Acoustic phoresis analysis apparatus and method

Abstract

A sample vessel having an outer surface, a microchannel within the range 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; a piezoelectric transducer coupled to the outer surface of the sample vessel to form an integral structure, having a first frequency configured to initiate separation of red blood cells and plasma in a blood sample, a second frequency configured to complete separation of red blood cells and plasma, and a third frequency configured to rupture the cell walls of blood cells to produce a lysed blood sample, and configured to emit ultrasonic acoustic waves having the first, second, and third frequencies into and / or towards a blood sample within the microchannel; and a piezoelectric transducer, wherein a fluid analysis device, method, and system including the analysis device are disclosed.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 367,347, filed on June 30, 2022, under 35 U.S.C. § 119(e). The entire content of the above patent application is hereby expressly incorporated by reference herein.

[0002] The present disclosure generally relates to devices, systems, and methods for testing blood samples. More particularly, the present disclosure relates to an analytical device configured to separate red blood cells from plasma in a sample vessel by ultrasonic acoustic waves generated in the vessel by a piezoelectric transducer driven at one or more first frequencies or frequency ranges. After separation and testing of the red blood cells and / or plasma, the analytical device is further configured to lyse the red blood cells in the vessel by ultrasonic acoustic waves, shear forces, pressure, and / or fluid motion generated in the sample vessel by a piezoelectric transducer driven at one or more second frequencies or frequency ranges. In some non-limiting embodiments, the ultrasonic acoustic waves are generated by a single piezoelectric transducer. The analytical device is used in conjunction with a blood sample test analyzer.

Background Art

[0003] Point-of-care testing generally refers to medical testing at or near the site of patient care, such as in an emergency treatment room. The desired result of such testing is often a rapid and accurate laboratory result for determining the next course of action in patient care. Such multiple point-of-care tests involve the analysis of blood samples from patients. Many of these tests use whole blood, plasma, or serum.

[0004] In some tests, the cell walls of red blood cells in the blood sample rupture (lyse) and hemoglobin is released. The lysis of red blood cells is sometimes referred to as hemolysis. Hemolysis is usually performed by chemical or mechanical means.

[0005] In some devices, ultrasound is used to lyse red blood cells. In some point-of-care test devices, spectrophotometric light absorption measurements are used to determine the oximetry parameters of a whole blood sample in which the red blood cells have been lysed. These devices are typically fluid systems that place a patient's blood sample in a special sample chamber for testing the blood sample. For example, in one system described in Patent Document 1 ("Apparatus for Hemolyzing a Blood Sample and for Measuring at Least One Parameter Thereof", issued on August 4, 2015), two piezoelectric elements with two balanced resonance elements symmetrically surrounding the sample chamber are used to lyse red blood cells by acoustic radiation force. However, these devices are difficult and expensive to manufacture and require high-precision symmetry by specially made resonance elements.

[0006] After the red blood cells are lysed, the blood sample is tested by a spectrophotometer that analyzes the intensity of light at a predetermined wavelength passing through the cartridge optical window. A spectrophotometer is a device for measuring the intensity of light in a part of the spectrum, particularly when transmitted or emitted by a specific substance. A spectrophotometer measures the degree to which a chemical substance absorbs light by measuring the intensity of light as it passes through a blood sample or other solution. Each compound in the sample or solution absorbs or transmits light over a specific range of wavelengths. Absorbance is determined using Lambert-Beer's law. Each compound in the sample or solution absorbs or transmits light over a specific set of wavelengths of interest governed by an absorption coefficient. [[ID= ]] [[ID= ]]

[0007] [[ID= ]] In such tests, critical care hematological parameters may be measured, which can include hematocrit, free hemoglobin, total hemoglobin, bilirubin, lipids, and oximetry (i.e., the E hemoglobin fraction). Physicians and clinicians rely on these measurements for decision-making during patient treatment. These measurements are often performed on large, complex-to-maintain analyzers in major blood laboratories. However, obtaining fast, accurate, and highly precise results in a point-of-care setting is preferred in many respects as it can save time in critical diagnostic situations and avoid specimen transportation problems in the critical care unit. Although some blood gas analyzers offer point-of-care functionality, they do not present a single solution that is simpler and easier to manufacture while providing the desired time-to-result (TTR), accuracy, precision, and reliability compared to existing devices.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] There is a need for an analytical device that improves the accuracy and precision of measurement parameters of samples within the desired TTR at the point-of-care for patients and is easy to manufacture and low-cost.

Means for Solving the Problems

[0010] An acoustic phoresis analysis device, method, and system are disclosed. The problems of complex, slow, low-precision, and inaccurate blood sample tests for point-of-care are addressed by an apparatus configured to separate red blood cells and plasma in a whole blood sample in a sample vessel by an ultrasonic acoustic wave generated in the sample vessel by a single acoustic transducer, such as a piezoelectric transducer, driven at one or more specific first excitation frequencies or excitation frequency ranges. After testing the separated plasma and / or red blood cells, the analyzer is configured to lyse the red blood cells by an ultrasonic acoustic wave generated in the sample vessel by a single piezoelectric transducer driven at one or more specific second excitation frequencies or excitation frequency ranges. Since the analyzer is configured to lyse the red blood cells after testing the separated plasma and / or red blood cells, the separated plasma and red blood cells do not mix to form whole blood within the analyzer or a separate device. The analyzer is configured to lyse the red blood cells immediately after testing the separated plasma and / or red blood cells. That is, the same analyzer is configured to separate red blood cells and plasma in a whole blood sample and then lyse the red blood cells immediately after testing the separated plasma and / or red blood cells.

[0011] The accompanying drawings, which are incorporated herein 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, for clarity and brevity, the specific configurations and specific figures of the drawings are exaggerated and shown out of scale or schematically. Also, not all components in all the drawings are labeled. The same reference numbers in the figures represent the same or similar elements or functions, and reference may be made thereto.

Brief Description of the Drawings

[0012]

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DETAILED DESCRIPTION OF THE INVENTION

[0013] In the following detailed description, reference is made to the accompanying drawings. The same reference numbers in different drawings can identify the same or similar elements.

[0014] The mechanism proposed in the present disclosure avoids the above problems. The present disclosure is for testing in a sample vessel by an ultrasonic acoustic wave generated in the sample vessel by a piezoelectric transducer connected to the sample vessel and driven at one or more first frequencies or a first excitation frequency range. After first separating red blood cells and plasma in a whole blood sample, it is configured to lyse red blood cells in the same sample vessel by an ultrasonic acoustic wave, shear force, pressure, cavitation, and / or fluid motion generated in the sample vessel by a piezoelectric transducer driven at one or more second frequencies or a second excitation frequency range. An acoustic electrophoresis analyzer, an analyzer, and a lysis method are described, including an acoustic electrophoresis analyzer. In one non-limiting embodiment, the acoustic transducer, such as a piezoelectric transducer, is a single piezoelectric transducer. The present disclosure further describes a method of use in addition to an analyzer configured to test a sample in a sample vessel by interacting with the acoustic electrophoresis analyzer.

[0015] As used herein, the terms "comprises" and "comprising", "includes" and "including", "has" and "having", or any other variation thereof, are intended to cover non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but may also include other elements not expressly listed or other elements inherent to such process, method, article, or apparatus. Further, unless otherwise expressly stated, "or" represents an inclusive "or" rather than an exclusive "or". For example, the condition "A or B" is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0016] Also, the use of "a" or "an" is employed in the description of elements and components of embodiments herein. This is merely for convenience and is done to give a general sense of the inventive concept. This description should be construed to include one or more, and the singular form includes the plural form unless it is clear otherwise.

[0017] Furthermore, the use of the term "plurality" conveys "more than one" unless otherwise expressly stated.

[0018] As used herein, qualifiers such as "about" and "approximately" and combinations and variations thereof are intended to cover not only the exact quantity or value being qualified, but also, for example, manufacturing tolerances, measurement errors, wear, stresses exerted on various components, and any slight deviations that may result from these and their combinations.

[0019] For use in this specification, the term "substantially" means that the parameter, event, or situation described thereafter occurs completely, or that most of the parameters, events, or situations described thereafter occur. For example, the term "substantially" means that the parameter, event, or situation described thereafter occurs with a probability of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or that the dimension or measurement result is within at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the reference dimension or measurement result.

[0020] The use of the terms "at least one" or "one or more" is understood to include, in addition to one, any quantity of two or more. Also, the use of the expression "at least one of X, V, and Z" is understood to include only X, only V, and only Z, as well as any combination of X, Y, and Z.

[0021] The use of ordinal terms (i.e., "first", "second", "third", "fourth", etc.) is for the sole purpose of distinguishing between two or more items and does not imply, unless otherwise expressly stated, any order, sequence, or precedence of one item over another, nor does it imply any additional order.

[0022] Finally, as used herein, any reference to "one embodiment" means that a particular element, configuration, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in this specification are not necessarily all referring to the same embodiment.

[0023] As described above, conventional general devices for point-of-care blood sample testing are complex, slow, inaccurate and imprecise. The present disclosure provides an apparatus, system, and method for separating plasma from red blood cells in a sample vessel by ultrasonic acoustic waves generated by a piezoelectric transducer driven by a first specific excitation frequency having sufficient power to initiate separation of red blood cells from plasma and then a second excitation frequency at a power level sufficient to substantially complete separation of red blood cells from plasma, and for lysing the red blood cells in the sample vessel by ultrasonic acoustic waves at a third excitation frequency at a power level sufficient to lyse the red blood cells after performing at least one first test, thereby generating a lysed blood sample in which one or more second tests are performed, to address the deficiencies as described above.

