Chemiluminescence analyzer

Abstract

The invention relates to a chemiluminescence analyzer, comprising: a sample preparation device for mixing a to-be-detected blood sample and a reaction reagent in a reaction container to obtain a to-be-detected sample liquid; the substrate providing device is used for providing a luminous substrate into a reaction container filled with a sample solution to be detected so as to carry out a chemiluminescence reaction; the light measuring device at least comprises a photometer and is used for detecting a light-emitting signal generated in the chemiluminescence reaction; and the data processing device is used for obtaining a detection result of the to-be-detected substance in the to-be-detected blood sample according to the light-emitting signal and a preset calibration model. The first linear detection range of the photometer is [A1, A2] photon counting / second, A1 is smaller than or equal to 2000, and A2 is larger than or equal to 108. The second linear detection range of the light-emitting substrate is [B1, B2] photon counting / second, B1 is smaller than or equal to 3000, and B2 is larger than or equal to 108. According to the embodiment of the invention, the chemiluminescence analyzer with high sensitivity and high linear detection upper limit can be obtained.

Description

Technical Field

[0001] The present disclosure relates to the field of chemiluminescence detection, and particularly to chemiluminescence analyzers. Background Art

[0002] Chemiluminescence Immunoassay (CLIA) is an immunoassay technique that combines a chemiluminescence system with an immune reaction for detecting antigens, antibodies, hormones, fatty acids, vitamins, drugs, etc. Chemiluminescence refers to the phenomenon that during a chemical reaction, a specific substance absorbs part of the chemical energy to reach an excited state, and the energy released during the return to the ground state is released in the form of photons, thus generating luminescence. Chemiluminescence immunoassay has both the high sensitivity of chemiluminescence and the characteristics of simple operation and rapid reaction of enzyme-linked immunoassay, and is easy to standardize experimental operations. It has now been widely used in biological research, medical research, and clinical diagnosis.

[0003] However, for chemiluminescence analyzers based on chemiluminescence immunoassay, it is difficult to achieve both high detection sensitivity and a wide linear detection range, especially for analytes with a large range span, such as human chorionic gonadotropin (HCG).

[0004] Human chorionic gonadotropin (HCG) is a glycoprotein mainly secreted by placental trophoblast cells. HCG consists of an α subunit and a β subunit, contains 237 amino acids, and has a molecular weight of approximately 38,000 daltons. Each subunit consists of a single polypeptide. Among them, the α subunit consists of 92 amino acids and has a molecular weight of approximately 14,900 daltons; the β subunit consists of 145 amino acids and has a molecular weight of approximately 23,000 daltons. In the HCG structure, the protein content accounts for 70%, and the sugar content accounts for 30%.

[0005] HCG can exist in urine, blood, and placenta, and the existence forms of HCG in each matrix are different. In blood, it mainly exists in the form of intact HCG. Among them, the α subunit has a similar structure to the α subunits of follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone secreted by the pituitary gland; the structures of the β subunits are different, and the differences in the β subunits result in their respective immunological and biological specificities.

[0006] The main function of human chorionic gonadotropin (HCG) is to stimulate the corpus luteum, which is beneficial for the continuous secretion of estrogen and progesterone, promoting the formation of the uterine decidua and enabling the placenta to grow and mature. HCG is produced by trophoblast transitional cells and syncytiotrophoblasts and is regulated by multiple factors including autocrine and paracrine. For normal pregnant women, HCG is produced 2 to 8 days after conception. In the early stage of pregnancy, the HCG content doubles approximately every 31 - 48 hours until it reaches a peak around 11 weeks of gestation, then slowly decreases in concentration until the 18th - 20th week, and then remains relatively constant. The detection of HCG is of great significance for the diagnosis of early pregnancy and pregnancy monitoring. In the early stage of pregnancy, a 35 - 50% increase in HCG within two days indicates a possible ectopic pregnancy. An abnormally low or rapidly decreasing HCG level indicates a high risk of ectopic pregnancy. On the other hand, in the second trimester of pregnancy, an increase in the HCG level indicates an adverse reaction during pregnancy, which may be pregnancy-induced hypertension, fetal growth restriction, etc.

[0007] The concentration distribution of HCG is relatively wide among non-pregnant, pregnant, and trophoblastic cell disease populations in normal women: For pregnant populations, the HCG can increase from several hundred mIU / mL in early pregnancy to about 200,000 mIU / mL at most, and then decrease to a level within about 10,000 mIU / mL; For patients with trophoblastic cell diseases (including choriocarcinoma), the HCG concentration can be as high as 1,000,000 mIU / mL or even higher; For non-pregnant women, the HCG concentration is usually below 5 mIU / mL, and even some are as low as 0.5 mIU / mL.

[0008] That is to say, HCG is secreted by placental trophoblast cells and has a very low content in non-pregnant populations, less than 5 mIU / ml. Therefore, high detection sensitivity is the key performance to ensure the use of HCG test results in clinical practice to assist in judging pregnancy or not. At the same time, in the pregnancy monitoring scenario, it is necessary to continuously measure the HCG concentration in the early stage of pregnancy (about 200,000 mIU / ml). Therefore, a wide linear detection range is the key performance to ensure the use of HCG test results in clinical practice for pregnancy status monitoring.

[0009] Therefore, for chemiluminescence analyzers, in the HCG detection scenario, the linear detection range that needs to be accurately detected spans 6 - 7 orders of magnitude. However, the current linear detection upper limit that chemiluminescence analyzers and their supporting reagents can achieve is at most no greater than 15,000 mIU / mL, far from covering the conventional pregnancy test scenarios. Summary of the Invention

[0010] Based on this background, the task of the present disclosure is to provide a chemiluminescence analyzer based on chemiluminescence immunoassay with a wide linear detection range, which can balance sensitivity and the upper limit of linear detection. Especially for samples with a high concentration of the analyte (such as HCG or β-HCG), there is no need to dilute and retest, improving efficiency and saving reagent costs.

[0011] To achieve the above task, the first aspect of the present disclosure provides a chemiluminescence analyzer, including:

[0012] A sample preparation device for mixing a blood sample to be tested with a reaction reagent in a reaction vessel so that the analyte in the blood sample to be tested reacts with the reaction reagent to obtain a sample solution to be tested, and the reaction reagent includes a capture reagent and a detection reagent with a marker;

[0013] A substrate providing device for providing a luminescent substrate into the reaction vessel containing the sample solution to be tested so that the marker in the sample solution to be tested undergoes a chemiluminescence reaction with the luminescent substrate;

[0014] A photometric device, the photometric device at least includes a photometer, and the photometer is used to detect the luminescence signal generated in the chemiluminescence reaction;

[0015] A data processing device for obtaining the detection result of the analyte in the blood sample to be tested according to the luminescence signal and a pre-set calibration model;

[0016] It is characterized in that the first linear detection range of the photometer is [A1, A2] photon counts per second, and the first linear detection range represents the range of photon counts output by the photometer. Within this first linear detection range, the intensity of the luminescence signal generated in the chemiluminescence reaction is linearly related to the photon counts output by the photometer, where A1 is less than or equal to 2000 and A2 is greater than or equal to 10 8 ; the second linear detection range of the luminescent substrate is [B1, B2] photon counts per second, and the second linear detection range represents the range of the intensity of the luminescence signal generated in the chemiluminescence reaction. Within this second linear detection range, the number of markers participating in the chemiluminescence reaction is linearly related to the intensity of the luminescence signal generated in the chemiluminescence reaction, where B1 is less than or equal to 3000 and B2 is greater than or equal to 10 8 .

[0017] The second aspect of the present disclosure provides another chemiluminescence analyzer, including:

[0018] A sample preparation device for mixing a blood sample to be tested with a reaction reagent in a reaction vessel so that a substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample solution to be tested, wherein the reaction reagent includes a capture reagent and a detection reagent with a marker;

[0019] A substrate providing device for providing a chemiluminescent substrate into the reaction vessel containing the sample solution to be tested so that the marker in the sample solution to be tested undergoes a chemiluminescent reaction with the chemiluminescent substrate;

[0020] A photometric device, the photometric device at least including a photometer, the photometer being used for detecting the luminescence signal generated in the chemiluminescent reaction;

[0021] A data processing device for obtaining a detection result of the substance to be tested in the blood sample to be tested according to the luminescence signal and a pre-set calibration model;

[0022] It is characterized in that the first linear detection range of the photometer is [A1, A2] photon counts per second, the first linear detection range represents the range of photon counts output by the photometer, within this first linear detection range, the intensity of the luminescence signal generated in the chemiluminescent reaction is linearly related to the photon counts output by the photometer, wherein the ratio A2 / A1 of A2 to A1 is greater than or equal to 30000; the second linear detection range of the chemiluminescent substrate is [B1, B2] photon counts per second, the second linear detection range represents the range of the intensity of the luminescence signal generated in the chemiluminescent reaction, within this second linear detection range, the number of the markers participating in the chemiluminescent reaction is linearly related to the intensity of the luminescence signal generated in the chemiluminescent reaction, wherein the ratio B2 / B1 of B2 to B1 is greater than or equal to 30000.

[0023] The third aspect of the present disclosure provides another chemiluminescent analyzer, including:

[0024] A sample preparation device for mixing a blood sample to be tested with a reaction reagent in a reaction vessel so that a substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample solution to be tested;

[0025] A substrate providing device for providing a chemiluminescent substrate into the reaction vessel containing the sample solution to be tested so that the sample solution to be tested undergoes a chemiluminescent reaction with the chemiluminescent substrate;

[0026] A photometric device, the photometric device at least including a photometer, the photometer being used for detecting the luminescence signal generated in the chemiluminescent reaction;

[0027] A data processing device for obtaining a detection result of a substance to be measured in the blood sample to be measured according to the luminescence signal and a pre-set calibration model;

[0028] It is characterized in that the photometer includes a receiving component and a processing component. The receiving component is configured to receive the optical signal generated in the chemiluminescence reaction and convert the optical signal into a corresponding electrical signal. The processing component is configured to be electrically connected to the receiving component and receive the electrical signal from the receiving component. The processing component includes a first photon counting module and a second photon counting module. The first photon counting module is configured to detect the number of pulses of the electrical signal by using a pulse recognition method to obtain a first photon counting result. The second photon counting module is configured to process the electrical signal to obtain a parameter characterizing the number of photons in the optical signal, and obtain a second photon counting result according to the parameter characterizing the number of photons in the optical signal and a preset calibration function. Wherein, the calibration function represents the mapping relationship between the parameter characterizing the number of photons in the optical signal and the photon counting result. The processing component is further configured to obtain the photon counting output by the photometer based on the first photon counting result and the second photon counting result, and output the photon counting; and

[0029] The second linear detection range of the luminescent substrate is [B1, B2] photon counts per second. The second linear detection range represents the range of the intensity of the luminescence signal generated in the chemiluminescence reaction. Within this second linear detection range, the number of the markers participating in the chemiluminescence reaction is linearly related to the intensity of the luminescence signal generated in the chemiluminescence reaction, where the ratio of B2 to B1 is greater than or equal to 30000 or B1 is less than or equal to 3000 and B2 is greater than or equal to 10 8 。

[0030] The fourth aspect of the present disclosure provides yet another chemiluminescence analyzer, including:

[0031] A sample preparation device for mixing a blood sample to be measured with a reaction reagent in a reaction container so that the substance to be measured in the blood sample to be measured reacts with the reaction reagent to obtain a sample solution to be measured;

[0032] A substrate providing device for providing a luminescent substrate into the reaction container containing the sample solution to be measured so that the sample solution to be measured undergoes a chemiluminescence reaction with the luminescent substrate;

[0033] A photometric device, the photometric device at least includes a photometer, and the photometer is used to detect the luminescence signal generated in the chemiluminescence reaction;

[0034] A data processing device for obtaining a detection result of a substance to be measured in the blood sample to be measured according to the luminescence signal and a preset calibration model;

[0035] It is characterized in that the first linear detection range of the photometer is [A1, A2] photon counts per second, and the first linear detection range represents the range of photon counts output by the photometer. Within this first linear detection range, the intensity of the luminescence signal generated in the chemiluminescence reaction is linearly related to the photon counts output by the photometer, where the ratio of A2 to A1 is greater than or equal to 30,000, or A1 is less than or equal to 2,000 and A2 is greater than or equal to 10 8 and the luminescent substrate includes a chemiluminescent substrate and a chemiluminescence enhancer, the chemiluminescence enhancer includes a fluorescent agent and a surfactant, and the fluorescent agent includes one or more carboxyfluoresceins.

