阅读说明：本技术 用于通过光检测进行定点照护凝固测定的方法及系统 (Method and system for point-of-care coagulation assays by optical detection ) 是由 约瑟夫·克里莫 曾寒松 罗恩·沙尔拉克 格特·布兰肯施泰因 于 2015-04-27 设计创作，主要内容包括：本发明涉及一种测定系统,包括：反应腔,其用于保持样品；激发光源,其使激发光导向通过光学基准物并进入到反应腔,在光学基准物与反应腔之间的界面处所述激发光的第一部分被导向通过反应腔并且激发光的第二部分被导向返回到光学基准物中,其中,光学基准物吸收激发光的所述第二部分,并且光学基准物响应于激发光的第二部分的吸收来发射发射光；以及光检测器,其用于检测来自光学基准物的光信号,光信号借助发射光来传送。(The present invention relates to an assay system comprising: a reaction chamber for holding a sample; an excitation light source directing excitation light through an optical reference and into the reaction chamber, a first portion of the excitation light being directed through the reaction chamber and a second portion of the excitation light being directed back into the optical reference at an interface between the optical reference and the reaction chamber, wherein the optical reference absorbs the second portion of the excitation light and the optical reference emits emission light in response to absorption of the second portion of the excitation light; and a light detector for detecting a light signal from the optical reference, the light signal being transmitted by means of the emitted light.)

1. An assay system, comprising:

A reaction chamber (88) for holding a sample;

An excitation light source (96) that directs excitation light (95) through an optical reference (78) and into the reaction chamber (88), a first portion of the excitation light (95) being directed through the reaction chamber (88) and a second portion of the excitation light (95) being directed back into the optical reference (78) at an interface between the optical reference (78) and the reaction chamber, wherein the optical reference (78) absorbs the second portion of the excitation light and the optical reference emits emission light in response to absorption of the second portion of the excitation light; and

A light detector for detecting a light signal from the optical reference, the light signal being transmitted by means of the emitted light.

2. The assay system of claim 1, wherein the optical reference comprises a fluorescent element.

3. The assay system of claim 2, wherein the fluorescent element and the sample are positioned to provide varying light energy to or from the optical reference in response to a motive force of a coagulation process of the sample, such that the light signal comprises a change in a fluorescent signal indicative of the motive force of the coagulation process.

4. The assay system of claim 1, wherein the reaction chamber further comprises a sample entry port and a reaction fluid exit port.

5. The assay system of claim 1, wherein the reaction chamber comprises an internal cavity, a first planar wall, and a second planar wall opposite and parallel to the first planar wall.

6. the assay system of claim 1, wherein the reaction chamber holds the sample in the absence of a colorimetric reagent.

7. the assay system of claim 1, wherein the excitation light source provides a specific wavelength in the range of 20nm to 5000 nm.

8. The assay system of claim 1, wherein the excitation light is in a wavelength range of about 20nm to about 2000 nm.

9. The assay system of claim 1, wherein the light detector and the excitation light source are integral.

10. The assay system of claim 5, wherein the first planar wall and the second planar wall are each optically transparent to the excitation light in a wavelength range from about 20nm to about 5000 nm.

11. The assay system of claim 1, wherein the sample is selected from the group consisting of plasma and blood.

12. The assay system of claim 1, wherein the optical reference is achievable with an optical technique selected from the group consisting of photometry, fluorescence, raman spectroscopy time resolved fluorescence, and surface enhanced raman spectroscopy.

13. The assay system of claim 1, wherein the optical reference is embedded in one or more walls of the optical reference.

14. The assay system of claim 1, wherein the optical reference is chemically or physically coated on a surface of a substrate.

15. The assay system of claim 1, wherein the optical reference is selected from the group consisting of glass doped with a fluorescent, fluorescent colored glass, fluorescent stained glass, and a fluorescent material exhibiting raman effect.

16. A method for detecting coagulation, comprising:

(i) Providing an optical construction system comprising an optical reference for generating an optical signal, wherein the optical reference comprises a fluorescent element;

(ii) A reaction chamber configured to hold a fluid;

(iii) Transmitting excitation light from an excitation light source into the reaction chamber (88) through an optical reference, a first portion of the excitation light (95) being directed through the reaction chamber (88) and a second portion of the excitation light (95) being directed back into the optical reference (78) at an interface between the optical reference (78) and the reaction chamber, wherein the optical reference (78) absorbs the second portion of the excitation light and the optical reference emits emission light in response to absorption of the second portion of the excitation light;

(iv) (iv) transmitting the emitted light in step (iii) from the optical reference to a light detector, wherein the light detector is positioned to detect a calibration light signal from the optical reference, the light signal being transmitted by the emitted light passing through the fluid in the reaction chamber; and

(vi) comparing the light signal to a predetermined standard to determine a clotting time in the system,

Wherein the fluorescent element and the sample are positioned to provide varying light energy to or from the optical reference in response to a motive force of a coagulation process of the sample, such that the light signal comprises a change in a fluorescent signal indicative of the motive force of the coagulation process.

17. the method of claim 16, wherein the reaction chamber holds the sample in the absence of a colorimetric reagent.