[0024] Reference is now made to the drawings, and in particular FIGS. 1-8, which illustrate an acoustic levitation device 10. Generally, the acoustic levitation device 10 includes a sample vessel 12 and an acoustic transducer 14 (a piezoelectric transducer 14 is contemplated). In this specification, the acoustic transducer 14 is referred to as the piezoelectric transducer 14. The piezoelectric transducer 14 is coupled to the sample vessel 12. In one embodiment, the acoustic levitation device 10 is an integral structure formed by the sample vessel 12 and the piezoelectric transducer 14 integrally coupled using a suitable bonding material such as epoxy. The sample vessel 12 is preferably permanently coupled to the piezoelectric transducer 14 in a non-fastening manner. This non-fastening minimizes the vibration loss between the sample vessel 12 and the piezoelectric transducer 14.

[0025] The sample vessel 12 is provided with a first substrate 15 coupled 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 range of the upper surface 20, a first port 24 extending from the lower surface 21 to the microchannel 22 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 can have an attachment area for the piezoelectric transducer 14.

[0026] 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, and 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 planar. In one embodiment, the first side 48 and the second side 50 are planar. In one embodiment, the first end 44 and the second end 46 are planar. 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 integrally form a three-dimensional rectangular parallelepiped. In some embodiments, the piezoelectric transducer 14 fits into engagement with the outer surface of the sample vessel 212. For example, the piezoelectric transducer 14 and the outer surface can have a plane configured to be disposed integrally.

[0027] The sample vessel 12 is considered to be partially, substantially, or completely transparent. In one embodiment, the sample vessel 12 is transparent at least above and below the microchannel 22 such that light rays can enter the microchannel 22 through the sample vessel 12, interact with any substances within the microchannel 22, and exit the sample vessel 12.

[0028] The sample vessel 12 is made of glass. In one embodiment, the sample vessel 12 is made of a material (glass or non - glass) having a Young's modulus in the range of about 50 GPa to about 90 GPa. The material property known as Young's modulus or modulus of elasticity is a measure of the ability of a material to withstand a change in length upon longitudinal tension or compression. Young's modulus is equal to the longitudinal stress divided by the strain. In one embodiment, the sample vessel 12 is made of a plastic having a rigidity and / or Young's modulus similar to that of glass. In one embodiment, the sample vessel 12 is made of alkali borosilicate glass. An example of alkali borosilicate glass is manufactured by Schott Advanced Optics located at 400 York Avenue, Duryea, PA 18642 and sold under the name "D 263 T ECO Thin Glass".

[0029] In one embodiment, the sample vessel 12 can have one or more conductive structures 30, which can be metal sputtered or bonded to the upper surface 17 of the first substrate 15. The one or more conductive structures 30 provide an electrical path that can connect elements such as the piezoelectric transducer 14. The one or more conductive structures 30 can include a first electrode 32 and a second electrode 34 that are located on both sides of the microchannel 22 and extend substantially parallel to the microchannel 22. The first electrode 32 and the second electrode 34 can extend within ±5° of parallel (preferably extending parallel). In the illustrated embodiment, both the first electrode 32 and the second electrode 34 are adjacent to the microchannel 22 in a linear configuration and do not cover the microchannel 22 so as not to block the light beam generated by the spectrophotometer as described above. In some embodiments, the first electrode 32 and the second electrode 34 can be either parallel or non-parallel as long as they do not block the light beam 116 and the capacitance measurements from the first electrode 32 and the second electrode 34 can be correlated with the expected channel components within the microchannel 22 during calibration. In some embodiments, the first electrode 32 and / or the second electrode 34 can have a meandering configuration that includes one or more portions that cross outside the expected path of the light beam 116 passing through the sample vessel 12 and above the microchannel 22.

[0030] 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 that defines the 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.

[0031] In one embodiment, the aspect ratio of the sample vessel 12 is in the range of about 0.5 to about 3.0. In one embodiment, the aspect ratio of the sample vessel 12 is in the range of 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.

[0032] The microchannel 22 is configured to receive a fluid sample (including, but not limited to, a blood sample, a "blank" sample, and / or a wash fluid sample) through the first port 24 and / or the second port 26. Connecting the inlet 52 to the first port 24 and the outlet 54 to the second port 26 enables fluid connection of each of the first tube 56 and the second tube 58 to the microchannel 22. 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 is understood that the microchannel 22 can have an orientation or offset at an angle from the longitudinal axis and / or the transverse axis of the sample vessel 12.

[0033] The microchannel 22 has an aspect ratio that defines the proportional relationship between the width and the height of the microchannel 22. In one embodiment, the width-to-height aspect ratio of the microchannel 22 is in the range of about 0.04 to about 0.175. In one embodiment, the width-to-height aspect ratio of the microchannel 22 is in the range of about 0.04 to about 0.125. In one embodiment, the width-to-height aspect ratio of the microchannel 22 is about 0.05.

[0034] In one embodiment, the width of the microchannel 22 is about 2 millimeters. In one embodiment, the width of the microchannel 222 is about 2.5 millimeters. In one embodiment, the width of the microchannel 22 is greater than the illumination width of the light collection area of the absorbance spectrophotometer 102. The illumination width is defined as the width of the cross-section of the light collection rate that intersects the microchannel 22 along the optical path from the absorbance spectrophotometer 102. For example, when the illumination diameter is 1 millimeter to 1.5 millimeters, the width of the microchannel 22 can be at least about 1.6 millimeters. The width of the microchannel 22 is determined so as to enable appropriate mechanical alignment between the microchannel 22 and the optical path. For example, when the illumination width is 1 millimeter to 1.5 millimeters, the width of the microchannel 22 can be about 2 millimeters.

[0035] In one embodiment, the length of the microchannel 22 can be about 10 millimeters to about 12 millimeters. In one embodiment, the length of the microchannel 22 can be at least about 4 millimeters. In one embodiment, the length of the microchannel 22 can be about 4 millimeters to about 20 millimeters.

[0036] In one embodiment, the length of the microchannel 22 can be based at least in part on a predetermined desired number of acoustic wave nodes to be generated in the microchannel 22. For example, when the width of the microchannel 22 is about 2 millimeters and the wave propagation speed of whole blood is about 1500 m / s, 350 kHz is calculated as a single acoustic wave node. The acoustic wave nodes are distributed at equal intervals (e.g., 2 × 2 mm = 4 mm) along the length of the microchannel 22, and the lysed blood is uniformly distributed by the high pressure. For example, in a region where the side walls of the microchannel 22 extend substantially parallel, when the predetermined desired number of acoustic wave nodes is six wave nodes per side wall (see FIG. 14C showing six wave nodes 68 including five antinode regions 67a and four wave nodes between two end wave nodes and five antinode regions 67a), the length of the microchannel 22 is set to about 17 millimeters including the tapered inlet and outlet regions.

[0037] The height of the microchannel 22 can vary as discussed below. The height of the microchannel 22 can be based on the absorption amount in the lysed blood of the light collection efficiency of the absorbance spectrophotometer 102 and the desired absorption accuracy. For example, about 1 absorbance (OD) can be considered as the desired absorption.

[0038] In one embodiment, the height of the microchannel 22 is about 100 micrometers. In one embodiment, the height of the microchannel 22 is about 150 micrometers. In one embodiment, the height of the microchannel 22 is about 250 micrometers. In one embodiment, the height of the microchannel 22 is about 300 micrometers. In one embodiment, the height of the microchannel 22 is from about 80 micrometers to about 300 micrometers. In one embodiment, the height of the microchannel 22 is from about 80 micrometers to about 150 micrometers.

[0039] 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, bottom 42, first end 44, second end 46, first side 48, and / or 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, bottom 42, first end 44, second end 46, first side 48, and / or 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, bottom 42, first end 44, second end 46, first side 48, and / or second side 50.

[0040] In one embodiment, the first port 24 and the second port 26 each have a diameter of from about 0.5 millimeter (500 micrometers) to about 1.5 millimeters (1500 micrometers). In one embodiment, the first port 24 and the second port 26 each have a diameter of about 0.8 millimeter (800 micrometers). As shown in FIG. 2, the microchannel 22 tapers toward the first port 24 and the second port 26. This taper assists in the supply of 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 smaller 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.

[0041] The sample vessel 12 is considered an integrated product in terms of being formed of a single piece of material or multiple pieces interconnected to form a single unit. As discussed with reference to FIG. 1, the sample vessel 212 is formed of two substrates integrally joined. Alternatively, the sample vessel 12 is formed of three substrates integrally joined, as shown in FIG. 9.

[0042] The second substrate 16 is laminated on the first substrate 15 to form an integrated structure. In one embodiment, the first substrate 15 and the second substrate 16 are annealed to each other. In one embodiment, the first substrate 15 and the second substrate 16 are 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.

[0043] The microchannel 22 is disposed in the first substrate 15, the second substrate 16, and / or is formed partly in the first substrate 15 and partly in the second substrate 16. In one embodiment, the microchannel 22, the first port 24, and the second port 26 are located in the first substrate 15. In one embodiment, the microchannel 22 is etched in the first substrate 15 and / or the second substrate 16. In one embodiment, the microchannel 22 is located in the first substrate 15 and one or both of the first port 24 and the second port 26 are located in the second substrate 16. Also, one or both of the first port 24 and the second port 26 are located in and / or extend through the first substrate 15 and / or the second substrate 16.