[0036] The chemiluminescence analyzer provided in various aspects of the present disclosure can achieve a chemiluminescence analyzer with high detection sensitivity and a high linear detection upper limit through the cooperative design of the photometer and the luminescent substrate. That is, the linear detection range of a single detection of the chemiluminescence analyzer according to the present disclosure is widened. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present disclosure will be described more clearly below in conjunction with the embodiments and the drawings. Through the detailed description of the embodiments of the present disclosure, the above advantages and other advantages will become clear to those of ordinary skill in the art. The drawings are only used to illustrate the preferred embodiments and should not be construed as a limitation of the present disclosure.

[0038] In the drawings:

[0039] Figure 1 A schematic block diagram of a chemiluminescence analyzer provided by an embodiment of the present disclosure is shown.

[0040] Figure 2 and Figure 3 A calibration curve of a chemiluminescence analyzer provided by an embodiment of the present disclosure is shown.

[0041] Figure 4 A schematic structural diagram of a chemiluminescence analyzer provided by an embodiment of the present disclosure is shown.

[0042] Figures 5 to 15 A schematic block diagram of a photometer provided by different embodiments of the present disclosure is shown.

[0043] Figures 16A to 16D It is a linear fitting curve of the luminescence values of chemiluminescence substrate solutions 1-1 to 1-4 in the alkaline phosphatase system in Example 1.

[0044] Figures 17A to 17DThe linear fitting curves of the luminescence values of the chemiluminescent substrate solutions 2-1 to 2-4 in Example 2 in the alkaline phosphatase system.

[0045] Figures 18A to 18D The linear fitting curves of the luminescence values of the chemiluminescent substrate solutions 3-1 to 3-4 in Example 3 in the alkaline phosphatase system.

[0046] Figures 19A to 19D The linear fitting curves of the luminescence values of the chemiluminescent substrate solutions 4-1 to 4-4 in Example 4 in the alkaline phosphatase system.

[0047] Figures 20A to 20D The linear fitting curves of the luminescence values of the chemiluminescent substrate solutions 5-1 to 5-4 in Example 5 in the alkaline phosphatase system. Detailed implementation manners

[0048] Next, the embodiments of the present disclosure will be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present disclosure without creative efforts belong to the scope of protection of the present disclosure.

[0049] It should be noted that the terms "first / second / third" involved in the embodiments of the present disclosure are only used to distinguish similar objects, and do not represent a specific order for the objects. It can be understood that "first / second / third" can be interchanged with a specific order or sequence when permitted.

[0050] Those skilled in the art of this technology can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used here have the same meaning as the general understanding of those of ordinary skill in the art belonging to the field of the present disclosure.

[0051] As mentioned in the background art, for some detection items, such as the HCG detection item, the requirement for the linear detection upper limit is relatively high, and the prior art cannot simultaneously meet the requirements of a high linear detection upper limit and a high detection sensitivity.

[0052] Based on this, the embodiments of the present disclosure propose to improve the linear detection upper limit and the detection sensitivity through the configuration design of a photometer and a luminescent substrate.

[0053] As Figure 1 shown, the embodiments of the present disclosure propose a chemiluminescent analyzer 100, including a sample preparation device 110, a substrate providing device 120, a photometric device 130, and a data processing device 140.

[0054] The sample preparation device 110 is used to mix a blood sample to be tested with a reaction reagent in a reaction container, so that the substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample solution to be tested. The reaction reagent includes a capture reagent and a detection reagent with a marker. In some embodiments, the capture reagent may be a magnetic bead reagent for capturing the substance to be tested, and the detection reagent with a marker may be a detection reagent with an enzyme marker, such as alkaline phosphatase.

[0055] The substrate providing device 120 is used to provide a luminescent substrate into the reaction container containing the sample solution to be tested, so that the marker in the sample solution to be tested undergoes a chemiluminescence reaction with the luminescent substrate.

[0056] The photometric device 130 at least includes a photometer, which is used to detect the luminescence signal generated in the chemiluminescence reaction.

[0057] The data processing device 140 is used to obtain the detection result, such as the concentration, of the substance to be tested in the blood sample to be tested according to the luminescence signal and a pre-set calibration model.

[0058] According to the first embodiment of the present disclosure, the first linear detection range of the photometer is [A1, A2] photon counts per second. The first linear detection range represents the range of photon counts output by the photometer. Within this first linear detection range, the intensity of the luminescence signal generated in the chemiluminescence reaction is linearly related to the photon counts output by the photometer, where A1 is less than or equal to 2000 and A2 is greater than or equal to 10 8 The second linear detection range of the luminescent substrate is [B1, B2] photon counts per second. The second linear detection range represents the range of the intensity of the luminescence signal generated in the chemiluminescence reaction. Within this second linear detection range, the number of markers participating in the chemiluminescence reaction is linearly related to the intensity of the luminescence signal generated in the chemiluminescence reaction, where B1 is less than or equal to 3000 and B2 is greater than or equal to 10 8

[0059] ​According to the second embodiment of the present disclosure, the first linear detection range of the photometer is [A1, A2] photon counts per second, and the first linear detection range represents the range of photon counts output by the photometer. Within this first linear detection range, the intensity of the luminescence signal generated in the chemiluminescence reaction is linearly related to the photon counts output by the photometer, where the ratio A2 / A1 of A2 to A1 is greater than or equal to 30,000; the second linear detection range of the luminescent substrate is [B1, B2] photon counts per second, and the second linear detection range represents the range of the intensity of the luminescence signal generated in the chemiluminescence reaction. Within this second linear detection range, the number of markers participating in the chemiluminescence reaction is linearly related to the intensity of the luminescence signal generated in the chemiluminescence reaction, where the ratio B2 / B1 of B2 to B1 is greater than or equal to 30,000.

[0060] Here, by designing the photometer and the luminescent substrate as in the first embodiment of the present disclosure or according to the first embodiment of the present disclosure, a chemiluminescence analyzer with a wide linear detection range can be obtained. In particular, a chemiluminescence analyzer with a linear detection range of [3000, 10 8 photon counts per second can be obtained.

[0061] In the embodiments of the present disclosure, the linear detection range refers to the linear detection range of a single detection, that is, the linear detection range that can be achieved in a single detection of a sample.

[0062] For example, for the HCG detection item, the chemiluminescence analyzer according to the first and second embodiments of the present disclosure can accurately and linearly detect HCG as low as 2 mIU / ml and as high as 200,000 mIU / ml without the need for dilution and retesting. That is, it is possible to accurately obtain HCG with a concentration between 2 and 200,000 mIU / ml through a single test, meeting the main requirements of clinical use of HCG for assisting in judging pregnancy and monitoring pregnancy status, avoiding redilution and retesting when the HCG concentration in the sample exceeds the detection upper limit, greatly improving the work efficiency of the laboratory department and saving costs. As Figure 2 and Figure 3 shown, for the HCG detection item, within the HCG concentration range of 2 to 200,000 mIU / ml, the calibration model or the calibration curve has good linearity in the low HCG concentration range and also has good linearity throughout the HCG concentration range.

[0063] Figure 4 The structural schematic diagram of a chemiluminescence analyzer 100 provided by the embodiments of the present disclosure is shown. As Figure 4As shown, the sample preparation device 110 includes a sample supply unit 111, a reagent supply unit 112, and a reaction incubation unit 113. The sample supply unit 111 is configured to aspirate a blood sample to be tested and supply it into a reaction vessel. The reagent supply unit 112 is configured to capture a reagent and a detection reagent with a marker and supply them into the reaction vessel. The reaction incubation unit 113 is configured to provide a place for reaction and incubation for the reaction vessel containing the blood sample to be tested and the reaction reagent, so that the blood sample to be tested and the reaction reagent in the reaction vessel form a sample solution to be tested.

[0064] The chemiluminescence analyzer 100 further includes a magnetic separation device 140 and a transfer device 150. The magnetic separation device 140 is configured to perform a magnetic separation operation on the reaction vessel containing the sample solution to be tested. A substrate supply device 120 (not shown) is configured to supply a luminescent substrate into the reaction vessel that has completed the magnetic separation operation. The transfer device 150 is configured to transfer the reaction vessel that has completed the magnetic separation operation from the magnetic separation device 140 to the reaction incubation unit 113. The incubated reaction vessel is transferred to the photometric device 130 for photometry.

[0065] In some embodiments, the chemiluminescence analyzer 100 further includes a sample processing system (not shown) for transporting a test tube containing a blood sample to be tested to a sampling position and transporting the test tube after sampling to a recovery area. Specifically, the sample processing system includes a sample loading area and a transport track. The sample loading area is used to store the test tubes containing the blood samples to be tested placed by the user, and the transport track is used to transport the test tubes in the sample loading area to the sampling position.

[0066] In addition, the sample processing system further includes a scanning module for scanning the sample barcode to achieve sample identification and management.

[0067] In some embodiments, the sample supply unit 111 may include a sample needle and a driving mechanism. The driving mechanism is used to drive the sample needle to move between the sampling position and the sample adding position. The sample needle aspirates the sample in the sample tube at the sampling position and injects the aspirated sample into the reaction vessel at the sample adding position. Here, the sampling position is, for example, the intersection of the movement track of the sample needle and the sample transport track of the transport track.

[0068] In addition, the sample supply unit 111 further includes a cleaning mechanism for cleaning the sample needle. Further, the sample supply unit 111 further includes a dilution position for placing the reaction vessel that has completed automatic dilution and pretreatment, so that the sample needle can aspirate the diluted sample or the pretreated sample in the reaction vessel at the dilution position to support the corresponding test mode.

[0069] In some embodiments, the chemiluminescence analyzer further includes a first robotic arm 160 for transporting reaction vessels. The first robotic arm is configured to move in three dimensions and is capable of gripping reaction vessels. The first robotic arm is configured to load a new reaction vessel into the sample addition position for the sample needle to inject the sample. The first robotic arm is also configured to discard empty reaction cups. The first robotic arm is further configured to transport reaction vessels between the sample addition position, the reaction incubation section 113, and the dilution position.

