transducer bonded to an outer surface of the sample vessel to form an integral structure, A controller configured to send signals to the piezoelectric transducer at a separation frequency and a lysis frequency, wherein the separation frequency is configured to cause the piezoelectric transducer to transmit separation sound waves into the sample vessel sufficient to separate red blood cells and plasma of a blood sample without rupturing the red blood cells, and the lysis frequency is configured to cause the piezoelectric transducer to transmit lysis sound waves into the sample vessel sufficient to induce shear forces in the microchannel, wherein the shear forces are configured to induce cavitation in the blood sample, thereby rupturing the cell walls of the red blood cells in the blood sample and releasing hemoglobin from the red blood cells.

2. The fluid analysis device according to claim 1, wherein: The sample vessels are constructed of glass.

3. The fluid analysis device according to claim 1, wherein: The separation frequency includes a pre-enrichment frequency and an enrichment frequency, wherein the controller is configured to send a signal to the piezoelectric transducer at a pre-enrichment frequency between approximately 950 kHz and approximately 1100 kHz, and wherein the controller is also configured to send a signal to the piezoelectric transducer at an enrichment frequency sufficient to cause the piezoelectric transducer to transmit enrichment sound waves into the sample vessel without rupturing the red blood cells.

4. The fluid analysis device according to claim 3, wherein: The enriched frequency is a frequency between approximately 320 kHz and approximately 500 kHz.

5. The fluid analysis device according to claim 1, wherein: The cracking frequency is a frequency between approximately 300 kHz and approximately 370 kHz.

6. The fluid analysis device according to claim 5, wherein: The cracking frequency is a frequency range between approximately 300 kHz and approximately 370 kHz, and the piezoelectric transducer is configured to scan the frequency range between approximately 300 kHz and approximately 370 kHz.

7. The fluid analysis device according to claim 1, wherein: The outer surface is a first outer surface having a mounting area, the mounting area having a first shape, and wherein the piezoelectric transducer has a second outer surface, the second outer surface having a second shape corresponding to the first shape, the second outer surface of the piezoelectric transducer being bonded to the mounting area.

8. The fluid analysis device according to claim 1, wherein: The piezoelectric transducer matingly engages an outer surface of the sample vessel.

9. The fluid analysis device according to claim 1, wherein: The separation acoustic waves are configured to cause separation of plasma into an antinode region located near an outer surface of the microchannel, and wherein the optical detector is positioned to receive light passing through the plasma located at the antinode region and is configured to detect plasma components based on measurements of the plasma at the antinode region located near the outer surface.

10. A fluid analyzer, comprising: A fluid analysis device, comprising: a sample vessel having an outer surface, a microchannel within the confines of the outer surface, a first port extending through the outer surface to the microchannel, and a second port extending through the outer surface to the microchannel such that a blood sample can be inserted into the microchannel through the first port; and a piezoelectric transducer coupled to an outer surface of the sample vessel to form an integral structure, the piezoelectric transducer being configured to generate a first ultrasonic sound wave having a first frequency, a second ultrasonic sound wave having a second frequency, and a third ultrasonic sound wave having a third frequency, the first ultrasonic sound wave being configured to initiate separation of red blood cells and plasma in a blood sample in a microchannel, the second ultrasonic sound wave being configured to substantially complete separation of red blood cells and plasma in a blood sample in a microchannel, and the third ultrasonic sound wave being configured to vibrate the sample vessel so as to induce shear force in the microchannel, the third ultrasonic sound wave and the shear force being configured to induce cavitation in the blood sample in the microchannel so as to rupture cell walls of red blood cells in the blood sample and release hemoglobin from the red blood cells; an absorbance spectrophotometer comprising an optical transmitter and an optical receiver positioned adjacent to the sample vessel, the optical transmitter positioned to transmit the optical medium through the microchannel, the optical receiver positioned to receive at least a portion of the optical medium after the portion has passed through the microchannel; a fluid distribution system having an outlet connected to the first port and an inlet connected to the second port; and A controller is electrically connected to the piezoelectric transducer, the controller being configured to send an electrical signal to the piezoelectric transducer, the electrical signal causing the piezoelectric transducer to emit the first ultrasonic sound wave, the second ultrasonic sound wave, and the third ultrasonic sound wave when received by the piezoelectric transducer.