[0044] As shown in FIG. 9, in one embodiment, the sample vessel 12 can 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 are stacked to form an integral structure. In one embodiment, the first substrate 70, the second substrate 72, and the third substrate 80 are thermally plasma bonded to each other. In one embodiment, the first substrate 70, the second substrate 72, and the third substrate 80 are annealed to each other. Also, one or both of the first port 74 and the second port 76 are located in the second substrate 72. In one embodiment, the microchannel 78 is a slot located through the third substrate 80. In one embodiment, the third substrate 80 can have the same thickness as the height of the microchannel 78. In one embodiment, the third substrate 80 can be made 100 micrometers thick. In one embodiment (not shown), the microchannel 78 is located in the second substrate 72.

[0045] Returning to FIG. 1, the piezoelectric transducer 14 is attached to the sample vessel 12 (such as the attachment area of the top 40) to form an integrated structure of the acoustic phoresis device 10. The piezoelectric transducer 14 can have an attachment surface for attachment to the attachment area of the top 40. In one embodiment, at least a part of the piezoelectric transducer 14 is attached to the top 40 of the sample vessel 12; however, it is understood that the piezoelectric transducer 14 can be attached 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 arranged with respect to the microchannel 22 so as not to block the movement of light through the microchannel 22 from the top 40 or the bottom 42 of the sample vessel 12. The piezoelectric transducer 14 is offset from the microchannel 22 so that light from the outside of the sample vessel 12 can be incident on the microchannel 22. In one embodiment, the piezoelectric transducer 14 has a length and a longitudinal axis that is oriented substantially parallel (e.g., within 5° of parallel) to the longitudinal axis of the sample vessel 12 along the length. In one embodiment, the width of the piezoelectric transducer 14 is smaller than the length of the piezoelectric transducer 14.

[0046] The piezoelectric transducer 14 is located on the sample vessel 12 on one or both sides 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.

[0047] The piezoelectric transducer 14 is coupled to the sample vessel 12. This coupling can be made thin relative to the thicknesses of the piezoelectric transducer 14 and the sample vessel 12. The piezoelectric transducer 14 is coupled to the sample vessel 12 by an adhesive. The adhesive is configured to enable propagation of acoustic waves with low attenuation. In one embodiment, after a liquid adhesive is applied to the piezoelectric transducer 14, the piezoelectric transducer 14 is attached to the sample vessel 12 via the liquid adhesive. For example, a liquid adhesive with a temperature stability up to 350°C and a high hardness (rigidity) with excellent adhesion to glass is applied. In one example, as the liquid adhesive, an epoxy adhesive such as EPO-TEK 353ND (manufactured by Epoxy Technology, Inc., located at 14 Fortune Drive, Billerica, MA) that enables ultrasonic propagation and has a Shore D hardness of about 85 can be considered. In one example, about 5 μl of the liquid adhesive is applied. The piezoelectric transducer 14 is fastened to the sample vessel 12 and the adhesive cures at about 150°C. In one embodiment, the thickness of the cured adhesive can be about 100 μm. In one embodiment, the thickness of the cured adhesive can be about 10 μm.

[0048] The piezoelectric transducer 14 is configured to convert an applied alternating electric field into another form of energy such as an acoustic pressure wave having one or more frequencies and / or frequency ranges. The piezoelectric transducer 14 is configured to vibrate when an alternating electric field is applied, generating an acoustic pressure wave that is introduced into the sample vessel 12, thereby creating one or more acoustic standing wave nodes within the blood sample in the sample vessel 12. As shown in FIG. 1, the piezoelectric transducer 14 can include a first terminal 90 and a second terminal 92 configured to be coupled to an alternating current power source. In one embodiment, the first terminal 90 and the second terminal 92 are electrically connected to a conductive structure 30 and can deliver an alternating voltage. In one embodiment, as the piezoelectric transducer 14, a piezoelectric ultrasonic transducer can be considered.

[0049] The piezoelectric transducer 14 is configured to generate mechanical activity and generate acoustic waves of a certain frequency by expansion and contraction when an alternating electric field is applied. FIG. 10 is a graph showing an example of the total displacement of the piezoelectric transducer 14 in an exemplary operation of the piezoelectric transducer 14.

[0050] In one embodiment, the piezoelectric transducer 14 is configured to generate ultrasonic waves having a first frequency that initiates separation of other components of a blood sample, such as plasma and red blood cells, over a first predetermined period within the range of 950 kHz to 1100 kHz. As used herein, the initiation of separation of plasma and other components of a blood sample suspended therein is referred to as preconcentration, and the first frequency is referred to as the preconcentration frequency. The first frequency is generated by a first voltage of 50 volts to 100 volts. Also, the first predetermined period can be 5 seconds to 15 seconds. In one embodiment, the first frequency can be in the range of 600 kHz to 630 kHz.

[0051] The piezoelectric transducer 14 is further configured to emit ultrasonic waves of a second frequency and / or frequency range at a second voltage for a second predetermined period to completely separate the red blood cells suspended in the plasma of the blood sample. As used herein, the complete separation of plasma and other components of a blood sample suspended therein is referred to as concentration, and the second frequency is referred to as the concentration frequency. In one embodiment, the second frequency and / or frequency range can be made different (e.g., lower) from the first frequency or range, the second charge can be made different (e.g., higher) from the first charge, the second predetermined period can be the same as the first predetermined period, or can be made different (e.g., longer). As an example, the second frequency and / or frequency range can be 320 kHz to 500 kHz, the second charge can be 70 volts to 100 volts. Also, the second predetermined period can be 10 to 25 seconds.

[0052] In one embodiment, the second frequency can generate one or more acoustic standing waves different from the acoustic standing wave at the first frequency. An acoustic standing wave, also known as a stationary wave, is a wave that has a peak amplitude profile that oscillates in time but does not move spatially. The acoustic standing wave in the microchannel 22 can form, with respect to other portions of the microchannel 22, a node region where the force is substantially zero and the particle motion is substantially zero, and an antinode region where the force is maximum and the particle motion is maximum. When the red blood cells and plasma in the blood sample are separated when the second frequency is applied, 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. The separation into known regions at specific repeatable positions within this microchannel 22 enables the measurement of analytes in the plasma and / or red blood cells, as described below.

[0053] It should be noted that, herein, the first frequency and the second frequency are collectively referred to as separation frequencies, that is, frequencies of ultrasonic or acoustic waves that are sufficient to separate red blood cells and plasma in a whole blood sample without rupture of the red blood cells and are transmitted to the sample vessel.

[0054] The piezoelectric transducer 14 is further configured to emit ultrasonic waves of a third frequency and / or frequency range at a third voltage for a third predetermined period following a first predetermined period and a second predetermined period to lyse red blood cells in a blood sample. As used herein, the third frequency and / or frequency range is referred to as the lysis frequency. When driven by a third charge, the piezoelectric transducer 14 is configured to rupture the cell walls of cells in the blood sample and generate acoustic standing waves in the microchannel 22 that are configured to release hemoglobin from within the cells and mix it with the plasma. In one embodiment, the third frequency and / or frequency range can be made different (e.g., lower) from the first and / or second frequencies or ranges, the third voltage can be made different (e.g., higher) from the first voltage, the third charge can be made the same as or different from (e.g., either higher or lower than) the second charge, and the third predetermined period can be made the same as or different from (e.g., shorter than) the first or second predetermined periods. As an example, the third frequency and / or frequency range can be from 330 kHz to 370 kHz, the third charge can be from 100 volts to 200 volts, and the third predetermined period can be less than 20 seconds (e.g., 0, 1, or 2 to 10 seconds, etc.).

[0055] In one non-limiting and exemplary embodiment, to cause pre-concentration, the first frequency can be from 1092 kHz to 1097 kHz, which is generated at a first voltage (which can be 70 volts) for a first predetermined period of 6 seconds. Also, to cause concentration, the second frequency and / or frequency range can be from 376 kHz to 377 kHz, which is generated at a second voltage (which can be 80 volts) for a second predetermined period of 12 seconds. Also, to cause lysis of red blood cells, the third frequency and / or frequency range can be from 349 kHz to 351 kHz, which is generated at a third voltage (which can be 120 volts).

[0056] In another non-limiting and exemplary embodiment, to cause pre-concentration, the first frequency can be set to 1092 kHz to 1097 kHz, which is generated at a first voltage (which can be 90 volts) for a first predetermined period of 6 seconds. Also, to cause concentration, the second frequency and / or frequency range can be set to 376 kHz to 377 kHz, which is generated at a second voltage (which can be 100 volts) for a second predetermined period of 12 seconds. Also, to cause lysis of red blood cells, the third frequency and / or frequency range can be set to 349 kHz to 351 kHz, which is generated at a third voltage (which can be 120 volts).

[0057] In another non-limiting and exemplary embodiment, to cause pre-concentration, the first frequency can be set to 600 kHz to 630 kHz, more particularly 612 kHz to 613 kHz, which is generated at a first charge (which can be 5 to 12 volts (e.g., 6 or 10 volts)) for a first predetermined period of 8 seconds. Also, to cause concentration, the second frequency and / or frequency range can be set to 470 kHz to 500 kHz, more particularly 486 kHz to 487 kHz, which is generated at a second charge (which can be 13 to 20 volts (e.g., 13 or 18 volts)) for a second predetermined period of 15 seconds. Also, to cause lysis of red blood cells, the third frequency and / or frequency range can be set to 343 kHz to 348 kHz, which is generated at a third charge (which can be 13 volts).