[0070] In some embodiments, the reagent supply section 112 includes a reagent tray 114 and a reagent needle 115. The reagent tray 114 is used to place reagent bottles containing capture reagents and detection reagents with markers and has the function of refrigerating reagent bottles. The reagent tray 114 is configured to be rotatable so as to convey the reagent bottle that needs to aspirate the reagent to the reagent aspiration position. The reagent needle 115 is used to aspirate the reagent from the reagent bottle located at the reagent aspiration position and discharge the aspirated reagent into the reaction vessel located at the sample addition position.

[0071] Further, the reagent tray has a mixing mechanism (not shown) for mixing magnetic bead reagents. This mixing mechanism realizes the mixing of capture reagents, for example, by rotating the reagent bottle containing capture reagents such as magnetic bead reagents.

[0072] Further, the reagent supply section 112 also includes a cleaning mechanism (not shown) for cleaning the reagent needle.

[0073] In some embodiments, the magnetic separation device 140 has a disc-shaped rotatable magnetic separation disc, a magnetic mechanism, and a temperature control mechanism. The magnetic separation disc is used to drive the reaction vessels therein to sequentially pass through the magnetic mechanism for magnetic separation operations. The temperature control mechanism is used to maintain the temperature of the magnetic separation disc within a preset range.

[0074] In some embodiments, the substrate providing device has a heating mechanism for preheating the luminescent substrate. The substrate providing device is used to inject the preheated luminescent substrate into the reaction vessel that has completed magnetic separation and is located at the substrate injection position, where the substrate injection position is located on the magnetic separation disc.

[0075] In some embodiments, the reaction incubation section 113 includes a disc-shaped rotatable reaction disc. The photometric device 130 is arranged on the outer peripheral side of the reaction disc. The reaction vessel after injecting the luminescent substrate is transported into the reaction disc by the transport device 150. The reaction disc drives the reaction vessel that has been incubated for a certain time into the photometric device 130 to complete the light measurement.

[0076] In some embodiments, the transport device 150 is configured as a second robotic arm. The second robotic arm is used to grip the reaction vessel and transport the reaction vessel between the reaction disc and the magnetic separation disc. The second robotic arm is configured to be capable of horizontal rotation and two-dimensional up-and-down movement.

[0077] In some embodiments, the chemiluminescence analyzer may further include a mixer 170 disposed between the reaction disk and the magnetic separation disk. The mixer is configured to mix the reaction vessel that has already been added with the reagent and the sample. The mixer may be configured, for example, as a vortex mixer that achieves non-contact mixing, which can effectively avoid cross-contamination.

[0078] In addition, the chemiluminescence analyzer may further include a hardware control system (not shown) and software systems running on a hardware board and a PC, respectively, to control the above-mentioned various devices of the chemiluminescence immunoassay analyzer to work in coordination. The chemiluminescence analyzer may further include a mechanical system (not shown) composed of a frame, a front shell, etc., to provide support, positioning, and protection for the above-mentioned various devices.

[0079] In some embodiments, the data processing device 140 includes, but is not limited to, a Central Processing Unit (CPU), a Micro Controller Unit (MCU), a Field-Programmable Gate Array (FPGA), a Digital Signal Processing (DSP) device, etc., which are used to interpret computer instructions and process data in computer software. For example, the data processing device is used to execute each computer application program in a computer-readable storage medium, so that the chemiluminescence analyzer 100 executes the corresponding detection process and analyzes in real time the luminescence signal detected by the photometry device 130.

[0080] A specific detection process of the chemiluminescence analyzer is as follows: The sample supply unit 111 aspirates the blood sample to be tested and adds it to the reaction vessel; the reagent supply unit 112 aspirates the capture reagent and the detection reagent and adds them to the reaction vessel that has already been added with the sample for mixing with the sample; then the reaction vessel is placed in the reaction incubation unit 113 for reaction, incubation, and magnetic separation and washing; then the substrate providing device adds a luminescent substrate to the reaction cup that has completed the reaction, incubation, and magnetic separation and washing, and incubates for a period of time; finally, the photometry device 130 detects the photons emitted by the analyte in the sample to be tested under the action of the luminescent substrate, so as to calculate the concentration level of the analyte through the measured number of photons.

[0081] In some embodiments, the lower limit B1 of the second linear detection range is less than or equal to 2000. Thereby, the sensitivity of the chemiluminescence analyzer can be further improved, and thus the linear detection range of a single detection of the chemiluminescence analyzer can be further broadened.

[0082] In some embodiments, the substance to be measured is human chorionic gonadotropin (HCG). At this time, the chemiluminescence analyzer 100 can be designed such that when the concentration of human chorionic gonadotropin in the blood sample to be measured is greater than or equal to 200,000 mIU / ml, the intensity of the luminescence signal generated in the chemiluminescence reaction is less than or equal to the smaller value of A2 and B2. Thus, it is possible to accurately detect HCG concentrations up to 200,000 mIU / ml.

[0083] In some embodiments, the substance to be measured is human chorionic gonadotropin (HCG). At this time, the chemiluminescence analyzer 100 can be designed such that when the concentration of human chorionic gonadotropin in the blood sample to be measured is less than or equal to 0.5 mIU / ml, the intensity C1 of the luminescence signal generated in the chemiluminescence reaction is greater than or equal to the larger value of A1 and B1. Thus, it is possible to accurately detect HCG concentrations as low as 0.5 mIU / ml.

[0084] Furthermore, the chemiluminescence analyzer 100 can be designed such that when the concentration of human chorionic gonadotropin in the blood sample to be measured is greater than 0.5 mIU / ml and less than or equal to 5 mIU / ml, preferably less than or equal to 2 mIU / ml, the intensity of the luminescence signal generated in the chemiluminescence reaction is C2, where the ratio C2 / C1 of C2 to C1 is greater than 2.

[0085] In some embodiments, the upper limit B2 of the second linear detection range is greater than the upper limit A2 of the first linear detection range.

[0086] In some embodiments, the sample preparation device 110 is further configured to: for all blood samples to be measured containing human chorionic gonadotropin as the substance to be measured, pre-dilute the blood samples to be measured before mixing the blood samples to be measured with the reaction reagent, for example, pre-dilute by 2 times, 5 times or 10 times. Thus, the re-inspection rate of high HCG concentration samples can be further reduced.

[0087] Furthermore, the calibration model can be obtained by the chemiluminescence analyzer 100 using pre-diluted calibration products. The pre-dilution multiple of the blood sample to be measured is preferably equal to the pre-dilution multiple of the calibration product.

[0088] Here, calibration is performed using pre-diluted calibration products, that is, the pre-diluted calibration products are directly traced back to the reference substance, and a gradient concentration response curve of the pre-diluted calibration products is established, so as to eliminate systematic errors caused by random deviations and matrix effects during the dilution process itself, and improve the accuracy of measurement results.

[0089] In some embodiments in China, the calibration model or calibration curve is obtained by multi-point calibration.

[0090] In the embodiments of the present disclosure, the multi-point calibration method may be as follows: using a series of samples (greater than or equal to 5 samples) pre-assigned by the supplier of the chemiluminescence analyzer, measuring the luminescence signal values of this series of samples on the chemiluminescence analyzer, establishing a curve of sample concentration and luminescence signal, and performing 4PLC fitting regression on this curve to obtain a fitting equation, that is, a calibration model.

[0091] It can be understood that the luminescence signal value measured for the blood sample to be tested on the chemiluminescence analyzer is substituted into the calibration model to calculate the concentration of the substance to be tested in the blood sample to be tested.

[0092] In some embodiments, the calibration model is obtained through the following formula

[0093]

[0094] Wherein, RLU is the reactivity, that is, the measured luminescence signal value, C is the concentration of the substance to be measured, and P1 to P4 are 4 fitting parameters. Among them, P1 represents the luminescence signal corresponding to zero concentration (asymptote), P4 represents the luminescence signal corresponding to the maximum concentration (asymptote), P2 represents the inflection point where the slope direction of the curve changes, and P3 reflects the slope at P2 (non-real slope).

[0095] It can be understood here that the 4PLC calibration model uses at least 5 (the number of fitting parameters + 1) points for 4PLC regression, and obtains the optimal solution through a numerical iteration process to obtain four fitting parameters P1, P2, P3, and P4.

[0096] In some embodiments of the present disclosure, the calibration model is recalibrated when the reagent batch is changed, or recalibrated at fixed intervals of days, or recalibrated when the instrument quality control gets out of control.

[0097] Next, some embodiments of the luminescent substrate used in the chemiluminescence analyzer of the present disclosure will be described, but the present disclosure is not limited thereto.

[0098] In some embodiments, the luminescent substrate is designed such that the background of the luminescent substrate is less than 3000. Thereby, the sensitivity of the chemiluminescence analyzer can be further improved.

[0099] Alternatively or additionally, the luminescent substrate may be designed such that the signal-to-noise ratio of the luminescent substrate is greater than 40000. Thereby, the sensitivity of the chemiluminescence analyzer can be further improved.

[0100] In some embodiments, the luminescent substrate includes a chemiluminescent substrate and a chemiluminescent enhancer. The chemiluminescent enhancer includes a fluorescent agent and a surfactant. The fluorescent agent includes one or more carboxyfluoresceins. Using such a luminescent substrate can achieve high sensitivity of the chemiluminescent analyzer.

[0101] As some implementation manners, the luminescent substrate may be selected from dioxetane compounds, preferably selected from one or more of AMPPD, CSPD, CDP-STAR, and more preferably CDP-STAR.

[0102] As some implementation manners, the luminescent substrate may be selected from chloro derivatives of AMPPD, fluorescein or its carboxyl-substituted derivatives, and water-soluble polymeric quaternary ammonium salt cationic surfactants.

[0103] The chloro derivative of AMPPD may be, for example, ADP-STAR or CDP-STAR. Alternatively, the fluorescein or its carboxyl-substituted derivative may be, for example, fluorescein or 5(6)-carboxyfluorescein. Alternatively, the water-soluble polymeric quaternary ammonium salt cationic surfactant may be, for example, polyvinylbenzyltrimethylammonium chloride.

[0104] In some embodiments, the substrate providing device is used to provide a chemiluminescent substrate solution containing a luminescent substrate or a chemiluminescent substrate. The chemiluminescent substrate solution includes a chemiluminescent substrate, fluorescein, and a water-soluble polymeric quaternary ammonium salt. Among them, the chemiluminescent substrate is selected from chloro-substituted dioxetane compounds having a spiro-adamantyl substituent.

[0105] A chemiluminescent substrate refers to a compound that participates in energy transfer in a chemiluminescent reaction and finally releases energy in the form of emitting photons, and is also called a chemiluminescent agent or a luminescent substrate. Dioxetane compounds are enzyme-catalyzed glow-type chemiluminescent substrates and are a class of substrates for ultrasensitive alkaline phosphatase (AP). In a suitable buffer solution, with the catalytic hydrolysis of alkaline phosphatase, the signal emitted by the decomposition of dioxetane compounds can last for more than 20 hours, which is an ideal chemiluminescent substance.