18. The method of claim 16, wherein the excitation light source provides a specific wavelength in the range of 20nm to 5000 nm.

19. The method of claim 16, wherein the excitation light is in a wavelength range of about 20nm to about 2000 nm.

20. the method of claim 16, wherein the light detector and the excitation light source are integral.

21. the method of claim 16, wherein the reaction chamber further comprises a sample entry port and a reaction fluid exit port.

Technical Field

The invention relates to an optical system and a method for detecting coagulation of plasma or blood, the optical system comprising: a standard optical reference, a sample processing structure, a light source, and a light detection unit.

Background

Coagulation assays are important tools for monitoring a patient's risk of bleeding or thrombosis, both of which can have fatal consequences if the intervention is not performed quickly and properly. This is particularly critical in emergency and operating rooms, where the patient's hemostatic health needs to be known before proper blood therapy can be performed. Among all coagulation assays, the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) assays are currently the most popular coagulation tests used in clinics and hospitals.

Instruments for performing PT and APTT assays typically include a blood sample preparation mechanism, such as a clotting reagent, and a spectroscopic measurement unit. Despite the advantages such as high throughput and good accuracy, these assays have some disadvantages that prevent their application to point-of-care testing. First, (1) the sampling time varies from several days to several weeks until the result is obtained due to the complicated sample preparation and measurement process. This slow turnaround time does not meet the near real-time requirements of emergency rooms or other near-patient applications. Second, (2) these instruments for proper sample processing and accurate measurement require large volumes of blood, i.e., more than one milliliter.

the most advanced fluorescence-based techniques of microfluidic sample preparation (e.g., lab-on-a-chip immunoassays) have been developed to address the above deficiencies. Common methods in the art are recognized to use thrombin or plasmatase (which generate both elements during the coagulation reaction pathway) specific substrates containing immunoreactive fragments. The substrate is cleaved after exposure to thrombin or plasmalase and the immunoreactive fragments are released from the substrate, which generates a fluorescent signal as a kinetic indicator of the clotting process. These techniques have poor reliability due to inefficient chemical reactions and the stability of immunoreactive fragments. Furthermore, the industry needs for quality control, instrumentation and end use for chemical production increase the cost of these prior art coagulation assays.

The invention disclosed herein was developed to successfully solve the following problems: slow turnaround times in prior art coagulation assays, sample size requirements, excessive production costs, lack of reagent stability, and the inability of prior art coagulation assays to meet near real-time requirements for emergency rooms or other applications proximate to the patient to obtain timely coagulation assay results.

Disclosure of Invention

The fluorescence-based and other coagulation assays according to the invention described below can be used in a wide variety of clinical situations. Large centralized instruments or point-of-care instruments can be developed based on these methods to achieve high throughput coagulation assays. This technique can be used to achieve a variety of assays specific to certain factors involved in, for example, the coagulation cascade (coagulation cascade).

More importantly, a compact point of care device according to the invention described herein can be developed for emergency rooms, surgical suites, intensive care units or doctor's offices. The rapid response and small sample size requirements of the disclosed invention allow the use of the technology for continuous monitoring of, for example, coagulation kinetics, when blood therapy is required. At the same time, the present invention can use existing immunoassay systems and/or microfluidic systems currently used for diagnosing heart disease and cancer in patients without the need for extensive new instrument development.

In one aspect, the invention relates to an assay system comprising: a reaction chamber for holding a sample; an excitation light source; an optical reference for providing an optical signal; and an optical receiver. The optical reference is positioned to absorb the excitation light and generate an optical signal to the optical receiver.

The reaction chamber according to the invention is positioned to suppress or enhance the signal generated from the optical reference and detected by the optical receiver. In one embodiment, the reaction chamber holds the sample in the absence of a colorimetric reagent.

The excitation light source provides specific wavelengths in a range such as, but not limited to, 20nm to 5000nm, 50nm to 2000nm, or 100nm to 1000 nm.

The optical reference according to the assay system is selected from the group comprising, for example, glass doped with a fluorescent substance, colored glass, dyed glass and a material exhibiting raman effect. The reaction chamber includes an inner cavity, a first planar wall, and a second planar wall. In one embodiment of the reaction chamber, the second planar wall is opposite and parallel to the first planar wall.

In one embodiment of the invention, the first planar wall and the second planar wall are each optically transparent to light in a wavelength range, for example, from about 20nm to about 5000nm, or alternatively, a wavelength range from about 20nm to about 2000 nm.

In various embodiments of the invention, the reaction chamber is positioned between the optical reference and the light receiver and the excitation light source, or the optical reference is positioned between the reaction chamber and the light receiver and the excitation light source, alternatively the optical reference is positioned between the excitation light source and the reaction chamber, and the reaction chamber is positioned between the optical reference and the light receiver.

The assay system also includes a light receiver having a light detector for detecting emitted light emitted from the light source or the optical reference or for detecting reflected or secondary light. In one embodiment, the light receiver assembly and the light source assembly are integral.

In one embodiment, each of the first and second planar walls of the reaction chamber comprises a facet, and the first planar facet is coated with one or more reagents. The reaction chamber may further comprise a sample entry port and a reaction fluid exit port. The first access port may feature a V-shape.