[0058] As the third frequency, a resonance frequency that causes resonance in the blood sample introduced into the microchannel 22 of the sample vessel 12 so that the walls of red blood cells in the blood sample rupture can be considered. In one embodiment, the piezoelectric transducer 14 has a first resonance frequency, and the integrated structure of the acoustophoresis device 10 has a second resonance frequency that is spectrally separated from the first resonance frequency. The second resonance frequency is generated by the piezoelectric transducer 14 and introduced into the sample vessel 12, causing cavitation in the blood sample that collapses in a high-pressure region to generate a shock wave, rupture the walls of red blood cells, and release hemoglobin from inside the red blood cells of the blood sample into the plasma to generate bubbles. It is the frequency of the acoustic wave.

[0059] In one example, at the main 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 microchannel 22 has a width of about 2 millimeters with an aspect ratio of 0.05 to 0.125, and the sample vessel 12 has a width of about 12 millimeters with an aspect ratio of 1.4 to 1.9. The piezoelectric transducer 14 is configured to generate ultrasonic waves in the range of 330 kHz to 370 kHz with a peak pressure of 5 MPa and a peak velocity of up to 8 m / s in the microchannel 22.

[0060] However, the ultrasonic acoustic waves inside the microchannel 22 and the ultrasonic piezoelectric transducer 14 are considered to generate unnecessary heat (including unnecessary heat in the blood sample in the microchannel 22). To avoid overheating of the blood sample, the piezoelectric transducer 14 operates to generate a resonance frequency over a predetermined period. For example, the piezoelectric transducer 14 operates to generate an acoustic wave having a second resonance frequency for about 1 second to about 2 seconds. In one embodiment, the piezoelectric transducer 14 operates to generate an acoustic wave having a second resonance frequency for less than about 1.5 seconds. In one example, the acoustic electrophoresis device 10 is configured to lyse 99.99% of red blood cells by operating the piezoelectric transducer 14 for 1.5 seconds or less. In one example, the acoustic electrophoresis device 10 is configured to operate the piezoelectric transducer 14 for 10 seconds or less.

[0061] In one embodiment, since the ultrasonic waves inside the microchannel 22 destroy blood cells and cell walls into fine particles, light scattering during optical measurement of the blood sample is less than that of larger particles.

[0062] In one embodiment, the piezoelectric transducer 14 is configured to generate ultrasonic acoustic waves in a certain frequency range, and the second resonance frequency can be within this frequency range.

[0063] In one embodiment, the piezoelectric transducer 14 is configured to generate ultrasonic waves in a certain frequency range within the frequency range of 300 kHz to 1.5 MHz.

[0064] The resonance frequency and / or frequency range is 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; 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.

[0065] For example, if the sample vessel 12 is made of glass, the microchannel 22 has an aspect ratio of about 0.05 to about 0.125, the sample vessel 12 has an aspect ratio of about 1.4 to about 1.9, and the piezoelectric transducer 14 is configured to generate ultrasonic acoustic waves in the range of about 330 kHz to about 370 kHz.

[0066] The width of the microchannel 22 is determined based at least on the acoustic wave propagation speed inside the blood sample (e.g., about 1500 m / s) to have a frequency of about 330 kHz to about 370 kHz by using a predetermined desired number of acoustic nodes as one node in the center of the microchannel 22. The first acoustic node inside the microchannel 22 (an exemplary width of 2000 μm and depth of 100 μm) can be at least partially determined by using the following equation, without considering any reflections such as minute reflections:

[0067] f=v / λ

[0068] where f is the frequency, v is the wave speed in the fluid, and lambda is the wavelength (for a half wavelength resonant mode with a single node across the full width of the channel, the wavelength is twice the width of the microchannel 22).

[0069] Because the resonant frequency of sample vessel 12 may be difficult to accurately calculate due to manufacturing and / or material variations, in one embodiment, piezoelectric transducer 14 is configured to sweep a third frequency within a frequency range having multiple frequencies, from a first sweep frequency to one or more second sweep frequencies of the multiple frequencies, up to the third sweep frequency. In one embodiment, piezoelectric transducer 14 is configured to sweep the third frequency range in frequency steps, such as 1 kHz frequency steps. In one embodiment, sweeping the third frequency range from the first sweep frequency to the third sweep frequency reaches the resonant frequency upon addition of a blood sample to acoustophoresis device 10, even when considering variations in the geometry and materials of acoustophoresis device 10.

[0070] In one embodiment, the piezoelectric transducer 14 is configured to sweep a third frequency range from about 330 kHz to about 370 kHz in steps such as about 1 kHz. The piezoelectric transducer 14 is configured to sweep the third frequency range, for example, from about 330 kHz to about 370 kHz and / or from about 370 kHz to about 330 kHz.

[0071] In one embodiment, the piezoelectric transducer 14 is configured to sweep the third frequency range over a period greater than zero 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 is configured to sweep the third frequency range over a period of about 1 to about 2 seconds.

[0072] In one embodiment, as an addition or alternative to the above, the acoustophoresis 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 of the rigidly coupled ultrasonic piezoelectric transducer 14 (primarily considered as lateral displacement) causes vibration and movement of the sample vessel 12 coupled to the piezoelectric transducer 14. Upon activation, the ultrasonic piezoelectric transducer 14 changes shape as shown in FIG. 12, causing contraction and elongation (lateral displacement). The movement of the ultrasonic piezoelectric transducer 14 transfers to the sample vessel 12, changing the shape and / or volume of the microchannel 22, thereby inducing shear and vibration in the microchannel 22 of the sample vessel 12. FIG. 12 is a graph showing an example of the total displacement of the piezoelectric transducer 14 in an exemplary operation of the piezoelectric transducer 14.

[0073] Due to the displacement of the piezoelectric transducer 14, vibrations and internal shear forces are generated in the sample vessel 12, which are considered to cause and / or result in the dissolution of the blood sample in the microchannel 22 due to the high pressure, shear force, and / or fluid motion inside the microchannel 22 of the sample vessel 12. Therefore, in some embodiments, it is considered that the blood sample in the microchannel 22 is dissolved by a combination of acoustic standing waves, pressure, cavitation, shear force, and / or fluid motion in the blood sample.

[0074] When the piezoelectric transducer 14 is activated, it is considered that shear stress is generated at the junction between the piezoelectric transducer 14 and the sample vessel 12. This shear stress is considered to increase the pressure inside the microchannel 22. For example, in one embodiment, a preferred high pressure is considered to be about 5 MPa. In one embodiment, the pressure is considered to be in the range of about 3 MPa to about 7 MPa. The pressure level is controlled by the level of contraction / expansion of the piezoelectric transducer 14, which is considered to be determined by the electric field strength of the piezoelectric transducer 14.

[0075] By combining the acoustic standing wave inside the microchannel 22 with the shear and / or vibration of the sample vessel 12, significant cavitation and the resulting shock waves occur in the whole blood sample in the microchannel 22, causing the cell walls of red blood cells to rupture and hemoglobin to be released from the red blood cells.

[0076] In one embodiment shown in FIGS. 2-8, the acoustophoretic device 10 is coupled or connected to a printed circuit board (PCB) 94. The acoustophoretic device 10 is configured in the same manner as the acoustophoretic device 204 described in U.S. Provisional Patent Application No. 63 / 366,552, the entire contents of which are incorporated herein by reference. As is known in the art, the PCB 94 has a conductive structure (not shown) provided on the outer surface or embedded therein. The conductive structure of the PCB 94 can electrically connect the conductive structure 30 of the acoustophoretic device 10 to one or more electrical connectors 96 (only one of which is numbered). One or more electrical connectors 96 are configured to connect the acoustophoretic device 10 to an analyzer such as the analyzer 100, which is described separately herein. The PCB 94 is configured in the same manner as the support substrate 202 described in U.S. Provisional Patent Application No. 63 / 366,552. Also, the acoustophoretic device 10 is attached to the PCB 94 by soldering attachment pads on the sample vessel 12 to attachment pads on the PCB 94, as described in U.S. Provisional Patent Application No. 63 / 366,552.

[0077] Referring now to FIGS. 11 and 12, in some embodiments, the acoustophoretic device 10 can be a component of the analyzer 100. The analyzer 100 can include the acoustophoretic device 10, an absorbance spectrophotometer 102, a fluid dispensing system 104 (e.g., including a peristaltic pump), and / or a controller 106. In one embodiment, the acoustophoretic device 10 is removable and / or replaceable from the other components of the analyzer 100. In one embodiment, the analyzer 100 can further include a fixture 108 configured to receive the PCB 94 that supports the other components of the acoustophoretic device 10. In one embodiment, the acoustophoretic device 10 is held by the fixture 108 such that it can vibrate and / or move within a range of vibration and / or movement.

[0078] In one embodiment, the analyzer 100 can further include one or more processors 140 coupled to one or more non-transitory computer-readable media 142 that store instructions, which, when provided to and executed by the one or more processors 140, cause the one or more processors 140 to perform the functions described below. In one embodiment, one or more processors 140 and one or more non-transitory computer-readable media 142 can be part of the controller 106. However, it is understood that one or more of the processor 140 and / or the non-transitory computer-readable media 142 can be located external to the controller 106 and / or external to other components of the analyzer 100. In one embodiment, the analyzer 100 can include one or more sensor cartridges 143 (FIG. 12) having a blood gas sensor 144 and / or one or more reagent cartridges 145 and / or can be connectable thereto.

[0079] In one embodiment, the analyzer 100 can include a light input 112 and a light receiver 114 positioned adjacent to the sample vessel 12. The light input 112 is arranged to emit a light beam 116 through the top 40, the bottom 42, and the microchannel 22. The light receiver 114 is arranged to receive a 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 light input 112 is coupled to a first optical fiber bundle 118 that couples a light source 120 to the light input 112. The light receiver 114 is coupled to a second optical fiber bundle 119 that couples the light receiver 114 to an absorbance spectrophotometer 102. Examples of the light source 120 include one or more light-emitting diodes or tungsten halogen light. In one embodiment, the light can be white light having a wavelength in the range of about 450 to 700 nanometers.