[0106] The inventors of the present invention found that a chloro-substituted dioxetane compound having a spiro-adamantyl substituent, in combination with a specific chemiluminescent enhancer, such as fluorescein or its carboxylic acid derivative, in the presence of a water-soluble polymeric quaternary ammonium salt cationic surfactant, can obtain a chemiluminescent substrate solution in which the ratio of the photon count per second of the upper detection limit to the lower detection limit reaches more than 30,000, and even more than 50,000. Thus, it can be conveniently applied to immunoassays in which the concentration span of analytes in samples such as HCG is as high as 10 5 ~10 6 orders of magnitude.

[0107] The "dioxetane compounds with spiro - adamantyl substituents" mentioned in this article refer to a class of compounds based on the following structure:

[0108]

[0109] For example, it includes the following compounds, but is not limited to:

[0110] AMPPD - (3-(2'-spiroadamatane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane, CAS = 122341-56-4),

[0111] CSPD - (3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane, CAS = 142456-88-0),

[0112] ADP-STAR - (3-(2'-spiroadamatane)-4-methoxy-4-(3"-phosphoryloxy-4"-chloro)-phenyl-1,2-dioxetane, CAS = 189942-84-5),

[0113] CDP-STA - (3-(2'-(spiro-5-chloroadamantane))-4-methoxy-4-(3"-phosphoryloxy-4"-chloro)-phenyl-1,2-dioxetane, CAS = 160081-62-9),

[0114] TFE-AMPPD - (3-(2'-spiroadamatane)-4-trifluoroethoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxetane).

[0115] The "chlorinated dioxetane compounds with spiro - adamantyl substituents" mentioned in this article refer to those containing at least one chlorine atom in the structure of AMPPD.

[0116] In some embodiments, the chlorinated dioxetane compounds with spiro - adamantyl substituents are one of ADP-STAR and CDP-STAR.

[0117]

[0118] In a specific embodiment, in the chemiluminescent substrate solution, the chemiluminescent substrate may exist in the form of a salt of the above compound. According to some embodiments, the salt may be an alkali metal salt, such as sodium salt.

[0119] These dioxetane compounds having a chloro group, especially ADP-STAR and CDP-STAR, in combination with fluorescein or its carboxyl derivatives and a water-soluble polymeric quaternary ammonium salt in an alkaline phosphatase catalytic system, unexpectedly can provide a sufficiently low sensitivity, and at the same time can maintain linearity within a relatively high luminescence value range.

[0120] The fluorescent agent and the surfactant can improve the chemiluminescence efficiency. Among them, the surfactant forms micelles in the solution to protect the chemiluminescent substrate, thereby reducing the quenching reaction of the chemiluminescent substrate in the aqueous solution. The fluorescent agent, as a photon acceptor, receives the photon energy in the system through the energy transfer effect and is excited to generate a light signal, thereby enhancing the luminescence efficiency.

[0121] In the chemiluminescent substrate solution of the present disclosure, the fluorescent agent is at least one of fluorescein and carboxyl-substituted fluorescein.

[0122] The carboxyl-substituted fluorescein includes compounds selected from those shown in General Formula I:

[0123]

[0124] Specifically, the carboxyfluorescein shown in General Formula I may be 5-carboxyfluorescein, 6-carboxyfluorescein, or 5(6)-carboxyfluorescein. Among them,

[0125] The 5-carboxyfluorescein is

[0126]

[0127] The 6-carboxyfluorescein is

[0128]

[0129] The 5(6)-carboxyfluorescein is a mixture of the 5-carboxyfluorescein and the 6-carboxyfluorescein in any ratio.

[0130] In the chemiluminescent substrate solution, the fluorescent agent is fluorescein. In other embodiments, the fluorescent agent may be any carboxyl-substituted fluorescein, especially one of 5-carboxyfluorescein, 6-carboxyfluorescein, and 5(6)-carboxyfluorescein.

[0131] Fluorescein and its carboxyl derivatives can improve the chemiluminescence efficiency of the chemiluminescence system. In addition to effectively improving the chemiluminescence efficiency of the chemiluminescence system, the above carboxyl derivatives can also reduce the time required to reach the plateau period and improve the sensitivity.

[0132] In the chemiluminescent substrate solution, the surfactant is a water-soluble polymer quaternary ammonium salt cationic surfactant. According to one embodiment, the water-soluble polymer quaternary ammonium salt is selected from quaternary ammonium salts containing vinyl groups. Such quaternary ammonium salts are, for example, vinylbenzyl-trialkylammonium halides. The three alkyl groups substituted on the N atom can be the same or different, and can be independently selected from, for example, methyl, ethyl, propyl, butyl, pentyl, benzyl, etc. In a specific embodiment, the water-soluble polymer quaternary ammonium salt is selected from at least one of vinylbenzyl-trimethylammonium chloride, vinylbenzyl-benzyldimethylammonium chloride, and vinylbenzyl-tributylammonium chloride.

[0133] The water-soluble polymer quaternary ammonium salt cationic surfactant can also form micelles in an aqueous solution. The chemiluminescent substrate is inside the hydrophobic micelles, and the fluorescent agent is in the hydrophilic part outside the micelles, which is more conducive to the chemiluminescent substrate transferring energy to the fluorescent agent to enhance the light signal. Compared with small molecule quaternary ammonium salts, due to the longer alkane chain of the polymer quaternary ammonium salt, it can more effectively prevent the chemiluminescent substrate from quenching in an aqueous solution. In addition, the polymer quaternary ammonium salt can also suppress the background signal and enhance the detection signal. For small molecule quaternary ammonium salts under the same conditions, the background signal value is high and the detection signal is low, and a wide linear detection range cannot be achieved.

[0134] In a specific embodiment, the chemiluminescent substrate solution includes ADP-STAR or CDP-STAR as the chemiluminescent substrate, fluorescein, and a water-soluble polymer quaternary ammonium salt.

[0135] In another specific embodiment, the chemiluminescent substrate solution includes ADP-STAR, fluorescein or carboxyl-substituted fluorescein as the fluorescent agent, and a water-soluble polymer quaternary ammonium salt.

[0136] The specific combination of the above-mentioned chemiluminescent substrate, fluorescent agent and surfactant provides a chemiluminescent substrate solution with a wide linear luminescence value. The chemiluminescent substrate solution can obtain a linear detection range with a lower limit B1 of less than 3,000 photon counts per second and an upper limit B2 of more than 100,000,000 (100M) photon counts per second in an alkaline phosphatase system. Exemplarily, the chemiluminescent substrate solution can obtain a linear detection range with a lower limit B1 of 1,000 to 3,000 photon counts per second and an upper limit B2 of 100,000,000 (100M) to 200,000,000 (200M) photon counts per second in an alkaline phosphatase system. As shown in the following examples, the chemiluminescent substrate solution can obtain a background luminescence value of 1,000 to 2,000 photon counts per second and a linear detection range with an upper limit B2 of 120,000,000 (120M) to 200,000,000 (200M) photon counts per second in an alkaline phosphatase system. It should be understood that the above lower limit range and upper limit range are only exemplary. By adjusting the detection device or signal processing method, etc., a lower lower limit of photon counts per second or a higher upper limit of photon counts per second can be obtained.

[0137] In some embodiments, the ratio of the photon counts per second of the detection upper limit B2 to the detection lower limit B1 of the chemiluminescent substrate solution is more than 30,000, for example, more than 50,000, and even more than 60,000. In some embodiments, the ratio of the photon counts per second of the detection upper limit B2 to the detection lower limit B1 of the chemiluminescent substrate solution is less than 1,000,000, for example, less than 800,000, less than 600,000, less than 500,000, and even less than 400,000. Exemplarily, the ratio of the photon counts per second of the detection upper limit B2 to the detection lower limit B1 of the chemiluminescent substrate solution is 30,000 to 1,000,000, 30,000 to 800,000, 30,000 to 500,000, 50,000 to 1,000,000, 50,000 to 800,000, 50,000 to 500,000, etc.

[0138] In some embodiments, the chemiluminescent substrate solution contains 50 to 500 mg / L of the chemiluminescent substrate. Preferably, the chemiluminescent substrate solution contains 150 to 250 mg / L of the chemiluminescent substrate. More preferably, it contains 200 mg / L of the chemiluminescent substrate. Exemplarily, the chemiluminescent substrate solution contains 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L of the chemiluminescent substrate.

[0139] In some embodiments, the chemiluminescent substrate solution comprises 30 to 500 mg / L of a fluorescent agent. Preferably, the chemiluminescent substrate solution comprises 150 to 250 mg / L of a fluorescent agent, and more preferably, 200 mg / L of a fluorescent agent. Exemplarily, the chemiluminescent substrate solution comprises 150 mg / L, 160 mg / L, 170 mg / L, 180 mg / L, 190 mg / L, 200 mg / L, 210 mg / L, 220 mg / L, 230 mg / L, 240 mg / L, 250 mg / L of a fluorescent agent.

[0140] In some embodiments, the chemiluminescent substrate solution comprises 1 to 10 g / L of a water-soluble polymeric quaternary ammonium salt. Preferably, the chemiluminescent substrate solution comprises 3 to 7 g / L of a water-soluble polymeric quaternary ammonium salt, and more preferably, 5 g / L of a water-soluble polymeric quaternary ammonium salt. Exemplarily, the chemiluminescent substrate solution comprises 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L of a water-soluble polymeric quaternary ammonium salt.

[0141] In a specific embodiment, the chemiluminescent substrate solution comprises 50 - 500 mg / L, preferably 150 - 250 mg / L of ADP-STAR or CDP-STAR, 30 - 500 mg / L, preferably 150 - 250 mg / L of fluorescein, and 1 - 10 g / L, preferably 3 - 7 g / L of a water-soluble polymeric quaternary ammonium salt.

[0142] In another specific embodiment, the chemiluminescent substrate solution comprises 50 - 500 mg / L, preferably 150 - 250 mg / L of ADP-STAR, 30 - 500 mg / L, preferably 150 - 250 mg / L of fluorescein or carboxyl-substituted fluorescein, and 1 - 10 g / L, preferably 3 - 7 g / L of a water-soluble polymeric quaternary ammonium salt.

[0143] The chemiluminescent substrate solution further contains additives such as a buffer and a preservative.

[0144] The present disclosure does not particularly limit the type of buffer, and any buffer conventionally applicable can be used in the chemiluminescent substrate solution of the present disclosure. In some embodiments, the buffer can be selected from Tris buffer, AMP buffer (AMP is 2-amino-2-methylpropanol), AMPD buffer (AMPD is 2-amino-2-methyl-1,3-propanediol), DEA buffer (DEA is diethanolamine), CHES buffer (CHES is 2-(N-cyclohexylamino)ethanesulfonic acid), Mopso buffer, imidazole buffer, phosphate buffer, carbonate buffer, malic acid buffer, glycine buffer, but not limited thereto. Exemplarily, the buffer can be selected from at least one of Tris-HCl, AMP-HCl, AMPD-HCl, DEA-HCl, CHES-HCl, boric acid-NaOH, glycine-NaOH buffer systems. Preferably, the buffer solution is selected from the AMP-HCl buffer system. The AMP-HCl buffer system can play a better buffering role in the range of pH 9.0 to 10.0, and it is found that it is beneficial to the stability of chemiluminescent substrate molecules.

[0145] According to some embodiments, the pH of the buffer is 7.1 to 10.6, preferably about 9.0 to about 10.0, and particularly preferably about 9.5.