11. The fluid analyzer according to claim 10, wherein: The sample vessel is constructed of glass.

12. The fluid analyzer according to claim 10, wherein: The first frequency is a frequency between approximately 950 kHz and approximately 1100 kHz.

13. The fluid analyzer according to claim 10, wherein: The second frequency is a frequency between approximately 320 kHz and approximately 500 kHz.

14. The fluid analyzer according to claim 10, wherein: The third frequency is a frequency between approximately 300 kHz and approximately 370 kHz.

15. The fluid analyzer according to claim 14, wherein: The third frequency is a frequency range between approximately 300 kHz and approximately 370 kHz, and the piezoelectric transducer is configured to scan the frequency range between approximately 300 kHz and approximately 370 kHz.

16. The fluid analyzer according to claim 10, wherein: The outer surface of the sample vessel is a first outer surface having a mounting area, the mounting area having a first shape, and wherein the piezoelectric transducer has a second outer surface, the second outer surface having a second shape corresponding to the first shape, and the second outer surface of the piezoelectric transducer is bonded to the mounting area.

17. The fluid analyzer according to claim 10, wherein: The piezoelectric transducer matingly engages an outer surface of the sample vessel.

18. The fluid analyzer according to claim 10, wherein: The second ultrasonic sound wave is configured to cause separation of plasma into an antinode region located near an outer surface of the microchannel, and wherein the absorbance spectrophotometer is configured to perform measurements on the plasma at the antinode region located near the outer surface to determine a plasma analyte.

19. A blood analysis method comprising the following steps: transferring a whole blood sample having red blood cells and plasma into a microchannel of a sample vessel; Separating red blood cells from plasma in a microchannel; obtaining a first absorbance spectrum reading of the plasma separated from the red blood cells; lysing red blood cells within the microchannel to provide a lysed blood sample; as well as A second absorbance spectrum reading is obtained of the lysed blood sample within the microchannel.

20. The method according to claim 19, wherein: Separating red blood cells from plasma within the microchannel is further defined as inducing a first acoustic wave within the microchannel at a sufficient frequency and duration to initiate separation of the red blood cells from the plasma, and inducing a second acoustic wave within the microchannel at a sufficient frequency and duration to substantially complete separation of the red blood cells from the plasma.

21. The method according to claim 20, wherein: A sufficient frequency of the first sound wave is a frequency between approximately 950 kHz and approximately 1100 kHz and a duration of a time period between approximately 5 seconds and approximately 15 seconds.

22. The method according to claim 20, wherein: A sufficient frequency of the second sound wave is a frequency between approximately 320 kHz and approximately 500 kHz and a duration of a time period between approximately 10 seconds and approximately 25 seconds.

23. The method according to claim 19, wherein: Lysing red blood cells within the microchannel to provide a lysed blood sample is further defined as inducing a third acoustic wave within the microchannel at a sufficient frequency and duration to induce a shear force within the microchannel, the shear force being configured to induce cavitation in the blood sample, causing cell walls of the red blood cells to be lysed and hemoglobin to be released from the red blood cells.

24. The method according to claim 23, wherein: The third sound wave has a sufficient frequency of between approximately 320 kHz and approximately 370 kHz and a duration of between approximately 2 seconds and approximately 20 seconds.

25. The method of claim 19, wherein: The separation step enriches the plasma in an antinode region near the surface of the microchannel, and wherein the step of obtaining a first absorbance spectrum reading is performed on the plasma located at the antinode region near the surface of the microchannel.

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