[0080] The absorbance spectrophotometer 102 is configured to measure the intensity of light in a part of the spectrum when transmitted or emitted by a specific substance in a fluid sample, particularly in the microchannel 22 of the sample vessel 12. The absorbance spectrophotometer 102 is configured to measure the degree to which a chemical substance absorbs light by measuring the intensity of light as the light beam passes through a blood sample or other fluid sample. Each compound in the sample or solution absorbs or transmits light over a specific range of wavelengths.

[0081] The fluid distribution system 104 can have an inlet 130 that is fluidly connectable to the first port 24 of the sample vessel 12 of the acoustic levitation device 10 and an outlet 132 that is fluidly connectable to the second port 26. The fluid distribution system 104 can move one or more fluid samples, such as a blank sample, a blood sample, or a cleaning solution, from the inlet 130 and the first port 24 into the microchannel 22 of the sample vessel 12. In one embodiment, the fluid distribution system 104 can flush the microchannel 22 and discharge the material in the microchannel 22 from the outlet 132 through the second port 26 of the sample vessel 12. As the fluid distribution system 104, for example, a peristaltic pump that operates automatically, manually, or in a combination of automatic and manual can be considered. The fluid distribution system 104 is operated by the controller 106. In one embodiment, the first tube 56 can be considered as the inlet 130, the second tube 58 can be considered as the outlet 132, 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.

[0082] The controller 106 is electrically connected to the piezoelectric transducer 14 of the acoustic levitation device 10. The controller 106 is configured to supply an electrical signal to the piezoelectric transducer 14 that causes the piezoelectric transducer 14 to emit an ultrasonic acoustic wave at one or more frequencies and / or frequency ranges, including the resonance frequency of the integrated structure with the addition of a fluid sample to the acoustic levitation device 10, when the piezoelectric transducer 14 receives it.

[0083] As shown in FIG. 11, in one embodiment, the controller 106 can have a first electrical contact 134 and a second electrical contact 136. The first electrical contact 134 and the second electrical contact 136 can be electrically connected to the first terminal 90 and the second terminal 92 of the piezoelectric transducer 14 of the acoustic phoresis device 10, respectively, so that a potential is applied to the piezoelectric transducer 14 of the acoustic phoresis device 10.

[0084] The fixture 108 can hold the acoustic phoresis device 10 in place between the light input 112 and the light receiver 114, and arrange the acoustic phoresis device 10 to be operably connected to the fluid distribution system 104 and the controller 106. The fixture 108 is configured to stabilize the acoustic phoresis device 10 in place without applying a force that would significantly change the acoustic impedance of the integrated structure of the acoustic phoresis device 10. For example, the fixture 108 can include one or more fasteners that apply a fastening force of about 20 newtons (N) or less.

[0085] In one embodiment, the analyzer 100 can further 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 (not shown) such as a Peltier element.

[0086] Referring now to FIGS. 13 through 14D, in one embodiment, a method 200 for analyzing blood can include a step 202 of obtaining or receiving a whole blood sample 60 that includes red blood cells 62 and plasma 64 for analysis.

[0087] In step 204, the whole blood sample 60 is placed in the microchannel 22 by pumping it through the inlet 130 and the first port 24 by the fluid distribution system 104 into the microchannel 22 of the acoustic levitation device 10. The controller 106 can measure, for example, the magnitude and phase of admittance by detecting the timing when the whole blood sample 60 is placed in the microchannel 22 using the first electrode 32 and the second electrode 34 disposed on both sides of the microchannel 22.

[0088] 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 is programmed to close or seal the inlet 130 after the blood sample is placed 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 is pressurized to about 5 psi. In other embodiments, the whole blood sample 60 is pressurized to a pressure of about 1 psi to about 10 psi.

[0089] In step 208, the controller 106 supplies a first electrical signal to the piezoelectric transducer 14 that causes the piezoelectric transducer 14 to emit ultrasonic waves of a first frequency and / or frequency range to cause preliminary concentration of the red blood cells 62 and the plasma 64. In particular, the first frequency is generated by a first voltage over a first predetermined period and can initiate the separation of the red blood cells 62 and the plasma 64 in the whole blood sample 60. For example, the first frequency and / or frequency range can be 950 kHz to 1100 kHz, the first voltage can be 50 volts to 100 volts, and the first predetermined period can be 5 seconds to 15 seconds.

[0090] (For example, following step 208) In step 210, the controller 106 supplies a second electrical signal to the piezoelectric transducer 14 that causes ultrasonic waves of a second frequency and / or frequency range to be emitted at a second voltage for a second predetermined period of time to completely separate red blood cells 62 and plasma 64 in the whole blood sample 60. As an example, the second frequency and / or frequency range can be from 320 kHz to 500 kHz, the second voltage can be from 70 volts to 100 volts, and the second predetermined period of time can be from 10 seconds to 25 seconds. In an exemplary embodiment, at least a portion of the plasma 64 (separated from the red blood cells 62) is separated / moved or concentrated toward the surface or outer edge of the microchannel 22 so as to be disposed in an antinode region near the surface of the microchannel 22.

[0091] (For example, following steps 208 and 210) In step 212, controller 106 activates light source 120 during a second predetermined period (during which red blood cells 62 and plasma 64 are completely separated), and sends light beam 116 from first optical fiber bundle 118 to light input 112. Light input 112 guides at least a portion of light beam 116 to a predetermined position that coincides with an antinode region within microchannel 22 that is plasma 64 and substantially free of red blood cells 62 through microchannel 22. Light receiver 114 receives a portion of light beam 116 guided to the predetermined position through the microchannel, and delivers it through second optical fiber bundle 119 to absorbance spectrophotometer 102 that acquires plasma spectrum measurement values. In an exemplary embodiment, as the predetermined position of microchannel 22 where light beam 116 is guided, an antinode region arranged along the outer edge of microchannel 22 and having plasma 64 and substantially free of red blood cells 62 is considered, and for plasma 64 arranged in the antinode region along the outer edge of microchannel 22, acquisition or execution of plasma spectrum measurement values is to be made. For example, a portion of light beam 116 passing through plasma 64 arranged in the antinode region along the outer edge / surface of microchannel 22 is measured by the light receiver (of the absorbance spectrophotometer), and by comparison of the intensity of light beam 116 that has been received by plasma 64 and output by plasma 64 within a desired frequency range, etc., the absorbance of plasma 64 (in the antinode region near the outer edge / surface of microchannel 22) is determined. Thereafter, by using the determined absorbance, the amount or concentration of a plasma component of particular interest, such as bilirubin or free hemoglobin, can be determined.

[0092] In step 214, the microchannel is pressurized again.

[0093] In step 216, after the absorbance spectrophotometer 102 obtains the plasma spectrum measurement value, the controller 106 causes the piezoelectric transducer 14 to emit ultrasonic waves of a third frequency and / or frequency range including the resonance frequency when the whole blood sample 60 is added to the integrated structure of the acoustophoretic device 10, and / or supplies to the piezoelectric transducer 14 a third electrical signal that causes the piezoelectric transducer 14 to expand and contract to generate a shearing force in the whole blood sample 60 that ruptures or lyses the walls of the red blood cells 62 in the whole blood sample 60 in the microchannel 22 to generate a lysed blood sample 66.

[0094] In step 218, the controller 106 activates the light source 120 and sends the medium 116 to the light input 112. The light input 112 sends at least a portion of the light beam 116 through the lysed blood sample 66 to the light receiver 114. The light receiver 114 sends a portion of the light beam 116 that has passed through the lysed blood sample 66 to the absorbance spectrophotometer 102 to obtain the measurement result of the lysed blood sample 66.

[0095] The method 200 can further include determining one or more oximetry parameters of the lysed blood sample based at least in part on a signal indicative of a portion of the light beam 116 received by the absorbance spectrophotometer 102.

[0096] Note that method 200 can be performed without pressurizing whole blood sample 60 in steps 206 and 214. For example, in one embodiment, lysing red blood cells 62 in step 216 is performed immediately after obtaining plasma spectral measurements for separated plasma 64 in step 212. Further, note that method 200 can be performed such that no reconstitution of whole blood occurs, that is, red blood cells 62 and plasma 64 separated in step 208 or 210 do not mix to form whole blood and no reconstituted whole blood sample is generated. Thus, instead of generating a reconstituted whole blood sample, method 200 cannot include reversibility of plasma separation step 208 or 210 because it generates lysed blood sample 66 in step 216. Further, note that method 200 is performed within microchannel 22. Thereby, the steps of pre-concentrating red blood cells 62 and plasma 64 (step 208), complete separation and measurement of red blood cells 62 and plasma 64 in whole blood sample 60 (steps 210, 212), and lysis and measurement of red blood cells 62 of whole blood sample 60 (steps 216, 218) are all advantageously performed within the same single (e.g., linear) microchannel 22 of the same sample vessel 12, eliminating the need for additional downstream channels or chambers in sample vessel 12 or additional devices for transferring or collecting separated plasma or red blood cells, which is convenient.

[0097] As shown in FIG. 15, an absorption spectrum is calculated based on known calculations regarding absorption of a liquid medium. Further, as shown in FIG. 16, determining one or more oximetry parameters can further include analyzing spectral profile coefficients of one or more of the following hemoglobin species: oxyhemoglobin (O2HB), deoxyhemoglobin (HHB), carboxyhemoglobin (COHB), methemoglobin (METHB), plasma bilirubin (NBILI), interfering substance cyanomethemoglobin (CN_MET_B), sulfhemoglobin (SULF_HIGH), and methylene blue (METH_BLUE_A).