[0146] The present disclosure does not particularly limit the amount of the buffer, which can be determined according to the selected buffer system.

[0147] The present disclosure does not particularly limit the type of preservative, and any preservative conventionally used in detection reagents can be used in the present disclosure. Examples include, but are not limited to, sodium azide, Proclin series, potassium sorbate, sodium benzoate, BND, erythromycin, gentamicin, etc. Preferably, they are sodium azide and Proclin series.

[0148] The present disclosure does not particularly limit the amount of the buffer, which can be determined according to the selected type of preservative.

[0149] In some embodiments, the chemiluminescent substrate solution may further include magnesium chloride. For example, it may include 0.1 to 2 mM of magnesium chloride.

[0150] According to a specific embodiment, the chemiluminescent substrate solution comprises ADP-STAR or CDP-STAR at 50-500 mg / L, preferably 150-250 mg / L; fluorescein at 30-500 mg / L, preferably 150-250 mg / L; a water-soluble polymeric quaternary ammonium salt at 1-10 g / L, preferably 3-7 g / L; an appropriate amount of (such as 30-500 mM) AMP-HCl buffer; an appropriate amount of (such as 0.5-2 g / L) sodium azide, with a pH of about 9.0 to about 10.0, such as about 9.5.

[0151] In another specific embodiment, the chemiluminescent substrate solution comprises ADP-STAR at 50-500 mg / L, preferably 150-250 mg / L; fluorescein or carboxyl-substituted fluorescein at 30-500 mg / L, preferably 150-250 mg / L; a water-soluble polymeric quaternary ammonium salt at 1-10 g / L, preferably 3-7 g / L; an appropriate amount of (such as 30-500 mM) AMP-HCl buffer; an appropriate amount of (such as 0.5-2 g / L) sodium azide, with a pH of about 9.0 to about 10.0, such as about 9.5.

[0152] According to some embodiments, the chemiluminescent substrate solution of the present disclosure has a low background luminescence value, a high luminescence efficiency, a wide linear detection range, is suitable for an alkaline phosphatase-catalyzed luminescence system, and is particularly suitable for chemiluminescent immunoassay.

[0153] According to some specific embodiments, the chemiluminescent substrate solution provided by the present disclosure reacts with alkaline phosphatase in a concentration range of 0.03-400 ng / mL, and the luminescence value shows good linearity (R 2 ≥0.99). As shown in the examples, the background value of the chemiluminescent substrate solution can be as low as below 3,000 photon counts per second, and it can detect an ultra-low concentration of alkaline phosphatase as low as 10-19 mol / L, improving the detection sensitivity; at the same time, the detection value of the chemiluminescent substrate solution can be as high as above 100 M photon counts per second, such as 100 M-200 M photon counts per second, 120 M-200 M photon counts per second, showing a significantly wider linearity. Without additional dilution of the sample, the above chemiluminescent substrate solution can meet the detection of samples with a high analyte concentration.

[0154] The chemiluminescent substrate solution of the present disclosure is particularly suitable for detecting human chorionic gonadotropin HCG in a test sample.

[0155] HCG is a glycoprotein mainly secreted by placental trophoblast cells. Its content is very low in non-pregnant people, usually less than 3 mIU / ml, while during pregnancy, the HCG concentration can be as high as about 2,000,000 mIU / ml. Clinically, the HCG test results are used to assist in judging pregnancy. At the same time, the detection of HCG during pregnancy also has important significance. As mentioned above, the chemiluminescent substrate solution of the present disclosure has a wide linear luminescence characteristic and is suitable for samples with a large span of analyte concentration changes such as HCG.

[0156] According to some embodiments, the concentration range of HCG in the sample is 1 - 200,000 mIU / mL.

[0157] In some embodiments, the detection of HCG is by the double antibody sandwich method. According to specific embodiments, superparamagnetic microparticles coated with HCG antibody are used as the capture reagent in the detection, and an HCG antibody labeled with alkaline phosphatase is used as the label. In some embodiments, the HCG antibody is a monoclonal antibody.

[0158] The chemiluminescent substrate solution of the present disclosure combined with the double antibody sandwich method has high sensitivity and a wide linear detection range for HCG detection.

[0159] In the present disclosure, for the use of the chemiluminescent substrate solution in the immunoassay of HCG, there are no special restrictions on the specific forms and dosages of corresponding immunoassay reagents such as HCG antibody, specific capture reagent, alkaline phosphatase label, etc., and any suitable reagents can be used.

[0160] The "sample" mentioned herein, unless otherwise specified, refers to a biological sample, which can be from a mammal, preferably a blood sample from a human, and more preferably a serum sample.

[0161] In addition to HCG, the chemiluminescent substrate solution of the present disclosure is also applicable to samples with a large span of other analytes, such as the detection of HBsAg, TSH, etc., and is not limited to the detection of HCG.

[0162] In some embodiments, the chemiluminescent detection method is performed in the chemiluminescent analyzer of the embodiments of the present disclosure using the above chemiluminescent substrate solution. The chemiluminescent detection method includes the following steps:

[0163] Mix the sample to be tested with the detection reagent, where the detection reagent includes a capture reagent that can bind to the analyte and an alkaline phosphatase label, so as to obtain an alkaline phosphatase-labeled immune complex;

[0164] Mix the alkaline phosphatase-labeled immune complex with the chemiluminescent substrate solution in any of the above embodiments to obtain a mixed solution;

[0165] Measure the optical signal of the mixed solution and obtain the analysis result of the sample to be measured based on the optical signal. Some embodiments of the photometer of the chemiluminescence analyzer of the present disclosure will be described next, but the present disclosure is not limited thereto.

[0166] In some embodiments, as Figure 5 shown, the photometer includes a receiving component 10 and a processing component 20 that are electrically connected to each other.

[0167] The receiving component 10 is configured to receive the optical signal generated in the chemiluminescence reaction and convert the optical signal into a corresponding electrical signal. The receiving component 10 can be configured, for example, as a photomultiplier tube that converts a weak optical signal into an electrical signal.

[0168] The processing component 20 is configured to be electrically connected to the receiving component 10 to receive the electrical signal from the receiving component 10, and process the received electrical signal to obtain the number of photons. The processing component 20 includes a first photon counting module 21 and a second photon counting module 22. Among them, the first photon counting module 21 is configured to process the electrical signal using a first photon counting method to obtain a first photon counting result, while the second photon counting module 22 is configured to process the electrical signal using a second photon counting method different from the first photon counting method to obtain a second photon counting result. The processing component 20 is further configured to output the final photon counting result of the sample to be measured based on the first photon counting result and the second photon counting result and use it as the photon counting output by the photometer. This final photon counting result, that is, the photon counting output by the photometer, can subsequently be used to calculate the content of the substance to be measured in the sample to be measured.

[0169] In the embodiments of the present disclosure, two photon counting modules are used to perform photon counting simultaneously. Among them, the first photon counting module uses a first counting method for the weak light segment to obtain a first photon counting result, while the second photon counting module uses a second counting method for the strong light segment to obtain a second photon counting result. Finally, the first photon counting result and the second photon counting result are integrated and the final counting result is output. Thereby, the linear detection range of the photometer can be greatly expanded. In particular, it can be realized that the linear detection range of the photometer is expanded to at least [2000, 100 million (10 8 )] photon counts / second, and even expanded to [2000, 200 million (10 8 )] photon counts / second.

[0170] In the embodiments of the present disclosure, the low-light section can be understood as an optical signal with a photon count per second not higher than a predetermined threshold, and the high-light section can be understood as an optical signal with a photon count per second higher than the predetermined threshold. For example, the predetermined threshold can be selected from any value between 20 million and 30 million photon counts per second, such as 20 million or 30 million photon counts per second. For example, the first counting method is a photon counting method for optical signals with a photon count not higher than 30 million photon counts per second, and the second counting method is a photon counting method for optical signals with a photon count higher than 30 million photon counts per second.

[0171] In some embodiments, the first counting method can be a pulse recognition method that calculates the photon count by identifying the electrical pulses caused by photons entering the receiving component, while the second counting method does not calculate the photon count by identifying the electrical pulses caused by photons entering the receiving component, but estimates the photon count by processing the electrical signal into a parameter that can characterize the number of photons in the optical signal (i.e., the number of electrical pulses caused by photons entering the receiving component).

[0172] Here, those skilled in the art can understand that in the embodiments of the present disclosure, the pulse recognition method can be understood as a method for calculating the photon count by identifying the pulses in the electrical signal, that is, when the peak value of the pulse identified from the electrical signal is greater than the threshold, it is considered that a photon is identified.

[0173] In some embodiments, the first photon counting module 21 can be configured to detect the number of pulses of the electrical signal using the pulse recognition method to obtain a first photon counting result, while the second photon counting module 22 can be configured to process the electrical signal to obtain a parameter that characterizes the number of photons in the optical signal, and obtain, for example, calculate a second photon counting result based on the parameter characterizing the number of photons and a preset calibration function, where the calibration function represents the mapping relationship between the parameter characterizing the number of photons in the optical signal and the photon counting result.

[0174] In some embodiments, after obtaining the first photon counting result and the second photon counting result, the processing component 20 can be further configured:

[0175] When the first photon counting result is lower than the first threshold, use the first photon counting result as the final photon counting result, that is, the photon count output by the photometer; and

[0176] When the first photon counting result is not lower than the first threshold, use the second photon counting result as the final photon counting result, that is, the photon count output by the photometer.

[0177] In some embodiments, such as Figure 6As shown, the first photon counting module 21 may include a level discrimination circuit 211, a shaping and frequency division circuit 212, and a first counting circuit 213 that are electrically connected to each other. The level discrimination circuit 211 is configured to convert an electrical signal into a square wave signal. The shaping and frequency division circuit 212 is configured to perform frequency division and shaping on the square wave signal. The first counting circuit 213 is configured to count the signal output by the shaping and frequency division circuit 212, that is, identify the number of pulses, to obtain a first photon counting result. The first photon counting module 21 according to this example can achieve a linear detection range of 2,000 to 30 million photon counts per second.

[0178] In some alternative embodiments, as Figure 7 shown, the first photon counting module 21 may include at least one first AD conversion circuit 214 and a first counting circuit 213 that are electrically connected to each other. The first AD conversion circuit 214 is configured to collect an electrical signal at a sampling frequency greater than 1 GHz / s and convert the collected electrical signal into a digital signal and output it to the first counting circuit 213. The first counting circuit 213 is configured to analyze the received digital signal to identify the number of pulses, and then obtain a first photon counting result. Thus, through high-speed sampling at a sampling frequency of 1 GHz / s, the first counting circuit 213 can more accurately identify the number of pulses in the electrical signal, and then obtain a more accurate first photon counting result. Compared with Figure 6 the embodiment shown, the first photon counting module 21 according to this embodiment can achieve a linear detection range of 2,000 to 80 million photon counts per second.

[0179] In Figure 7 some modification schemes of the embodiment shown, the first AD conversion circuit may be configured to collect an electrical signal at a sampling frequency not less than 2 GHz / s. Thereby, the linear detection range of the first photon counting module 21 can be further broadened.