In another aspect, the invention relates to a method for detecting coagulation. In one embodiment of this aspect of the invention, the method entails:

(i) Providing a system comprising an optical reference, the optical reference comprising means for generating a calibration light signal;

(ii) Set up reaction chamber and entry, the reaction chamber includes: a cavity for holding a fluid, the cavity comprising a first planar wall and a second planar wall opposite and parallel to the first planar wall; and an inner chamber for holding a fluid, the first and second planar walls of the reaction chamber comprising a luminal surface, and the first planar luminal surface being coated with one or more reagents, the inlet (e.g., a V-shaped inlet) for introducing a bulk fluid sample into the reaction chamber;

(iii) transmitting excitation light from a light source through the fluid in the reaction chamber to an optical reference;

(iv) Measuring emitted light transmitted from an optical reference through the fluid in the reaction chamber to a light detector;

(v) The measured emitted light is compared to a predetermined standard to determine the clotting time in the system.

in another embodiment, the method entails:

(i) Providing a system comprising an optical reference, the optical reference comprising means for generating a calibration light signal;

(ii) Set up reaction chamber and entry, the reaction chamber includes: a cavity for holding a fluid, the cavity comprising a first planar wall and a second planar wall opposite and parallel to the first planar wall; and an inner chamber for holding a fluid, the first and second planar walls of the reaction chamber comprising a luminal surface, and the first planar luminal surface being coated with one or more reagents, an inlet (e.g., a V-shaped inlet) for introducing a bulk fluid sample into the reaction chamber;

(iii) Transmitting excitation light from a light source through an optical reference to a fluid in a reaction chamber;

(iv) Measuring reflected emission light transmitted from the optical reference to the light detector; and (v) comparing the measured emitted light with a predetermined standard to determine the clotting time in the system.

In another embodiment, the method entails:

(i) Providing a system comprising an optical reference, the optical reference comprising means for generating a calibration light signal;

(ii) Set up reaction chamber and entry, the reaction chamber includes: a cavity for holding a fluid, the cavity comprising a first planar wall and a second planar wall opposite and parallel to the first planar wall; and an inner cavity for holding a fluid, the first and second planar walls of the reaction chamber comprising a luminal surface, and the first planar luminal surface being coated with one or more reagents, the inlet (e.g., a V-shaped inlet) for introducing a bulk fluid sample into the reaction chamber.

(iii) Transmitting excitation light from a light source through an optical reference;

(iv) The optical reference generates secondary light that passes through the reaction chamber;

(v) Measuring the secondary light with a photodetector; and

(vi) the measured secondary light is compared to a predetermined standard to determine the coagulation time in the system.

The foregoing and other objects, features and advantages of the invention will be apparent from the following, more particular description of preferred embodiments of the invention.

Drawings

The invention is described with particular reference to the appended claims. Further advantages of the invention described herein may be better understood by reference to the following description taken in conjunction with the accompanying drawings.

Fig. 1 shows a double-absorption optical configuration for a coagulation system for absorbing excitation light from a light source and return light from an optical reference during coagulation of a plasma or blood sample according to one embodiment of the present invention.

Fig. 2 shows a reflective optical configuration for a coagulation system according to another embodiment of the present invention for capturing excitation light at an interface between a sample and an optical reference during coagulation of a plasma or blood sample to enhance an optical signal generated in the optical reference.

Fig. 3 shows a transmissive configuration for a coagulation system for absorbing light excited by a light source and emitted from an optical reference during coagulation of a plasma or blood sample according to another embodiment of the invention.

fig. 4 shows that the distance (d) between the optical reference and the sample fluid can vary from 0 to a large value (which is typically 0mm to 200mm) with all configurations described with reference to fig. 1, 2 and 3.

Fig. 5A-5E illustrate a method of making an optical reference from various compositions, including: (A) doping optical media such as, but not limited to, fluorescent molecules, particles, dyes, etc. into the interior of substrate materials such as, but not limited to, plastics, glass, and silicon; (B) chemically assembling an optical medium layer onto a first surface of a substrate; (C) chemically assembling an optical medium layer onto an opposite surface of a substrate; (D) coating an optical medium layer on the first surface of the substrate by a physical or chemical method; (E) coating an optical medium layer on the opposite surface of the substrate by a physical or chemical method;

Fig. 6A to 6G show exemplary configurations for integrating an optical reference with a reaction chamber of a sample processing device: (A) embedding an optical reference into a first wall of an integral reaction chamber; (B) combining a flat optical reference to form a first wall of the reaction chamber such that the cavity of the reaction chamber is located at a lower portion; (C) bonding an optical reference to the remaining portion of the reaction chamber shown in FIG. 6B except the bottom; (D) placing the optical reference as a separate part outside the closed reaction chamber; (E) embedding an optical reference into a wall portion of the integrated reaction chamber opposite the first wall; (F) combining a planar optical reference to form opposing walls of the reaction chamber such that the cavity of the reaction chamber is in the remainder; (G) the optical reference was bonded to the remaining part of the reaction chamber shown in fig. 6F except for the first wall.

Fig. 7A is a view of the bottom of a microfluidic plate with a reaction chamber showing one exemplary configuration of a reaction chamber with a fluid inlet for a sample, a fluid outlet and a dry reagent pre-stored in the chamber, while fig. 7B shows a side view of fig. 7A.