[0098] Determining one or more oximetry parameters can be based on spectrophotometric light absorption, i.e., measurement of light absorption by components in the lysed blood sample 66.

[0099] Determining one or more oximetry parameters can include measuring at least total hemoglobin (THB) and one or more of the following hemoglobin fractions: oxyhemoglobin (O2HB), deoxyhemoglobin (HHB), carboxyhemoglobin (COHB), methemoglobin (METHB).

[0100] In one embodiment, method 200 can include inputting and discharging a cleaning fluid into the microchannel 22 of the sample vessel 12 before and / or after introduction of the whole blood sample 60 into the microchannel 22. Method 200 can further include activating the piezoelectric transducer 14 to generate acoustic waves and / or shear forces that agitate the cleaning fluid in the microchannel 22. In one embodiment, the sample vessel 12 is used, cleaned, and reused. In one embodiment, the acoustic separation device 10 is not reusable and is replaced for each new whole blood sample 60. In such an embodiment, the acoustic separation device 10 is discarded after each use.

[0101] Method 200 can further include calibrating the analyzer 100 with a fluid sample. In one embodiment, the fluid sample can be a test sample known as a "blank sample" used for calibration of the analyzer 100. The blank sample can include a dye solution used for measuring scattering of transmission of the medium 116.

[0102] In one embodiment, the whole blood sample 60 can be about 12 microliters in volume. The whole blood sample 60 typically includes plasma 64 and red blood cells 62 (which can constitute 45% - 60% of the blood sample) and optionally lipids.

[0103] In one embodiment, the whole blood sample 60 is maintained at a constant temperature. In one embodiment, the temperature of the whole blood sample 60 is 37°C ± about 0.3°C. In one embodiment, the temperature of the whole blood sample 60 is less than 40°C to avoid damage to the whole blood sample 60. In one embodiment, the whole blood sample 60 is maintained at a substantially constant temperature using one or more temperature sensors (not shown) and / or one or more thermal control elements (not shown).

[0104] Here, an example of the analyzer 100 and the acoustophoresis device 10 during use will be described. In one example, the sample vessel 12 can be made of glass and 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 (for example, having a height of about 100 micrometers and a width of about 2 millimeters). The sample vessel 12 is inserted into the path where the medium 116 moves between the light input 112 and the light receiver 114 of the absorbance spectrophotometer 102. It should be noted that the analyzer 100 is provided with various instruments including mirrors and / or waveguides for guiding the medium along the path. The fluid distribution system 104 can insert the whole blood sample 60 into the microchannel 22 of the sample vessel 12.

[0105] The controller 106 is electrically connected to the piezoelectric transducer 14 of the sample vessel 12 and can emit a first ultrasonic wave in the frequency range of about 950 kHz to about 1100 kHz at steps of about 1 kHz to the piezoelectric transducer 14 by supplying an electrical signal to the piezoelectric transducer 14. This frequency range permeates within a period of about 5 seconds to about 15 seconds. Thereby, as shown in FIG. 14B, the separation of the red blood cells 62 from the plasma 64 in the whole blood sample 60 is initiated. In FIGS. 14A to 14D, for clarity, the red blood cells 62 are shown as circles and the plasma 64 is represented by lines.

[0106] Thereafter, the controller 106 can cause the piezoelectric transducer 14 to emit a second ultrasonic wave in a frequency range of approximately 320 kHz to approximately 500 kHz in steps of approximately 1 kHz by supplying an electrical signal to the piezoelectric transducer 14. This frequency range penetrates within a period of approximately 10 seconds to approximately 25 seconds. By the second ultrasonic wave penetrating the whole blood sample 60, as shown in FIG. 14C, the separation of the red blood cells 62 from the plasma 64 is completed.

[0107] Due to the standing wave generated by the second ultrasonic wave, as shown in FIG. 14C, the red blood cells 62 and the plasma 64 move to a known portion of the microchannel 22. The known portion of the microchannel 22 is formed by the antinode regions 67 (only numbered as 67a and 67b in the figure) and the node region 68 (only numbered for one of them in the figure) between the antinode regions 67. The separated plasma 64 is concentrated (e.g., separated / moved) in the antinode region 67 that substantially does not contain the red blood cells 62 in the microchannel 22, while the red blood cells 62 move to the node region 68. As shown in FIG. 14C, a plurality of alternately arranged node regions 68 and antinode regions 67 (exemplary antinode region 67a, etc.) are formed along the outer edge of the microchannel 22, and a plurality of antinode regions 67 (exemplary antinode region 67b, etc.) are formed along the central axis of the microchannel 22. Since the outer edge of the microchannel 22 is also referred to as the side, wall, or surface of the microchannel 22, the antinode region 67a is formed near the side, wall, or surface of the microchannel 22, while the antinode region 67b is formed at the center of the microchannel 22. The node region 68 is formed in other spaces of the microchannel 22. Also, by using the above-described technique, the desired pattern of the antinode regions 67 and the node regions 68 is calculated.

[0108] Thereafter, the controller 106 can cause the light source 120 to emit a light beam 116, which is guided by the light input 112 to a predetermined portion of the microchannel 22 where the plasma 64 has been separated from the red blood cells 62 of the whole blood sample 60 (i.e., one of the antinode regions 67). When at least a portion of the light beam 116 passes through the plasma 64, the light receiver 114 receives this and guides it to the absorbance spectrophotometer 102, thereby obtaining the measurement result of the plasma. The measurement regarding the plasma is also performed by absorbance spectroscopy. A patient's blood sample (i.e., a whole blood sample) contains plasma and red blood cells. As described above, by separating red blood cells (RBCs) from the plasma and physically separating the plasma, plasma components of particular interest, such as bilirubin or free hemoglobin, are measured. RBCs contain hemoglobin species such as: oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and methemoglobin. In some cases, free hemoglobin is found in the plasma. Free hemoglobin is caused by a patient's physiological conditions such as improper bloodletting (blood collection) or hemolytic anemia.

[0109] In an exemplary embodiment, a predetermined portion of the microchannel 22 through which the light beam 116 passes is an antinode region 67a disposed 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 so as to pass through the antinode region 67a on the side of the microchannel 22 where the plasma is separated. For this reason, at least a part of the light beam 116 passes through the plasma 64 in the antinode region 67a formed near the surface of the microchannel 22, and the light receiver 114 of the absorbance spectrophotometer 102 receives this, and the measurement result of the plasma component in the antinode region 67a (i.e., the side of the microchannel 22) is obtained. The light receiver 114 and / or the absorbance spectrophotometer 102 is aligned with the side of the microchannel 22, receives a part of the light beam 116 that has passed through the plasma 64 in the antinode region 67a, and performs light detection of the plasma formed along the side of the microchannel 22 (i.e., the antinode region 67a), so that components or analytes can be detected by measuring the plasma in 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), measuring or detecting the plasma component in the antinode region 67a along the side of the microchannel 22 is convenient because the accuracy of the plasma measurement is improved compared to the measurement in the antinode region 67b along the central axis of the microchannel 22.

[0110] Once the plasma measurement result is obtained, the controller 106 can cause the piezoelectric transducer 14 to emit a third ultrasonic wave in a frequency range of about 330 kHz to about 370 kHz in steps of about 1 kHz by supplying an electrical signal to the piezoelectric transducer 14. This frequency range passes within a period of about 2 seconds.

[0111] Due to the third ultrasonic frequency, intensity, and duration, red blood cells 62 within the whole blood sample 60 are lysed, and hemoglobin is released from within the red blood cells 62 as shown in FIG. 14D. Since the frequency range of the third ultrasonic wave includes the resonance frequency when the whole blood sample 60 is added to the integrated structure of the acoustic migration device 10, cavitation occurs in the whole blood sample 60, rupturing the cell walls of the red blood cells 62 in the whole blood sample 60. As an addition or alternative to this, the controller 106 causes one or more processors 140 to deliver to the piezoelectric transducer 14 a signal for expanding and contracting the piezoelectric transducer 14, thereby generating a shearing force in the whole blood sample 60 within the microchannel 22 that ruptures the cell walls of the red blood cells 62 in the whole blood sample 60 and generates a lysed blood sample 66. In the lysed blood sample 66, the lysed red blood cells are indicated by dots.

[0112] More than 50% of the cell walls of the red blood cells 62 rupture.

[0113] Once the lysed blood sample 66 is generated, the controller 106 activates the light source 120 of the analyzer 100 and sends a medium 116 such as light from the sample vessel 12 to the lysed blood sample 66. The light receiver 114 can receive at least a portion of the medium 116 exiting from the lysed blood sample 66 and the sample vessel 12. The light receiver 114 can include one or more photodiodes for generating an electrical signal, for example, due to the reception of the medium 116.

[0114] Analyzer 100 or one or more computer processors 140 can determine one or more analytes present in a lysed blood sample based at least in part on a signal indicative of light received by light receiver 114 of absorbance spectrophotometer 102. Analyzer 100 or one or more computer processors can further analyze spectral profile coefficients of one or more of the following hemoglobin species: carboxyhemoglobin (COHB), oxyhemoglobin (O2HB), methemoglobin (METHB), deoxyhemoglobin (HHB), neonatal bilirubin (NBILI), cyanmethemoglobin (CN_MET_B), sulfhemoglobin (SULF_HIGH), methylene blue dye (METH_BLUE_A).

[0115] Analyzer 100 or one or more computer processors 140 can measure total hemoglobin (THB) and / or one or more of the following hemoglobin fractions: oxyhemoglobin (O2HB), methemoglobin (METHB), deoxyhemoglobin (HHB), carboxyhemoglobin (COHB).