[0180] In Figure 7 some modification schemes of the embodiment shown, the first photon counting module 21 may include at least two, especially at least four, first AD conversion circuits 214. The sum of the sampling frequencies of each first AD conversion circuit is not less than 1 GHz / s, and each first AD conversion circuit collects the electrical signal by means of asynchronous sampling at a preset interval time. By using multiple first AD conversion circuits to achieve a sampling frequency not less than 1 GHz / s, the performance requirements for a single AD conversion circuit can be reduced, and thus the cost can be reduced.

[0181] Preferably, these first AD conversion circuits 214 have the same sampling frequency, and each first AD conversion circuit collects the electrical signal by means of asynchronous sampling at equal time intervals.

[0182] Accordingly, the first counting circuit 213 may be configured to rearrange and combine the data collected by the above-mentioned plurality of first AD conversion circuits 214, perform filtering processing, and then perform pulse identification on the filtered signal to obtain a first photon counting result.

[0183] Furthermore, the first counting circuit 213 may also be configured to correct the first photon counting result through a Poisson distribution compensation algorithm. Thereby, a more accurate photon counting result can be obtained.

[0184] In some embodiments, the first counting circuit 213 may include an FPGA chip and its surrounding circuits.

[0185] In some embodiments, the second photon counting module 22 may be configured to process the electrical signal, for example, obtain a DC component signal through integration processing. The parameter characterizing the number of photons in the optical signal includes a parameter related to the DC component signal, such as the DC component signal itself per unit time. The second photon counting module 22 is further configured to obtain a second photon counting result according to the parameter related to the DC component signal and a preset calibration function.

[0186] Alternatively or additionally, the second photon counting module 22 is configured to integrate the electrical signal within a predetermined time period to obtain an integration result, such as a DC component signal. The parameter characterizing the number of photons in the optical signal includes a parameter related to the integration result, such as the integration result per unit time. The second photon counting module 22 is further configured to obtain a second photon counting result according to the parameter related to the integration result and a preset calibration function.

[0187] In a specific example, as Figure 8 and Figure 9 shown, the second photon counting module 22 includes an integration circuit 221 and a second counting circuit 222 that are electrically connected to each other. The integration circuit 221 is configured to integrate the electrical signal within a predetermined time period to obtain a DC component signal. The parameter characterizing the number of photons in the optical signal includes a parameter related to the DC component signal. The second counting circuit 222 is configured to obtain a second photon counting result according to the parameter related to the DC component signal and a preset calibration function.

[0188] Furthermore, as Figure 10 shown, an AD conversion circuit 224 is further provided between the integration circuit 221 and the second counting circuit 222. The AD conversion circuit 224 is configured to convert the DC component signal output by the integration circuit 221 into a digital signal. The sampling frequency of the AD conversion circuit 224 may be set to less than 1 MHz / s, for example, several hundred kHz / s.

[0189] In some embodiments, the second counting circuit 222 may include an FPGA chip and its surrounding circuits.

[0190] Preferably, as Figure 11 and Figure 12 shown, the first counting circuit 213 and the second counting circuit 222 are the same counting circuit, and this same counting circuit includes, for example, an FPGA chip. That is to say, the counting functions of the first photon counting module 21 and the second photon counting module 22 are integrated in the same FPGA chip. Namely, on the one hand, this FPGA chip is configured to count the signal output by the shaping and frequency division circuit 212 to obtain the first photon counting result ( Figure 11 ) or analyze the digital signal collected by the first AD conversion circuit 214 to obtain the first photon counting result ( Figure 12 ), and on the other hand, it is configured to obtain the second photon counting result according to the parameter characterizing the number of photons in the optical signal and a preset calibration function.

[0191] In another alternative example, as Figure 13 and Figure 14 shown, the second photon counting module includes a second AD conversion circuit 223 and a second counting circuit 222 that are electrically connected to each other. The second AD conversion circuit is configured to convert the electrical signal output by the receiving component 10 into a digital signal, and the second counting circuit is configured to process the digital signal to obtain a parameter characterizing the number of photons in the optical signal, and obtain the second photon counting result according to the parameter characterizing the number of photons in the optical signal and a preset calibration function.

[0192] Here, a second AD conversion circuit 223 with a sampling frequency less than 10 MHz / s can be adopted. In some embodiments, the sampling frequency of the second AD conversion circuit 223 may be in the range of 1 MHz / s to 10 MHz / s, for example, 1, 2, or 3 MHz / s.

[0193] Preferably, as Figure 15 shown, the first counting circuit 213 and the second counting circuit 222 are the same counting circuit, and this same counting circuit includes, for example, an FPGA chip. That is to say, the counting functions of the first photon counting module 21 and the second photon counting module 22 are integrated in the same chip.

[0194] Also preferably, as Figure 15 shown, the first AD conversion circuit 214 serves as the second AD conversion circuit 223, that is, the first photon counting module 21 and the second photon counting module 22 use the same AD conversion circuit.

[0195] Further, the second counting circuit 222 may be configured to process a digital signal to obtain a DC component signal, and the parameter characterizing the number of photons in the optical signal includes a parameter related to the DC component signal.

[0196] For example, the second counting circuit 222 may be configured to perform a Fourier transform on the digital signal to obtain a DC component signal.

[0197] Alternatively or additionally, the second counting circuit 222 may be configured to integrate or sum the digital signal over a predetermined time period to obtain a parameter related to the integration result or the summation result and use it as the parameter characterizing the number of photons in the optical signal.

[0198] For more embodiments of the photometer of the present disclosure, reference may be made to the applicant's prior Chinese application CN202211510485.X, the content of which is incorporated herein by reference.

[0199] The third embodiment of the present disclosure provides another chemiluminescence analyzer, including: a sample preparation device for mixing a blood sample to be tested with a reaction reagent in a reaction container so that a substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample solution to be tested; a substrate providing device for providing a luminescent substrate into the reaction container containing the sample solution to be tested so that the sample solution to be tested undergoes a chemiluminescence reaction with the luminescent substrate; a photometric device, the photometric device at least includes a photometer for detecting the luminescence signal generated in the chemiluminescence reaction; and a data processing device for obtaining a detection result of the substance to be tested in the blood sample to be tested according to the luminescence signal and a pre-set calibration model.

[0200] Here, the photometer includes a receiving component and a processing component. The receiving component is configured to receive the optical signal generated in the chemiluminescence reaction and convert the optical signal into a corresponding electrical signal. The processing component is configured to be electrically connected to the receiving component and receive the electrical signal from the receiving component. The processing component includes a first photon counting module and a second photon counting module. The first photon counting module is configured to detect the number of pulses of the electrical signal using the pulse identification method to obtain a first photon counting result. The second photon counting module is configured to process the electrical signal to obtain a parameter characterizing the number of photons in the optical signal, and obtain a second photon counting result according to the parameter characterizing the number of photons in the optical signal and a preset calibration function. Wherein, the calibration function represents the mapping relationship between the parameter characterizing the number of photons in the optical signal and the photon counting result. The processing component is further configured to obtain the photon counting output by the photometer based on the first photon counting result and the second photon counting result, and output the photon counting; and the second linear detection range of the luminescent substrate is [B1, B2] photon counts / second. The second linear detection range represents the range of the intensity of the luminescent signal generated in the chemiluminescence reaction. Within this second linear detection range, the number of markers participating in the chemiluminescence reaction is linearly related to the intensity of the luminescent signal generated in the chemiluminescence reaction. Wherein, the ratio of B2 to B1 is greater than or equal to 30,000 or B1 is less than or equal to 3,000 and B2 is greater than or equal to 10 8 。

[0201] For more embodiments and advantages of the chemiluminescence analyzer according to the third embodiment of the present disclosure, reference may be made to the description of the chemiluminescence analyzers according to the first and second embodiments of the present disclosure above.

[0202] The fourth embodiment of the present disclosure provides yet another chemiluminescence analyzer, including: a sample preparation device for mixing a blood sample to be tested with a reaction reagent in a reaction container so that the substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample solution to be tested; a substrate providing device for providing a luminescent substrate to the reaction container containing the sample solution to be tested so that the sample solution to be tested undergoes a chemiluminescence reaction with the luminescent substrate; a photometric device, the photometric device at least includes a photometer for detecting the luminescent signal generated in the chemiluminescence reaction; and a data processing device for obtaining the detection result of the substance to be tested in the blood sample to be tested according to the luminescent signal and a preset calibration model.

[0203] Here, the first linear detection range of the photometer is [A1, A2] photon counts per second. The first linear detection range represents the range of the output photon counts of the photometer. Within this first linear detection range, the intensity of the luminescence signal generated in the chemiluminescence reaction is linearly related to the photon counts output by the photometer. Among them, the ratio of A2 to A1 is greater than or equal to 30,000. Or, A1 is less than or equal to 2,000 and A2 is greater than or equal to 10 8 and the luminescent substrate includes a chemiluminescent substrate and a chemiluminescence enhancer. The chemiluminescence enhancer includes a fluorescent agent and a surfactant. The fluorescent agent includes one or more carboxyfluoresceins.

[0204] For more embodiments and advantages of the chemiluminescence analyzer according to the fourth embodiment of the present disclosure, reference may be made to the descriptions of the chemiluminescence analyzers according to the first and second embodiments of the present disclosure above.

[0205] The features or combinations of features mentioned in the above description, drawings, and claims, as long as they are meaningful within the scope of the present disclosure and do not conflict with each other, can be combined with each other arbitrarily or used alone.

[0206] The following illustrates various embodiments and advantages of the chemiluminescent substrate of the present disclosure through specific examples, but the scope of the present disclosure is not limited by these.

[0207] Example 1: Linear performance of different chemiluminescent substrates

[0208] Chemiluminescent substrate solution formulation:

[0209] Chemiluminescent substrate: 200 mg

[0210] 5(6)-Carboxyfluorescein: 200 mg

[0211] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0212] AMP-HCl: 50 mM

[0213] Magnesium chloride: 200 mg

[0214] Sodium azide: 1 g

[0215] Water: Add to a total volume of 1 L

[0216] According to the above formula, where the chemiluminescent substrates are as shown in Table 1 below, a series of chemiluminescent substrate solutions 1-1 to 1-4 are prepared.

[0217] Table 1

[0218] Chemiluminescent substrate solution Chemiluminescent substrate 1-1 AMPPD 1-2 CSPD 1-3 ADP-STAR 1-4 CDP-STAR

[0219] Using the above chemiluminescent substrate solutions 1-1 to 1-4, tests were carried out on Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Alkaline phosphatase (AP enzyme) solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals were collected, and the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values at a 2-minute light collection time were obtained respectively. The concentration of alkaline phosphatase in the system and the detected signal values are shown in Table 2 below.

[0220] Table 2

[0221]

[0222] The light signals (photon counts per second) measured for the chemiluminescent substrate solutions 1-1 to 1-4 were plotted against the concentration of alkaline phosphatase in the system, and linear fitting was performed.

[0223] According to the data in Table 2 and Figures 16A to 16D It can be seen that the chemiluminescent substrate solutions 1-2, 1-3, and 1-4 have low background signals, while the chemiluminescent substrate solutions 1-3 and 1-4 simultaneously have the characteristics of high luminescence efficiency, and have good linearity (R2≥0.99) within the detected luminescence range, among which ADP-STAR performs the best.