8A-8D illustrate a liquid treatment apparatus including an exemplary configuration of a reaction chamber having a fluid outlet and two fluid inlets; one fluid inlet for the sample and one fluid inlet for the reagent, respectively; (A) is a perspective view of the reaction chamber; (B) is a top view of the reaction chamber; (C) is a cross-sectional view of fig. 8B; (D) is another cross-sectional view of fig. 8B. Fig. 8E to 8H show sequential filling of the reaction chamber with reagents from time-0 to time-3, while fig. 8I to 8L show sequential filling of the reaction chamber with sample fluid from time-4 to time-7.

Fig. 9 shows a cross-sectional view of a reaction chamber filled with a reagent and a sample fluid.

Fig. 10A and 10B are top and bottom views, respectively, of an exemplary microfluidic device including a plurality of reaction chambers according to the present invention.

FIG. 11 shows an embodiment of the present invention according to the optical configuration and exemplary assay results shown in FIG. 1, (A) in this particular embodiment, an LED is used as the light source, a glass doped with a fluorescent substance is used as the optical reference, and a quantitative fluorescence detector is used as the light detection unit; (B) shows fluorescence signals from a test group with abnormal plasma (b) and a control group with normal plasma (a) according to one embodiment of a dual absorption configuration of a coagulation system based on the invention shown in (a); the results of the abnormal assay group showed a delayed signal change compared to the results of the normal control assay group.

FIG. 12 shows one embodiment of the present invention according to the optical configuration of FIG. 2 and exemplary assay results; (A) in this particular embodiment, an LED is used as the light source, glass doped with a phosphor is used as the optical reference, and a quantitative fluorescence detector is used as the light detection unit; (B) shows the fluorescence signals from the assay group with coagulated plasma (a) and the control group with unclotted plasma (b) according to one embodiment of the dual absorption configuration of the coagulation system based on the invention shown in (a), the assay results show that: the signal change is greater for the coagulated plasma (a) compared to the signal change for the unclotted plasma (b).

fig. 13A-13D illustrate exemplary mathematical methods for processing optical data to obtain quantitative clotting times.

Detailed Description

In one aspect, the invention relates to a system for detecting coagulation of a patient plasma or blood sample in a reaction chamber (e.g., a chamber of a microfluidic device). The system includes an optical reference portion such as, but not limited to, a standard fluorescent element such as, but not limited to, a glass doped with a phosphor, a polymer film or sheet containing intrinsic fluorescence for generating a fluorescent reference signal. The positioning of the fluorescent element and the coagulation of the blood/plasma sample are configured to alter the amount of light energy reaching or leaving the optical reference. With this configuration, the system according to the invention decouples the fluorescent signal from the chemical reaction (decouple). The change in the fluorescence signal is indicative of the kinetics of the plasma/blood sample coagulation process.

The coagulation detection system according to the present invention is used for performing a coagulation assay using, for example, fluorescence detection. As a point of care (POC) coagulation immunoassay system, sample preparation can be carried out in a microfluidic cartridge, allowing for small sample volumes (i.e. less than 1 ml, preferably less than 100 microliters) and low manufacturing costs. The present invention can be used in many types of wet chemical assays where changes in absorbance, turbidity, etc. during the assay are used to detect and quantify analytes in a sample. Typical wet chemistry assays are enzymatic coagulation assays of immunochemistry, affinity-based and nucleic acid-based assays. Different light detection methods may be used in various embodiments such as, but not limited to, turbidity, absorption, reflection, fluorescence intensity, time resolved fluorescence, NIR, and the like. Compared to conventional coagulation assay tools, such as spectroscopy or lab-on-a-chip assay systems, the coagulation system according to the present invention has at least the following advantages:

(1) the enhanced portability and fast turnaround time of the system allows point-of-care applications;

(2) The processing of the sample by the system requires only a small amount (i.e., less than 1 ml, preferably less than 100 microliters) of patient blood or plasma;

(3) Indicators such as fluorescent or colorimetric reagents, which are typically required in the most advanced fluorescent assays, need not be added to the assay. This simplifies the assay protocol by reducing the assay processing steps that otherwise require immunoreactive reagents, intra-assay chemistry and chemical reactions. The fluorescence signal generated according to the invention is only a function of the coagulation reaction and no fluorescence needs to be added to the sample, which results in lower cost and less background interference;

(4) The decorrelation of the fluorescence signal and the chemical reaction and the use of standard fluorescent elements allow a simple and reliable quality management;

(5) The system according to the invention described herein can be implemented in any fluorescence system, a variety of liquid handling systems including microfluidics, machines and manual liquid transfer systems to allow rapid and cost-effective adoption and incorporation of other biomarker detection systems such as, but not limited to, solid phase immunoassays for quantifying other analytes in blood, such as cardiac markers (e.g., troponin I, etc.) or markers that provide additional information to coagulation parameters such as D-dimers. Arranging for D-dimer testing and other laboratory tests and imaging scans to help rule out the presence of thrombi;

(6) the cost of the cartridge (cartridge) including the various embodiments of the optical system according to the invention is sufficiently low to be disposable, which reduces the risk of cross-contamination. The cartridge is preferably made from a polymer such as polystyrene or a cyclic olefin using a manufacturing method preferably injection molding or hot embossing.