[0116] Analyzer 100 or one or more computer processors 140 can output the results of the analysis. The output is presented on one or more displays. Also, based on the output, treatment of a patient can be determined.

[0117] An assembly 300 configured in accordance with the present invention for use in analyzer 100 is shown in FIGS. 17A and 17B. The assembly 300 includes an acoustophoretic device 10, a PCB 94, and a flow cell holder 301. The flow cell holder 301 is attached to the PCB 94 and is configured to define a cavity 302 sized and dimensioned to receive the acoustophoretic device 10. When the acoustophoretic device 10 is positioned within the cavity 302, the flow cell holder 301 and the PCB 94 press against the acoustophoretic device 10 to firmly hold it within the cavity 302, thereby preventing movement of the acoustophoretic 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 fit and engage at least a portion of the acoustophoretic device 10 and a second surface 312 opposite the first surface 310. The body 308 also includes an inner wall 314 extending between the first surface 310 and the second surface 312 and defining an opening 316 that extends through the body 308. The opening 316 is disposed within the body 308 such that it is aligned with the microchannel 22 when the acoustophoretic device 10 is positioned within the cavity 302. By aligning the opening 316 with the microchannel 22, light rays can pass through the microchannel 22 and the opening 316, enabling measurement results (e.g., absorbance measurement results) of components (e.g., samples) within the microchannel 22 to be obtained. The body 308 of the flow cell holder 301 also includes an inlet aperture 320 and an outlet aperture 322 that extend from the first surface 310 to the second surface 312. The inlet aperture 320 firmly receives the first tube 56, and the outlet aperture 322 firmly receives the second tube 58. For example, the inlet aperture 320 can have an inner diameter smaller than the outer diameter of the first tube 56 in order to firmly receive the first tube 56. The outlet aperture 312 and the second tube 58 are configured similarly to the inlet aperture 320 and the first tube 56. In this embodiment, the inlet and outlet tube connectors 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.

[0118] The flow cell holder 301 is fixed to the PCB 94 without soldering and can hold the acoustic levitation device 10 with respect to the PCB 94. For example, the PCB 94 and the body 308 are provided with a series of alignment holes 340 adapted to receive a fastener 342 that connects the flow cell holder 301 to the PCB 94. In the illustrated example, the hole in the body 308 for receiving the fastener 342 is not shown. In the illustrated example, the PCB 94 and the body 308 are provided with four alignment holes 340 applied to receive the fastener 342 and / or adapted to receive the fastener 342. As the fastener 342, for example, a screw can be considered.

[0119] In this example, the mounting pads on the sample vessel 12 are pressed against the mounting pads on the PCB 94, and an electrical connection is formed therebetween. As the assembly 300, components of the analyzer 100 can be considered, and as described above, it may be used for performing absorbance measurement on a blood sample.

[0120] The following is a list of numbers of non - limiting exemplary embodiments of the inventive concept disclosed in this specification:

[0121] 1. A fluid analysis device comprising: [[ID=—18]]A sample vessel having an outer surface, a microchannel within 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, whereby a blood sample can be inserted from the first port into the microchannel; A piezoelectric transducer coupled to the outer surface of the sample vessel to form an integral structure; A controller configured to send a signal to a piezoelectric transducer, the signal including a separation frequency configured to cause the piezoelectric transducer to transmit to a sample vessel separation acoustic waves sufficient to separate red blood cells and plasma of a blood sample without rupturing the red blood cells, and a lysis frequency configured to cause the piezoelectric transducer to transmit to the sample vessel lysis acoustic waves sufficient to induce cavitation in the blood sample to rupture cell walls of the red blood cells in the blood sample and release hemoglobin from within the red blood cells. The fluid analysis device including the same.

[0122] 2. The fluid analysis device according to exemplary embodiment 1, wherein the sample vessel is made of glass.

[0123] 3. The separation frequency includes a pre-concentration frequency and a concentration frequency. The controller is configured to send a signal of the pre-concentration frequency substantially from 950 kHz to substantially 1100 kHz to the piezoelectric transducer, and the controller is further configured to send a signal of the concentration frequency to the piezoelectric transducer sufficient to cause the piezoelectric transducer to transmit concentration acoustic waves to the sample vessel without rupturing the red blood cells. The fluid analysis device according to exemplary embodiment 1 or 2.

[0124] 4. The fluid analysis device according to exemplary embodiment 3, wherein the concentration frequency is a frequency substantially from 320 kHz to substantially 500 kHz.

[0125] 5. The fluid analysis device according to any one of exemplary embodiments 1 to 4, wherein the lysis frequency is a frequency substantially from 300 kHz to substantially 370 kHz.

[0126] 6. The lysis frequency is in a frequency range substantially from 300 kHz to substantially 370 kHz, and the piezoelectric transducer is configured to sweep a frequency range substantially from 300 kHz to substantially 370 kHz. The fluid analysis device according to any one of exemplary embodiments 1 to 5.

[0127] 7. The outer surface is a first outer surface having an attachment area with a first shape, and the piezoelectric transducer has a second shape corresponding to the first shape and has a second outer surface coupled to the attachment area. The fluid analysis device according to any one of exemplary embodiments 1 to 6.

[0128] 8. The piezoelectric transducer fits and engages with the outer surface of the sample vessel. The fluid analysis device according to any one of exemplary embodiments 1 to 7.

[0129] 9. The acoustic wave is configured to separate plasma in a belly region near the outer surface of the microchannel, a photodetector is arranged to receive light that has passed through the plasma in the belly region, and is configured to detect plasma components based on the measurement results of the plasma in the belly region near the outer surface. The fluid analysis device according to any one of exemplary embodiments 1 to 8.

[0130] 10. A fluid analyzer comprising: A fluid analysis device: A sample vessel having an outer surface, a microchannel within 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, thereby enabling insertion of a blood sample from the first port into the microchannel; A piezoelectric transducer coupled to the outer surface of the sample vessel to form an integral structure, having a first frequency and configured to initiate separation of red blood cells and plasma in the blood sample in the microchannel, a second frequency and configured to substantially complete separation of red blood cells and plasma in the blood sample in the microchannel, and a third frequency and configured to vibrate the sample vessel so that a shear force is induced into the microchannel. The piezoelectric transducer is configured to generate a third ultrasonic acoustic wave, and the third ultrasonic acoustic wave and the shear force are configured to induce cavitation in the blood sample in the microchannel so that the cell wall of the red blood cells in the blood sample ruptures and hemoglobin is released from within the red blood cells. A fluid analysis device including; A light transmitter disposed adjacent to a sample vessel and configured to emit a light medium through a microchannel, and a light receiver disposed to receive a part of the light medium after at least a part of the light medium has passed through the microchannel, an absorbance spectrophotometer including; A fluid distribution system having an outlet connected to a first port and an inlet connected to a second port; A controller electrically connected to a piezoelectric transducer and configured to send an electrical signal to the piezoelectric transducer that causes the piezoelectric transducer to emit a first ultrasonic acoustic wave, a second ultrasonic acoustic wave, and a third ultrasonic acoustic wave when the piezoelectric transducer receives it; The fluid analyzer including.

[0131] 11. The fluid analyzer according to exemplary embodiment 10, wherein the sample vessel is made of glass.

[0132] 12. The fluid analyzer according to exemplary embodiment 10 or 11, wherein the first frequency is a frequency from substantially 950 kHz to substantially 1100 kHz.

[0133] 13. The fluid analyzer according to any one of exemplary embodiments 10 to 12, wherein the second frequency is a frequency from substantially 320 kHz to substantially 500 kHz.

[0134] 14. The fluid analyzer according to any one of exemplary embodiments 10 to 13, wherein the third frequency is a frequency from substantially 300 kHz to substantially 370 kHz.

[0135] 15. The fluid analyzer according to any one of exemplary embodiments 10 to 14, wherein the third frequency is in a frequency range from substantially 300 kHz to substantially 370 kHz, and the piezoelectric transducer is configured to sweep a frequency range from substantially 300 kHz to substantially 370 kHz.

[0136] 16. The outer surface of the sample vessel is a first outer surface having a mounting area with a first shape, and the piezoelectric transducer has a second shape corresponding to the first shape and has a second outer surface coupled to the mounting area, the fluid analyzer according to any one of exemplary embodiments 10 to 15.

[0137] 17. The piezoelectric transducer fits and engages with the outer surface of the sample vessel, the fluid analyzer according to any one of exemplary embodiments 10 to 16.

[0138] 18. The second ultrasonic acoustic wave is configured to separate plasma in a region of maximum amplitude near the outer surface of the microchannel, and the absorbance spectrophotometer is configured to perform measurements on the plasma in the region of maximum amplitude near the outer surface to determine a plasma analyte, the fluid analyzer according to any one of exemplary embodiments 1 to 17.

[0139] 19. A blood analysis method comprising: passing a whole blood sample having red blood cells and plasma through a microchannel of a sample vessel; separating red blood cells from plasma within the microchannel; acquiring a first absorbance spectroscopic measurement value of the plasma separated from the red blood cells; dissolving the red blood cells within the microchannel to prepare a lysed blood sample; acquiring a second absorbance spectroscopic measurement value of the lysed blood sample within the microchannel, and including the said blood analysis method.

[0140] 20. The step of separating red blood cells from plasma within the microchannel is further defined as inducing a first acoustic wave within the microchannel having a frequency and duration sufficient to initiate separation of red blood cells from plasma, and inducing a second acoustic wave within the microchannel having a frequency and duration sufficient to substantially complete separation of red blood cells and plasma, the method according to exemplary embodiment 19.