[0224] Example 2: Linear performance of ADP-STAR and different fluorescent agents

[0225] Chemiluminescent substrate solution formula:

[0226] ADP-STAR: 200 mg

[0227] Fluorescent agent: 200 mg

[0228] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0229] AMP-HCl: 50 mM

[0230] Magnesium chloride: 200 mg

[0231] Sodium azide: 1 g

[0232] Water: Add up to a total volume of 1 L

[0233] According to the above formula, with the fluorescent agents as shown in Table 3 below, a series of chemiluminescent substrate solutions 2-1 to 2-4 were prepared.

[0234] Table 3

[0235] Chemiluminescent substrate solution Fluorescent agent 2-1 Fluorescein 2-2 5-Carboxyfluorescein 2-3 6-Carboxyfluorescein 2-4 5(6)-Carboxyfluorescein

[0236] Using the above chemiluminescent substrate solutions 2-1 to 2-4, tests were conducted on the Mindray CL-6000i fully automated chemiluminescent immunoassay analyzer. Alkaline phosphatase solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals were collected, and the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values at a 2-minute light collection time were obtained respectively. The concentrations of alkaline phosphatase in the system and the detected signal values are shown in Table 4 below.

[0237] Table 4

[0238]

[0239]

[0240] The light signals (photon counts per second) measured for the chemiluminescent substrate solutions 2-1 to 2-4 were plotted against the concentration of alkaline phosphatase in the system, and linear fitting was performed.

[0241] According to the data in Table 4 and Figures 17A to 17D it can be seen that the chemiluminescent substrate solutions 2-1 to 2-4 all exhibit the characteristics of low background signal and high luminescence efficiency, and have good linearity (R2≥0.99) within the detected luminescence range.

[0242] Example 3: Linear performance of CDP-STAR and different fluorescent agents

[0243] Prepare the chemiluminescent substrate solution according to the following formula:

[0244] CDP-STAR: 200 mg

[0245] Fluorescent agent: 200 mg

[0246] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0247] AMP-HCl: 50 mM

[0248] Magnesium chloride: 200 mg

[0249] Sodium azide: 1 g

[0250] Water: Add to a total volume of 1 L

[0251] According to the above formula, with the fluorescent agents as shown in Table 5 below, a series of chemiluminescent substrate solutions 3-1 to 3-4 were prepared.

[0252] Table 5

[0253] Chemiluminescent substrate solution Fluorescent agent 3-1 Fluorescein 3-2 5-Carboxyfluorescein 3-3 6-Carboxyfluorescein 3-4 5(6)-Carboxyfluorescein

[0254] Using the above chemiluminescent substrate solutions 3-1 to 3-4, tests were conducted on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Alkaline phosphatase solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals were collected, and the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values at a 2-minute light collection time were obtained respectively. The concentrations of alkaline phosphatase in the system and the detected signal values are shown in Table 6 below.

[0255] Table 6

[0256]

[0257] The light signals (photon counts per second) measured for the chemiluminescent substrate solutions 3-1 to 3-4 were plotted against the concentration of alkaline phosphatase in the system, and linear fitting was performed.

[0258] According to the data in Table 6 and Figures 18A to 18D it can be seen that the chemiluminescent substrate solutions 3-1 to 3-4 all exhibit the characteristics of low background signal and high luminescence efficiency, and have good linearity (R2≥0.99) within the detected luminescence range.

[0259] Example 4: Linear performance of ADP-STAR and different quaternary ammonium salt cationic surfactants

[0260] Chemiluminescent substrate solution formula:

[0261] ADP-STAR: 200 mg

[0262] Fluorescein: 200 mg

[0263] Quaternary ammonium salt surfactant: 5 g

[0264] AMP-HCl: 50 mM

[0265] Magnesium chloride: 200 mg

[0266] Sodium azide: 1 g

[0267] Water: Add up to a total volume of 1 L

[0268] According to the above formula, where the quaternary ammonium salt cationic surfactants are shown in Table 7 below, a series of chemiluminescent substrate solutions 4-1 to 4-4 were prepared.

[0269] Table 7

[0270] Chemiluminescent substrate solution Quaternary ammonium cationic surfactant 4-1 Polyvinylbenzyltrimethylammonium chloride (TMQ) 4-2 Polyvinylbenzyltributylammonium chloride (TBQ) 4-3 Polyvinylbenzyl(benzyldimethyl)ammonium chloride (BDMQ) 4-4 Cetyltrimethylammonium bromide (CTAB)

[0271] Using the above chemiluminescent substrate solutions 4-1 to 4-4, tests were conducted on the Mindray CL-6000i fully automated chemiluminescent immunoassay analyzer. Alkaline phosphatase solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals were collected to obtain the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values under a 2-minute light collection time. The concentrations of alkaline phosphatase in the system and the detection signal values are shown in Table 8 below.

[0272] Table 8

[0273]

[0274]

[0275] The light signals (photon counts / second) measured for the chemiluminescent substrate solutions 4-1 to 4-4 were plotted against the concentration of alkaline phosphatase in the system, and linear fitting was performed.

[0276] According to the data in Table 8 and Figures 19A to 19D it can be seen that the chemiluminescent substrate solutions 4-1 to 4-3 have low background signals and high luminescence efficiencies, and have good linearity (R2≥0.99) within the detected luminescence range. The chemiluminescent substrate solution 4-4 has a high background signal and low luminescence efficiency, cannot maintain good linearity (R2<0.99) within the detected luminescence range, and the ratio of the light signals at the detection upper limit and the detection lower limit is also small (less than 3000).

[0277] Example 5: Linear performance of CDP-STAR and different quaternary ammonium salt cationic surfactants

[0278] Chemiluminescent substrate solution formula:

[0279] CDP-STAR: 200 mg

[0280] Fluorescein: 200 mg

[0281] Quaternary ammonium salt surfactant: 5 g

[0282] AMP-HCl: 50 mM

[0283] Magnesium chloride: 200 mg

[0284] Sodium azide: 1 g

[0285] Water: added to a total volume of 1 L

[0286] According to the above formula, where the quaternary ammonium salt cationic surfactants are shown in Table 9 below, a series of chemiluminescent substrate solutions 5-1 to 5-4 were prepared.

[0287] Table 9

[0288] Chemiluminescent substrate solution Quaternary ammonium cationic surfactant 5-1 Polyvinylbenzyltrimethylammonium chloride (TMQ) 5-2 Polyvinylbenzyltributylammonium chloride (TBQ) 5-3 Polyvinylbenzyl(benzyldimethyl)ammonium chloride (BDMQ) 5-4 Cetyltrimethylammonium bromide (CTAB)

[0289] Using the above chemiluminescent substrate solutions 5-1 to 5-4, tests were carried out on the Mindray CL-6000i fully automatic chemiluminescent immunoassay analyzer. Alkaline phosphatase solutions with different concentration gradients were mixed with each chemiluminescent substrate solution respectively. After incubation for a period of time, light signals were collected, and the background signal values (i.e., the alkaline phosphatase concentration was 0) and luminescence signal values at a 2-minute light collection time were obtained respectively. The concentrations of alkaline phosphatase in the system and the detected signal values are shown in Table 10 below.

[0290] Table 10

[0291]

[0292] The light signals (photon counts per second) measured for the chemiluminescent substrate solutions 5-1 to 5-4 were plotted against the concentration of alkaline phosphatase in the system, and linear fitting was performed.

[0293] According to the data in Table 10 and Figures 20A to 20D It can be seen that the chemiluminescent substrate solutions 5-1 to 5-3 have low background signals and high luminescence efficiencies, and have good linearity (R2≥0.99) within the detected luminescence range. The chemiluminescent substrate solution 5-4 has a high background signal and low luminescence efficiency, cannot maintain good linearity (R2<0.99) within the detected luminescence range, and the ratio of the light signals at the detection upper limit and the detection lower limit is also small (less than 3000).

[0294] Example 6: Wide linear detection of HCG

[0295] Prepare the chemiluminescent substrate solution 6-1 according to the following formula:

[0296] ADP-STAR: 200 mg

[0297] Fluorescein: 200 mg

[0298] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0299] AMP-HCl: 50 mM

[0300] Magnesium chloride: 200 mg

[0301] Sodium azide: 1 g

[0302] Water: Add to a total volume of 1 L

[0303] Prepare the chemiluminescent substrate solution 6-2 according to the following formula:

[0304] CDP-STAR: 200 mg

[0305] Fluorescein: 200 mg

[0306] Polyvinylbenzyltrimethylammonium chloride: 5 g

[0307] AMP-HCl: 50 mM

[0308] Magnesium chloride: 200 mg

[0309] Sodium azide: 1 g

[0310] Water: Add to a total volume of 1 L

[0311] Using the above chemiluminescent substrate solutions 6-1 to 6-2, serum samples containing different HCG concentrations were tested on the Mindray CL-6000i fully automated chemiluminescent immunoassay analyzer. The sample concentration range was 1 to 200,000 mIU / mL, and the HCG detection kit was the Mindray total β-human chorionic gonadotropin (TotalβHCG) assay kit (chemiluminescent immunoassay method). The photon count per second of the light signal collected when each sample was incubated with the chemiluminescent substrate solutions 6-1 and 6-2 for 2 minutes was recorded, and the concentration of HCG in the sample was calculated based on the light signal, denoted as the back-calculated concentration. The relative deviation of the back-calculated concentration was calculated relative to the nominal concentration of HCG in the sample, and the results are shown in Tables 11 - 12 below.

[0312] Table 11

[0313]

[0314]

[0315] Table 12

[0316]

[0317] As can be seen from Tables 11 and 12, the chemiluminescent substrate solutions of the present disclosure can be directly detected and analyzed without dilution in the concentration range of 1 to 200,000 mIU / mL of HCG in the sample during the detection of the HCG item, and the deviation of the detection results relative to the nominal concentration is within ±5%.

[0318] The above are only the preferred embodiments of the present disclosure, and do not limit the patent scope of the present disclosure. Any equivalent transformation made under the inventive concept of the present disclosure by using the content of the specification and drawings of the present disclosure, or directly / indirectly applied in other related technical fields, is included in the patent protection scope of the present disclosure.

Claims

1. A chemiluminescence analyzer, comprising: A sample preparation device, used to mix a blood sample to be tested with a reaction reagent in a reaction container so that the substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample liquid to be tested, wherein the reaction reagent includes a capture reagent and a detection reagent with a marker; A substrate providing device, used for providing a luminescent substrate into the reaction container containing the sample liquid to be tested, so that the marker in the sample liquid to be tested reacts with the luminescent substrate to undergo a chemiluminescent reaction; A light measuring device, the light measuring device at least comprising a photometer, the photometer being used to detect the luminescent signal generated in the chemiluminescent reaction; A data processing device, used for obtaining the detection result of the substance to be detected in the blood sample to be detected according to the luminescent signal and a preset calibration model; The invention is characterized in that the first linear detection range of the photometer is [A1, A2] photon counts / second, and the first linear detection range represents the range of photon counts output by the photometer. Within the first linear detection range, the intensity of the luminescent signal generated in the chemiluminescent reaction is linearly related to the photon counts output by the photometer, wherein A1 is less than or equal to 2000 and A2 is greater than or equal to 10 8 The second linear detection range of the luminescent substrate is [B1, B2] photon counts / second, and the second linear detection range represents the range of the intensity of the luminescent signal generated in the chemiluminescent reaction. Within the second linear detection range, the number of the markers participating in the chemiluminescent reaction is linearly related to the intensity of the luminescent signal generated in the chemiluminescent reaction, wherein B1 is less than or equal to 3000 and B2 is greater than or equal to 10 8 .