(7) Different wavelengths may be used for the light source and signal detection to reduce background interference. The light source may be selected from, but is not limited to, the group consisting of a laser, a mercury arc lamp, and an LED. The wavelength is for example in the range of about 20nm to about 5000nm, about 50nm to about 2000nm, about 100nm to about 1000 nm.

Optical structure

Various optical configurations of optical standards and sample reaction chambers having different configurations are disclosed for various turbidity measurements, such as blood coagulation measurements. Fig. 1, 2 and 3 show schematic diagrams of respective configurations according to an embodiment of the present invention, and the operational principle is described below.

Dual absorption optical structure

As shown in FIG. 1, a dual-absorption optical construction system 100 has a fluorescent assembly 98, a reaction chamber 88, and a fluorescent reference 78, according to one embodiment of the invention. In one embodiment, fluorescence assembly 98 integrates both light source 96 and fluorescence detection unit 94, fluorescence detection unit 94 being, for example and without limitation, a detection system that measures time-resolved fluorescence (TRF) using, for example, an excitation LED (360nm) and a photodetector such as a photodiode or multi-pixel photon counter (MPPC, for quantifying fluorescence emission).

With continued reference to FIG. 1, a dual absorption optical construction system 100 has a light source 96, a light detection unit (detector) 94, an optical reference 78, and a reaction chamber 88, in accordance with one embodiment of the present invention. During operation, both light 95 from the light source 96 and return light 93 from the optical reference 78 are transmitted through the sample in the reaction chamber 88 and are absorbed due to turbidity changes in the sample, e.g., plasma or blood. The source or excitation light 95 and the return or emission light 93 may have the same or different wavelengths. The optical reference 78 may be implemented using a variety of optical techniques such as, but not limited to, general photometry, fluorescence, Raman (Raman) spectroscopic time-resolved fluorescence, and surface-enhanced Raman spectroscopy. In one embodiment, a fluorescence-based method is used for blood clotting time measurement, wherein the light source is an LED, the optical reference is fluorescent glass, the returning light is emitted from a fluorescent element, the sample is blood plasma and the optical detection unit is a fluorescence detector. In this embodiment, when plasma is coagulated in the reaction chamber, the fluorescence signal read at the light detection unit is reduced due to the enhanced light absorption by the coagulated plasma.

With continued reference to FIG. 1, the reaction chamber 88 contains a plasma or blood sample and reagent(s) for a specific target coagulation assay. The reaction chamber 88 includes: a first wall 86; and a second wall 84 opposite the first wall and positioned between optical reference 78 and light source 96 and detector 94. The first wall 86 is optically transparent to light of a particular wavelength and is closer to the phosphor assembly 98 than the second wall 84. Second wall 84 is optically transparent to light of a particular wavelength, is positioned opposite and parallel to first wall 86, and is closer to fluorescent reference 78 than first wall 86. The reagent added to the plasma or blood sample in the reaction chamber 88 enables a coagulation reaction to occur in the reaction chamber 88.

The optical reference 78 is, for example but not limited to, a phosphor-doped glass or a fluorophore fixed on a surface of the opposing second wall 84 of the reaction chamber 88. In the dual absorption optical configuration embodiment, as shown in FIG. 1, the fluorescent reference 78 is positioned on the opposite side of the reaction chamber 88 from the fluorescent assembly 98. The purpose of the optical reference 78 is to provide a collimated optical signal at a particular wavelength.

During operation, once the plasma or blood sample coagulation process begins, more and more fibrin forms, thereby increasing the turbidity of the plasma or blood sample in the reaction chamber 88. As a result, the excitation light 95 transmitted through the sample is reduced, and the excitation of fluorescent molecules on the optical reference 78 is suppressed. In addition, the reduced emitted light 93 from the optical reference 78 is further absorbed as it passes through the sample in the reaction chamber 88 to the fluorescent assembly 98, where the emitted light 93 is detected and measured by the fluorescent detector 94. The combined effect of the two absorption processes, i.e., the first absorption, excitation light 95 is expected to pass through the reaction chamber 88 to the fluorescent reference 78; and a second absorption, emission light 93 passes from the optical reference 78 through the reaction chamber 88, the combined effect producing a change in signal detected by the photodetector 94. The change in signal is indicative of the coagulation of the sample in the reaction chamber 88. Thus, a decrease in the fluorescence signal detected by photodetector 94 in this dual absorption optical configuration indicates that the coagulation process has begun. The relative change of the signal over time gives information about the coagulation process (dynamic, slope). For a suitable calculation of different coagulation parameters, such as PT, APTT, etc., maximum and minimum signals are determined.

Reflected light signal

FIG. 2 shows a reflective optical configuration of a turbidity system 100' according to another embodiment of the invention, wherein a fluorescent reference 78 is positioned between the reaction chamber 88 and the fluorescent assembly 98.