[0141] 21. The sufficient frequency of the first acoustic wave is a frequency of substantially 950 kHz to substantially 1100 kHz, and the duration is a period of substantially 5 seconds to substantially 15 seconds, according to the method described in exemplary embodiment 20.

[0142] 22. The sufficient frequency of the second acoustic wave is a frequency of substantially 320 kHz to substantially 500 kHz, and the duration is a period of substantially 10 seconds to substantially 25 seconds, according to the method described in exemplary embodiment 20 or 21.

[0143] 23. The step of lysing red blood cells in the microchannel to prepare a lysed blood sample is further defined as inducing a third acoustic wave with a frequency and duration sufficient to induce cavitation in the blood sample such that the cell wall of the red blood cells lyses and hemoglobin is released from the red blood cells into the microchannel, according to the method described in any one of exemplary embodiments 19 to 22.

[0144] 24. The sufficient frequency of the third acoustic wave is a frequency of substantially 320 kHz to substantially 370 kHz, and the duration is a period of substantially 2 seconds to substantially 20 seconds, according to the method described in exemplary embodiment 23.

[0145] 25. The step of separating concentrates the plasma in the antinode region near the surface of the microchannel, and the step of obtaining the first absorbance spectroscopic measurement value is performed on the plasma in the antinode region near the surface of the microchannel, according to the method described in any one of exemplary embodiments 19 to 24.

[0146] Conclusion Conventionally, blood analysis could not be used at the patient's point of care, was time-consuming, and expensive. According to the present disclosure, an analyzer 100 that is easy to manufacture and low-cost is disclosed while improving the accuracy and precision of measurement parameters of a blood sample within a desired TTR at the patient's point of care. The controller 106 of the analyzer 100 sends a predetermined signal to the piezoelectric transducer 14 to transmit acoustic waves to the sample vessel 12, first separates the plasma 64 from other components of whole blood such as red blood cells 62 and obtains a plasma measurement result, and then, by the single piezoelectric transducer 14 driven at one or more specific excitation frequencies or frequency ranges, the ultrasonic acoustic waves, pressure, cavitation, fluid motion, and / or shear force generated in the sample vessel 12 dissolve the red blood cells 62 in the sample vessel 12.

[0147] The above description provides examples and explanations, but is not intended to be exhaustive or to limit the inventive concept to the exact form of the disclosure. Improvements and modifications are possible in light of the above teachings, and can also be obtained by implementing the methods described in the present disclosure.

[0148] The enumeration in the claims and / or the disclosure in the present specification show specific combinations of components and steps, but these combinations are not intended to limit the present disclosure. In fact, many of these components and steps are combined in ways not specifically enumerated in the claims and / or not specifically disclosed in the present specification. Each dependent claim listed below directly depends on only one other claim, but the present disclosure also includes combinations of each dependent claim with all other claims in the claims.

[0149] None of the elements, operations, or instructions used in this application should be construed as important or essential to the present invention unless explicitly stated otherwise outside the preferred embodiments. Further, the expression "based on" is intended to mean "based, at least in part, on" unless otherwise explicitly stated.

Claims

1. A fluid analysis device comprising: a sample vessel having an outer surface, microchannels within the scope of the outer surface, a first port extending through the outer surface to the microchannels, and a second port extending through the outer surface to the microchannels, whereby a blood sample can be inserted from the first port into the microchannels; a piezoelectric transducer coupled to the outer surface of the sample vessel to form an integral structure; a controller configured to send signals of a separation frequency to the piezoelectric transducer to transmit separation acoustic waves sufficient to separate red blood cells and plasma of the blood sample without rupturing the red blood cells to the sample vessel, and signals of a lysis frequency to the piezoelectric transducer to transmit lysis acoustic waves sufficient to induce cavitation in the blood sample to induce shear forces within the microchannels such that the cell walls of the red blood cells in the blood sample rupture and hemoglobin is released from within the red blood cells; the fluid analysis device comprising the above.

2. The fluid analysis device according to claim 1, wherein the sample vessel is made of glass.

3. The separation frequency includes a pre-concentration frequency and a concentration frequency, and the controller is configured to send a signal of the pre-concentration frequency substantially between 950 kHz and substantially 1100 kHz to the piezoelectric transducer, and the controller is further configured to send a signal of the concentration frequency to the piezoelectric transducer sufficient to transmit concentration acoustic waves to the sample vessel through the piezoelectric transducer without rupturing the red blood cells. The fluid analysis device according to claim 1.

4. The fluid analysis device according to claim 3, wherein the concentration frequency is a frequency substantially between 320 kHz and substantially 500 kHz.

5. The fluid analysis device according to claim 1, wherein the lysis frequency is a frequency substantially between 300 kHz and substantially 370 kHz.

6. The fluid analysis device according to claim 5, wherein the lysis frequency is in a frequency range substantially between 300 kHz and substantially 370 kHz, and the piezoelectric transducer is configured to sweep a frequency range substantially between 300 kHz and substantially 370 kHz.

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

8. 10. The fluid analyzer of claim 1, wherein the piezoelectric transducer matingly engages an exterior surface of the sample vessel.

9. 2. The fluid analysis device of claim 1, wherein the separating acoustic wave is configured to separate plasma into antinode regions near the outer surface of the microchannel, and a photodetector is positioned to receive light that has passed through the plasma in the antinode regions, and the device is configured to detect plasma components based on measurement results of the plasma in the antinode regions near the outer surface.

10. 1. A fluid analyzer comprising: A fluid analysis device comprising: a sample vessel having an exterior surface, a microchannel within the exterior surface, a first port extending through the exterior surface to the microchannel, and a second port extending through the exterior surface to the microchannel, thereby allowing a blood sample to be inserted through the first port into the microchannel; a piezoelectric transducer coupled to an exterior surface of the sample vessel to form an integral structure, the piezoelectric transducer configured to generate: first ultrasonic acoustic waves having a first frequency configured to initiate separation of red blood cells and plasma in the blood sample in the micro-channel; second ultrasonic acoustic waves having a second frequency configured to substantially complete separation of red blood cells and plasma in the blood sample in the micro-channel; and third ultrasonic acoustic waves having a third frequency configured to vibrate the sample vessel such that a shear force is induced in the micro-channel, the third ultrasonic acoustic waves and the shear force being configured to induce cavitation in the blood sample in the micro-channel such that cell walls of red blood cells in the blood sample rupture and hemoglobin is released from within the red blood cells; a fluid analysis device comprising: an absorbance spectrophotometer including an optical transmitter positioned adjacent the sample vessel to emit an optical medium through the microchannel, and an optical receiver positioned to receive a portion of the optical medium after at least a portion of the optical medium 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; A controller electrically connected to a piezoelectric transducer and configured to send an electrical signal to the piezoelectric transducer that, when received by the piezoelectric transducer, causes the piezoelectric transducer to emit a first ultrasonic acoustic wave, a second ultrasonic acoustic wave, and a third ultrasonic acoustic wave. The fluid analyzer including the same.

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

12. The fluid analyzer according to claim 10, wherein the first frequency is a frequency from substantially 950 kHz to substantially 1100 kHz.

13. The fluid analyzer according to claim 10, wherein the second frequency is a frequency from substantially 320 kHz to substantially 500 kHz.

14. The fluid analyzer according to claim 10, wherein the third frequency is a frequency from substantially 300 kHz to substantially 370 kHz.

15. The fluid analyzer according to claim 14, wherein the third frequency is in a frequency range from substantially 300 kHz to substantially 370 kHz, and the piezoelectric transducer is configured to sweep a frequency range from substantially 300 kHz to substantially 370 kHz.

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

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

18. The second ultrasonic acoustic wave is configured to separate plasma in a region of maximum amplitude near an outer surface of the microchannel, and the absorbance spectrophotometer is configured to perform a measurement on the plasma in the region of maximum amplitude near the outer surface to determine a plasma analyte. The fluid analyzer according to claim 10.

19. A blood analysis method comprising: Passing a whole blood sample having red blood cells and plasma through a microchannel of a sample vessel; Separating red blood cells from plasma within the microchannel; Obtaining a first absorbance spectroscopic measurement value of the plasma separated from the red blood cells; Dissolving the red blood cells within the microchannel to prepare a lysed blood sample; Obtaining a second absorbance spectroscopic measurement value of the lysed blood sample within the microchannel. The blood analysis method including the above steps.

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

21. The method according to claim 20, wherein the sufficient frequency of the first acoustic wave is a frequency of substantially 950 kHz to substantially 1100 kHz, and the duration is a period of substantially 5 seconds to substantially 15 seconds.

22. The method according to claim 20, wherein the sufficient frequency of the second acoustic wave is a frequency of substantially 320 kHz to substantially 500 kHz, and the duration is a period of substantially 10 seconds to substantially 25 seconds.

23. The step of lysing red blood cells within the microchannel to prepare a lysed blood sample is further defined as inducing a third acoustic wave having a frequency and duration sufficient to induce cavitation in the blood sample such that the cell wall of the red blood cells lyses and hemoglobin is released from the red blood cells into the microchannel, the method according to claim 19.

24. The method according to claim 23, wherein the sufficient frequency of the third acoustic wave is a frequency of substantially 320 kHz to substantially 370 kHz, and the duration is a period of substantially 2 seconds to substantially 20 seconds.

25. The step of separating concentrates the plasma in the antinode region near the surface of the microchannel, and the step of obtaining the first absorbance spectroscopic measurement value is performed on the plasma in the antinode region near the surface of the microchannel, the method according to claim 19.

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