2. A chemiluminescence analyzer, comprising: A sample preparation device, used to mix a blood sample to be tested with a reaction reagent in a reaction container so that the substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample liquid to be tested, wherein the reaction reagent includes a capture reagent and a detection reagent with a marker; A substrate providing device, used for providing a luminescent substrate into the reaction container containing the sample liquid to be tested, so that the marker in the sample liquid to be tested reacts with the luminescent substrate to undergo a chemiluminescent reaction; A light measuring device, the light measuring device at least comprising a photometer, the photometer being used to detect the luminescent signal generated in the chemiluminescent reaction; A data processing device, used for obtaining the detection result of the substance to be detected in the blood sample to be detected according to the luminescent signal and a preset calibration model; It is characterized in that the first linear detection range of the photometer is [A1, A2] photon counts / second, the first linear detection range represents the range of photon counts output by the photometer, within the first linear detection range, the intensity of the luminescent signal generated in the chemiluminescent reaction is linearly related to the photon counts output by the photometer, wherein the ratio of A2 to A1 A2 / A1 is greater than or equal to 30000; the second linear detection range of the luminescent substrate is [B1, B2] photon counts / second, the second linear detection range represents the range of the intensity of the luminescent signal generated in the chemiluminescent reaction, within the second linear detection range, the number of the markers participating in the chemiluminescent reaction is linearly related to the intensity of the luminescent signal generated in the chemiluminescent reaction, wherein the ratio of B2 to B1 B2 / B1 is greater than or equal to 30000.

3. The chemiluminescence analyzer according to claim 1 or 2, characterized in that: The lower limit B1 of the second linear detection range is less than or equal to 2000.

4. The chemiluminescence analyzer according to any one of claims 1 to 3, characterized in that: The luminescent substrate is designed so that the background of the luminescent substrate is less than 3000; and / or The luminescent substrate is designed such that the signal-to-noise ratio of the luminescent substrate is greater than 40,000.

5. The chemiluminescence analyzer according to any one of claims 1 to 4, characterized in that: The substance to be tested is human chorionic gonadotropin, wherein the chemiluminescence analyzer is designed so that: when the concentration of human chorionic gonadotropin in the blood sample to be tested is greater than or equal to 200,000 mIU / ml, the intensity of the luminescent signal generated in the chemiluminescent reaction is less than or equal to the smaller value of A2 and B2.

6. The chemiluminescence analyzer according to any one of claims 1 to 5, characterized in that: The substance to be tested is human chorionic gonadotropin, wherein the chemiluminescence analyzer is designed so that: when the concentration of human chorionic gonadotropin in the blood sample to be tested is less than or equal to 0.5mIU / ml, the intensity C1 of the luminescent signal generated in the chemiluminescent reaction is greater than or equal to the larger value of A1 and B1.

7. The chemiluminescence analyzer according to claim 6, characterized in that: The chemiluminescence analyzer is designed so that: when the concentration of human chorionic gonadotropin in the blood sample to be tested is greater than 0.5mIU / ml and less than or equal to 5mIU / ml, preferably less than or equal to 2mIU / ml, the intensity of the luminescent signal generated in the chemiluminescence reaction is C2, wherein the ratio of C2 to C1, C2 / C1, is greater than 2.

8. The chemiluminescence analyzer according to any one of claims 1 to 7, characterized in that: An upper limit B2 of the second linear detection range is greater than an upper limit A2 of the first linear detection range.

9. The chemiluminescence analyzer according to any one of claims 1 to 8, characterized in that: The sample preparation device is further used for: for all blood samples to be tested that contain human chorionic gonadotropin as the substance to be tested, pre-diluting the blood samples to be tested before mixing the blood samples to be tested with the reaction reagent.

10. The chemiluminescence analyzer according to claim 9, characterized in that: The calibration model is obtained by using a pre-diluted calibrator on the chemiluminescence analyzer.

11. The chemiluminescence analyzer according to claim 10, characterized in that: The pre-dilution multiple of the blood sample to be tested is equal to the pre-dilution multiple of the calibrator.

12. The chemiluminescence analyzer according to any one of claims 1 to 11, characterized in that: The luminescent substrate comprises a chemiluminescent substrate and a chemiluminescent enhancer, the chemiluminescent enhancer comprises a fluorescent agent and a surfactant, and the fluorescent agent comprises one or more carboxyfluoresceins.

13. The chemiluminescence analyzer according to any one of claims 1 to 12, characterized in that: The luminescent substrate is selected from dioxetane compounds, preferably one or more selected from AMPPD, CSPD, CDP-STAR, and more preferably CDP-STAR.

14. The chemiluminescence analyzer according to any one of claims 1 to 13, characterized in that: The luminescent substrate is selected from chlorinated derivatives of AMPPD, fluorescein or its carboxyl substituted derivatives, and water-soluble polymer quaternary ammonium salt cationic surfactants.

15. The chemiluminescence analyzer according to claim 14, characterized in that: The chlorinated derivative of AMPPD is ADP-STAR or CDP-STAR, or The fluorescein or its carboxyl substituted derivative is fluorescein, or 5(6)-carboxyfluorescein, or The water-soluble polymer quaternary ammonium salt cationic surfactant is polyvinylbenzyltrimethylammonium chloride.

16. The chemiluminescence analyzer according to any one of claims 1 to 15, characterized in that: The photometer comprises: a receiving component configured to receive the optical signal generated in the chemiluminescent reaction and convert the optical signal into a corresponding electrical signal; and A processing component is configured to be electrically connected to the receiving component and receive the electrical signal from the receiving component, the processing component includes a first photon counting module and a second photon counting module, the first photon counting module is configured to detect the number of pulses of the electrical signal using a pulse recognition method to obtain a first photon counting result, the second photon counting module is configured to process the electrical signal to obtain a parameter characterizing the number of photons in the optical signal, and obtain a second photon counting result based on the parameter characterizing the number of photons in the optical signal and a preset calibration function, wherein the calibration function represents a mapping relationship between the parameter characterizing the number of photons in the optical signal and the photon counting result, and the processing component is further configured to obtain a photon count output by the photometer based on the first photon counting result and the second photon counting result, and output the photon count.

17. The chemiluminescence analyzer according to claim 16, characterized in that: The processing component is further configured to: When the first photon counting result is lower than a first threshold, using the first photon counting result as the photon counting output by the photometer; and When the first photon counting result is not lower than the first threshold, the second photon counting result is used as the photon counting output by the photometer.

18. The light measuring device according to claim 16 or 17, characterized in that: The first photon counting module includes at least one first AD conversion circuit and a first counting circuit electrically connected to each other, the first AD conversion circuit is configured to collect the electrical signal at a sampling frequency greater than 1 GHz / s and convert the collected electrical signal into a digital signal and output it to the first counting circuit, the first counting circuit is configured to analyze the received digital signal to identify the number of pulses, and thereby obtain the first photon counting result.

19. The chemiluminescence analyzer according to any one of claims 16 to 18, characterized in that: The second photon counting module is configured to process the electrical signal to obtain a DC component signal, and the parameters characterizing the number of photons in the optical signal include parameters related to the DC component signal; preferably, the second photon counting module is configured to integrate the electrical signal within a predetermined time period to obtain an integration result, and the parameters characterizing the number of photons in the optical signal include parameters related to the integration result.

20. The light measuring device according to claim 19, characterized in that: The second photon counting module includes a second AD conversion circuit and a second counting circuit electrically connected to each other, the second AD conversion circuit is configured to convert the electrical signal into a digital signal, the second counting circuit is configured to process the digital signal to obtain a parameter characterizing the number of photons in the optical signal, and obtain the second photon counting result based on the parameter characterizing the number of photons in the optical signal and a preset calibration function; preferably, the second counting circuit is configured to integrate or sum the digital signal within a predetermined time period to obtain a parameter related to the integration result or the summation result and use it as the parameter characterizing the number of photons in the optical signal.

21. A chemiluminescence analyzer, comprising: A sample preparation device, used to mix the blood sample to be tested with the reaction reagent in a reaction container, so that the substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample liquid to be tested; A substrate providing device, used for providing a luminescent substrate into the reaction container containing the sample liquid to be tested, so that the sample liquid to be tested and the luminescent substrate undergo a chemiluminescent reaction; A light measuring device, the light measuring device at least comprising a photometer, the photometer being used to detect the luminescent signal generated in the chemiluminescent reaction; A data processing device, used for obtaining the detection result of the substance to be detected in the blood sample to be detected according to the luminescent signal and a preset calibration model; Characterized in that the photometer includes a receiving component and a processing component, the receiving component is configured to receive the light signal generated in the chemiluminescent reaction and convert the light signal into a corresponding electrical signal, the processing component is configured to be electrically connected to the receiving component and receive the electrical signal from the receiving component, the processing component includes a first photon counting module and a second photon counting module, the first photon counting module is configured to detect the number of pulses of the electrical signal using a pulse recognition method to obtain a first photon counting result, the second photon counting module is configured to process the electrical signal to obtain a parameter characterizing the number of photons in the light signal, and obtain a second photon counting result according to the parameter characterizing the number of photons in the light signal and a preset calibration function, wherein the calibration function represents a mapping relationship between the parameter characterizing the number of photons in the light signal and the photon counting result, the processing component is further configured to obtain the photon count output by the photometer based on the first photon counting result and the second photon counting result, and output the photon count; and The second linear detection range of the luminescent substrate is [B1, B2] photon counts / second, and the second linear detection range represents the range of the intensity of the luminescent signal generated in the chemiluminescent reaction. Within the second linear detection range, the number of the markers participating in the chemiluminescent reaction is linearly related to the intensity of the luminescent signal generated in the chemiluminescent reaction, wherein the ratio of B2 to B1 is greater than or equal to 30,000 or B1 is less than or equal to 3,000 and B2 is greater than or equal to 10 8 .

22. A chemiluminescence analyzer, comprising: A sample preparation device, used to mix the blood sample to be tested with the reaction reagent in a reaction container, so that the substance to be tested in the blood sample to be tested reacts with the reaction reagent to obtain a sample liquid to be tested; A substrate providing device, used for providing a luminescent substrate into the reaction container containing the sample liquid to be tested, so that the sample liquid to be tested and the luminescent substrate undergo a chemiluminescent reaction; A light measuring device, the light measuring device at least comprising a photometer, the photometer being used to detect the luminescent signal generated in the chemiluminescent reaction; A data processing device, used for obtaining the detection result of the substance to be detected in the blood sample to be detected according to the luminescent signal and a preset calibration model; The invention is characterized in that the first linear detection range of the photometer is [A1, A2] photon counts / second, the first linear detection range represents the range of photon counts output by the photometer, within which the intensity of the luminescent signal generated in the chemiluminescent reaction is linearly related to the photon counts output by the photometer, wherein the ratio of A2 to A1 is greater than or equal to 30000 or A1 is less than or equal to 2000 and A2 is greater than or equal to 10 8 , and the luminescent substrate includes a chemiluminescent substrate and a chemiluminescent enhancer, the chemiluminescent enhancer includes a fluorescent agent and a surfactant, and the fluorescent agent includes one or more carboxyfluoresceins.

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