With continued reference to FIG. 2, as with the dual-absorption optical construction system 100 'described above, the reflective optical construction system 100' includes a fluorescent assembly 98, a reaction chamber 88, and a fluorescent reference 78. In one embodiment of a reflective optical configuration, fluorescence assembly 98 integrates both light source 96 and fluorescence detection unit 94, with fluorescence detection unit 94 being, for example and without limitation, a fluorescence reader having an LED (360nm) light source and an MPPC (Multi-pixel photon counter) detector, such as, but not limited to, Horiba instruments Inc. (Horiba instruments, Inc.) (Kyoto, Japan).

The reaction chamber 88 holds a plasma or blood sample and reagent(s) for a given target coagulation assay and typically has a plurality of planar walls, at least two of which are parallel and opposing. The optical reference 78 is positioned between the reaction chamber 88 and the excitation light source 96 and the light receiver 94. For example, the reaction chamber 88 includes a first wall 86 and a second wall 84 opposite the first wall 86. In a preferred embodiment, the first wall 86 and the second wall 84 are parallel to each other. Alternatively, the first and second walls may be positioned at an angle to each other, for example at an angle of 45 °. In the reflective optical configuration, first wall 86 is optically transparent to light of a particular wavelength and is positioned closer to fluorescent reference 78 than second wall 84. Second wall 84 is positioned opposite and parallel to first wall 86 and is further from fluorescent reference 78 than first wall 86. The second wall 84 may or may not be optically transparent. The reagent added to the plasma or blood sample in the reaction chamber 88 enables a coagulation reaction to occur in the reaction chamber 88.

Optical reference 78 is, for example but not limited to, a glass doped with a phosphor or a fluorophore fixed on the surface of first wall 86 of reaction chamber 88. In this embodiment, as shown in FIG. 2, the fluorescent reference 78 is positioned between the reaction chamber 88 and the fluorescent assembly 98. The purpose of the fluorescent reference 78 is to provide a calibrated fluorescent signal.

during operation, once the plasma or blood sample coagulation process begins, more and more fibrin forms, thereby increasing the turbidity of the plasma or blood sample in the reaction chamber 88.

as shown in fig. 2, in the reflective optical configuration, excitation light 95 first reaches optical reference 78 and is subsequently transmitted through the sample in reaction chamber 88. In other words, a portion of the excitation light 95 excites fluorescence of the optical reference 78 before transmitting through the reaction chamber 88, while the remainder of the excitation light 95 is transmitted through the sample in the reaction chamber 88. While coagulation of the plasma or blood sample in the reaction chamber 88 begins and expands and the amount of fibrin in the sample increases, the energy distribution of the two light portions (i.e., the transmitted light and the reflected light) changes due to a change in the transmission characteristics of the sample. That is, transmission of the excitation light 95 is suppressed and more light is captured at the interface of the fluorescent reference 78, and more fluorescence is excited by the first wall 86 of the reaction chamber 88. Thus, an increase in the fluorescence signal detected by photodetector 94 in this configuration indicates that the coagulation process has begun. As shown in fig. 12, the clotting time can be determined using, for example, the slope of the clotting curve, which is calculated from the first derivative of the clotting curve (the maximum of the first derivative gives the start time of coagulation). Maximum (reaction start, time point zero) and minimum (coagulation completion) signals are required to determine clotting time.

Transmissive optical construction

Fig. 3 shows another optical configuration of the system 100 ". The optical reference 78 is disposed between the light source 96 and the sample reaction chamber 88, and the reaction chamber 88 is placed between the optical reference 78 and the light detection unit 94. During operation, the optical reference 78 is excited by the light source 96 and emits secondary light (secondary light), such as a fluorescent signal. The secondary light 93 passes through the reaction chamber 88 and is absorbed due to the turbidity change of the sample. The photodetector 94 reads a signal of the secondary light 93 from the optical reference 78. The quantitative value of the signal is indicative of the kinetics of the coagulation reaction.

fig. 4 shows: in the various configurations described above with reference to fig. 1, 2, and 3, the distance (d) between the optical reference 78 and the sample in the inner cavity 83 of the reaction chamber 88 may vary from about 0mm to about 200 mm.

fig. 5 shows an exemplary configuration of optical fiducials 78. Using optical fiducials with fluorescent properties as a non-limiting example, the optical medium 61 may be made by embedding fluorescent molecules, particles or other carriers into a plastic, glass or silicon material substrate (fig. 5A). Alternatively, the optical fluorescent medium may be chemically or physically coated on the upper surface 60 or the lower surface 62 (i.e., the first surface 60 or the second surface 62 opposite the first surface 60) or both surfaces of the substrate. For example, as shown in FIG. 5(B), the layer of optical media 61 may be chemically assembled, either physically or chemically, onto the first surface 60 of the substrate; in fig. 5(C), the optical medium 61 layer may be chemically assembled on the second surface 62 of the substrate by physical means or chemical means; in fig. 5(D), a layer of optical media 61 may be chemically or physically coated on the first surface 60 of the substrate; alternatively, in fig. 5(E), the layer of optical media 61 may be chemically or physically coated on the second surface 62 of the substrate.

FIG. 6 illustrates various exemplary configurations of reaction chamber 88 and optical reference 78. The optical reference 78 may be an integral part of the reaction chamber 88, for example by being embedded in the upper or bottom portion of the closed wall 63 of the reaction chamber 88, or alternatively, the optical reference may be a separate part positioned above or below the exterior of the reaction chamber 88, thereby forming a suitable optical configuration in accordance with the present invention. Preferably, the long axis of the optical reference 78 is perpendicular to the excitation light. Alternatively, the excitation light may be at an angle to the long axis of the optical reference 78.

FIG. 6A illustrates an exemplary planar optical reference 78 embedded in the first wall 65 of the enclosed wall portion 63 of the reaction chamber 88, according to one embodiment. Alternatively, FIG. 6B shows the planar optical reference 78 bonded to the first wall 65 of the reaction chamber 88 and forming the first wall 65 of the reaction chamber 88 such that the inner cavity 83 of the reaction chamber 88 is located inside the first wall 65 of the reaction chamber 88. In a preferred embodiment, the long axis of optical reference 78 is perpendicular to the light source, or alternatively at an angle of up to about 45 ° to the light source.

In another embodiment, as shown in FIG. 6C, optical reference 78 forms three wall portions 65 ', 65 ", 65 '" of reaction chamber 88, and only a second wall 65 ' opposite wall portion 65 is not part of optical reference 78.

in another embodiment, as shown in FIG. 6D, the optical reference 78 is positioned as a separate element from any wall of the reaction chamber 88, and the long axis of the optical reference 78 is parallel to at least one wall of the reaction chamber 88; as shown in fig. 6E, the optical reference 78 is embedded in the second wall 65' of the reaction chamber 88; as shown in fig. 6F, the optical reference 78 is planar and is bonded to the second wall 65' of the reaction chamber 88; as shown in fig. 6G, the optical reference 78 forms three wall portions 65 ', 65 ", and 65 '", and only the first wall 65 opposite the wall portion 65 ' is not part of the optical reference 78.

Sample preparation cartridge

According to the embodiments of the coagulation systems 100, 100' and 100 "shown in fig. 1, 2 and 3, sample preparation in the present invention can be achieved in a variety of ways, from manual pipetting to automated fluidic control systems. Non-limiting examples of microfluidic devices and methods suitable for the coagulation assay systems described above are given below. These devices and methods are not limited to use in coagulation assays and may be used in a variety of wet chemistry assays where measurement, reagent addition, mixing, incubation and quantification of the assay reaction product is required. Typical assays use enzymatic reactions to measure metabolites such as lactate or creatinine or to perform turbidity assays. An example of such a turbidity assay is an agglutination assay such as latex agglutination, where monodisperse immune particles are complexed in the presence of an analyte (which can be monitored using turbidity changes).

flow chamber with dry reagents

Referring now to fig. 7A and 7B, in one embodiment, a reaction chamber 88 of a liquid handling device 120 having a defined volume is formed by covering a microchannel plate 90 with a cover portion 91. The reaction chamber is used to meter the sample volume, one fluid inlet 68 is used to introduce a sample, such as plasma, from the bottom side 60b of the reaction chamber 88, and one fluid outlet 66 at the bottom side 60b of the reaction chamber 88 is used to drain excess liquid from the inner cavity 83 of the reaction chamber 88. Dry reagents such as lyophilized wt/wt reagents, biotin and the like are pre-stored in the reaction chamber 88 and are uniformly coated, for example, on the luminal face of the first wall 86. As the plasma fills the reaction chamber 88, the dry reagent begins to dissolve and then diffuses into the sample in a vertical direction (i.e., from the bottom side 60b of the reaction chamber 88 toward the top side 60a of the chamber body). The dry reagent has a relatively large contact area with the liquid sample and a relatively short diffusion distance along the vertical direction. This configuration provides a uniform solidification process across the transverse plane of the reaction chamber 88. During operation, once the cavity 88 is filled with sample, the assay process begins and the acquisition of the fluorescence signal begins to follow the reaction kinetics.

Flow chamber with liquid reagent

Fig. 8A to 8D show an embodiment of the invention showing a liquid handling device 120 for investigating a sample fluid. The liquid processing apparatus 120 includes: a reaction chamber 88; two inlets 66, 68 for delivering sample fluid and reagent fluid, respectively, into the inner cavity 83 of the reaction chamber; and an outlet 64 for venting the reaction chamber 88 during filling. The device 120 may comprise, for example, one or more fluidic structures 68a and 64a, the fluidic structures 68a and 64a being configured to provide controlled bubble-free filling of the reaction chamber lumen 83.

According to one embodiment of the liquid handling device 120 shown in fig. 8A-8D, the reaction chamber 88 of the device 120 is first filled with a metered amount of liquid reagent via the first inlet 66. Bubble-free liquid filling may be achieved with a fluidic structure 64a, i.e., a capillary stop feature, proximate the outlet 64. In fig. 8A, for example, a cylindrical groove is used as a capillary stop feature. The capillary stop feature is defined by the abrupt channel opening and the bending of the fluidic structure 64a, or by making the outlet 64 hydrophobic. Fig. 8E to 8H show that the reaction chamber 88 is filled with the reagents in sequence at different time points from time 0 to time 3. After filling the metered amount of reagent into the inner cavity 83 of the reaction chamber 88, a metered amount of sample fluid (e.g., plasma and whole blood) is filled into the reaction chamber inner cavity 83 via the second inlet 68 as shown in fig. 8I-8L.

Additional features of the embodiment described in fig. 8A-8D are described below. The liquid treatment device 120 is oriented in a horizontal direction, i.e., a top view of the liquid treatment device 120 is shown in fig. 8B. The V-shape 68a at the second inlet 68 has an opening angle (opening angle) of, for example, 30 °. The V-shape 68a may have an angle in the range from 0 ° to 180 ° (typically 15 ° to 120 °). It should also be noted that according to this embodiment of the liquid treatment device 120, the second inlet 68 and the outlet 64 are positioned on the top side 60a of the liquid treatment device 120, while the first inlet 66 is positioned on the bottom side 60b of the liquid treatment device 120. Other configurations of inlets and outlets on the top and bottom sides of the liquid handling structure are possible and are not limited by the illustrated embodiment. The flow rate of the sample and reagent may be in the range of about 0.5. mu.l/s to 200. mu.l/s (typically 2. mu.l/s to 100. mu.l/s).

Fig. 9 schematically shows the reaction chamber 88 after the filling of the reagents and sample is completed. Two layers are shown: a reagent layer and a sample fluid layer. The sample layer spreads over the reagent layer across the entire fluid surface in the inner cavity 83 of the reaction chamber 88. Thus, a large contact area is formed between the two liquids (i.e., the reagent and the sample). With this large contact area, the mixing of the reagent and the sample fluid and thus the reaction is efficient.

in the illustrated embodiment in fig. 8, the V-shaped geometry of the second inlet 68 is used to support even distribution of the sample fluid into the reaction chamber. As shown in fig. 8c and 8d, the second inlet 68 is connected to the top side 60a of the reaction chamber 88, while the first inlet 66 is positioned opposite the bottom side 60b of the reaction chamber 88.

Referring to fig. 10A, a top view of an embodiment of a microfluidic device 50 having four reaction chambers 88 a-88 d is shown. In the illustrated embodiment, the reaction chambers 88 a-88 d are positioned toward one side of the microfluidic device 50, but may be positioned at other locations in the microfluidic card.

Figure 10B shows a bottom view of microfluidic card 50 including a plurality of channels 67 in fluid communication with reaction chambers 88.

Example/verification of the principle

The embodiments of the clotting systems 100, 100' and 100 "discussed above and related assay methods for detecting clotting of blood or plasma samples were evaluated with controlled plasma samples and reagents for FT and APTT assays. In the example of the dual absorption configuration described above with reference to fig. 1 and as shown in fig. 11A, the fluorescent component applied in this method is a PMT-based time-resolved fluorescence (TRF) cell, the fluorescent reference 78 is a europium-doped glass that contains a precisely controlled amount of europium and has no photobleaching during excitation, and the LED 96 is used as the light source. A filter 95a is placed between the LED 96 and the dichroic mirror 97. A second filter 95b is placed between the detector 94 and the dichroic mirror 97. Plasma samples included normal control plasma (a) and high abnormal control plasma (b) from the instrumentation laboratory Company (Orangeburg, NY, new york). The coagulation is started by introducing a coagulation initiator.

Referring to fig. 11B, the curves for normal control plasma (a) and abnormal control plasma (B) represent the intensity of the fluorescence signal emitted from the fluorescent reference and transmitted to the fluorescence detector in the dual absorption coagulation system described above with reference to fig. 1 and 11A, in the normal plasma sample and the abnormal plasma sample, the fluorescence signal decreases at the beginning of coagulation, expands, and reaches a stable value at the completion of coagulation. Abnormal plasma takes longer to initiate and complete the clotting process than normal plasma.

Referring to fig. 12A, an embodiment of the present invention is implemented using the reflective construction described with reference to fig. 2. As shown in fig. 12A, an LED 96 is used as a light source, glass doped with a fluorescent substance is used as the optical reference 78, and a quantitative fluorescence detector is used as the light detection unit 94. A filter 95a is placed between the LED 96 and the dichroic mirror 97. A second filter 95b is placed between the detector 94 and the dichroic mirror 97. The plasma samples included a normal plasma sample (a) with a clotting reagent introduced and a normal control sample (b) with water (no clotting reagent) introduced. Fig. 12B shows the optical signals obtained from plasma (a) with clotting reagent and plasma (B) without clotting reagent. The fluorescence signal of sample (a) reaches a steady value as coagulation begins and spreads and reaches a steady value when coagulation is complete. Control (b) used the same plasma sample but with the addition of deionized water (no coagulation).

Fig. 13 shows an exemplary method for processing optical data to obtain quantitative clotting times, in four steps, initial data (fig. 13A) is first normalized (fig. 13B) and filtered to eliminate redundant data and noise. When the fastest change in the light signal occurs, the first derivative of the initial data (fig. 13C) is applied to identify the point in time. The peak position of the first derivative (fig. 13D) was used as the coagulation start time. Other methods can also be used to quantitatively investigate the coagulation process.

Various modifications and other embodiments of the invention described and illustrated herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. The invention is not limited by the foregoing description or drawings.