High sensitivity troponin i detection method

Abstract

This invention discloses a highly sensitive method for detecting troponin I. This method utilizes the reusable nature of a biosensor to perform multiple fluorescence signal reads. Based on these multiple reads, it selects the most accurate concentration range for determining concentrations in the low-to-medium concentration range, achieving higher resolution detection results in both low and medium concentration areas. Furthermore, it incorporates chemiluminescence detection, employing higher-resolution chemiluminescence to determine the final concentration in the high-concentration region. Ultimately, this method achieves better resolution detection results across high, medium, and low concentration regions, enabling accurate identification of sample concentrations across all concentration ranges. It boasts a sensitivity of 0.824 ng / L and a detection range of 0.824 ng / L–100,000 ng / L. The method significantly reduces sample volume, requiring only 20 μL for accurate detection, thus reducing blood volume and improving blood collection efficiency in physical examinations.

Description

Technical Field

[0001] This invention relates to the field of detection, and in particular to a method for detecting high-sensitivity troponin I. Background Technology

[0002] High-sensitivity troponin I (hs-cTnI) is a sensitive biomarker for myocardial injury, and its detection is the basis for various myocardial injury studies.

[0003] When detecting the concentration of high-sensitivity troponin I, commonly used detection systems are fluorescence or chemiluminescence detection systems. Fluorescence detection combines the specificity and sensitivity of antigen-antibody reactions with the precision of microscopic tracing. Using fluorescein as a label, the antigen-antibody complex exhibiting specific fluorescence and its location can be directly observed under a fluorescence microscope. Patent document CN118409098B discloses a fluorescence detection method.

[0004] Chemiluminescence detection is a novel labeled immunoassay technique that combines luminescence analysis and immunoreaction to detect trace amounts of antigens or antibodies. Chemiluminescence refers to the emission of light that accompanies a chemical reaction process. For example, patent application CN116068199A discloses a chemiluminescence detection method.

[0005] Although both of these detection methods can be applied to the detection of high-sensitivity troponin I, they both have the problem that the accuracy of the detection results needs to be improved within a certain concentration range. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems existing in the prior art and to provide a highly sensitive method for detecting troponin I.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The high-sensitivity troponin I detection method includes the following steps:

[0009] S1, the first reading is obtained by pre-reading the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1.

[0010] S2, After reacting the biosensor with a high-sensitivity troponin I sample, the biosensor is cleaned with a cleaning solution.

[0011] S3, the cleaned biosensor is reacted with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain the first fluorescence signal reading.

[0012] S4, the biosensor is reacted with streptavidin-cyanine dye 5 and streptavidin-acridone ester, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain a second fluorescence signal reading.

[0013] S5, the biosensor is reacted sequentially with the pre-excitation solution and the excitation solution, and the chemiluminescence signal generated when the biosensor reacts with the excitation solution is read to obtain the chemiluminescence reading;

[0014] S6, determine the first difference, second difference, and third difference between the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading and the first reading, and determine the concentration of high-sensitivity troponin I corresponding to them;

[0015] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the low concentration region and not in the intersection region between the low concentration region and the medium concentration region, the concentration corresponding to the second difference is determined as the final concentration of high-sensitivity troponin I.

[0016] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the medium concentration region and not in the intersection region of the low concentration region and the medium concentration region, the concentration corresponding to the first difference is determined as the final concentration of high-sensitivity troponin I.

[0017] When it is determined that at least one of the concentrations of high-sensitivity troponin I corresponding to the first difference and the second difference is in the intersection region of the low concentration region and the medium concentration region, the concentration of high-sensitivity troponin I is determined according to the concentrations corresponding to the first difference and the second difference.

[0018] When it is determined that the concentration of high-sensitivity troponin I corresponding to the third difference is in the high concentration region, the concentration corresponding to the third difference is determined as the final concentration of high-sensitivity troponin I.

[0019] Preferably, the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1 is obtained by sequentially immersing the probe in PBS buffer, R1 working solution, blocking buffer including milk powder and HS-PEG, PBST washing solution, R2 working solution, blocking solution, PBST washing solution and sucrose aqueous solution, and then drying it.

[0020] Preferably, the R1 working solution is obtained by diluting the fluorescein antibody-conjugated polymer to 30-40 ug / ml with 0.01M PBS buffer;

[0021] The R2 working solution was obtained by diluting fluoresceinized goat anti-human troponin I monoclonal antibody 1 to 20-30 ug / ml with 0.01M PBST solution.

[0022] Preferably, the amounts of the R1 working solution and the R2 working solution are between 180 and 240 ml, the reaction time of the R1 working solution with the biosensor is between 500 and 800 seconds, and the reaction time of the R2 working solution with the biosensor is between 180 and 240 seconds.

[0023] Preferably, the amount of the high-sensitivity troponin I sample is 20 μL.

[0024] Preferably, the concentration of the mouse anti-human troponin I monoclonal antibody in the anthocyanin-labeled mouse anti-human troponin I monoclonal antibody is between 0.5 and 1 ug / ml.

[0025] Preferably, the concentration of streptavidin in the streptavidin-cyanine dye 5 is between 5-8 ug / ml.

[0026] Preferably, the concentration of streptavidin in the streptavidin-acridone ester is between 2-4 ug / mL.

[0027] Preferably, the concentration in the low concentration region is less than or equal to 45.7 ng / L, the concentration in the medium concentration region is greater than or equal to 15.2 ng / L and less than 1235 ng / L, and the concentration in the high concentration region is greater than or equal to 1235 ng / L.

[0028] Preferably, the determination of the concentration of high-sensitivity troponin I based on the concentrations corresponding to the first difference and the second difference is to take the average of the concentrations corresponding to the first difference and the second difference.

[0029] The high-sensitivity troponin I detection method includes the following steps:

[0030] S1, the first reading is obtained by pre-reading the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1.

[0031] S2, After reacting the biosensor with a high-sensitivity troponin I sample, the biosensor is cleaned with a cleaning solution.

[0032] S3, the cleaned biosensor is reacted with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain the first fluorescence signal reading.

[0033] S4, after reacting the biosensor with streptavidin-cyanine dye 5, it is cleaned with a cleaning solution, and the fluorescence signal of the biosensor is read after cleaning to obtain a second fluorescence signal reading;

[0034] S5, after reacting the biosensor with streptavidin-acridone ester and cleaning it, it is reacted sequentially with pre-excitation solution and excitation solution, and the chemiluminescence signal generated by the biosensor when reacting with the excitation solution is read to obtain the chemiluminescence reading;

[0035] S6, determine the first difference, second difference, and third difference between the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading and the first reading, and determine the concentration of high-sensitivity troponin I corresponding to them;

[0036] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the low concentration region and not in the intersection region between the low concentration region and the medium concentration region, the concentration corresponding to the second difference is determined as the final concentration of high-sensitivity troponin I.

[0037] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the medium concentration region and not in the intersection region of the low concentration region and the medium concentration region, the concentration corresponding to the first difference is determined as the final concentration of high-sensitivity troponin I.

[0038] When it is determined that at least one of the concentrations of high-sensitivity troponin I corresponding to the first difference and the second difference is in the intersection region of the low concentration region and the medium concentration region, the concentration of high-sensitivity troponin I is determined according to the concentrations corresponding to the first difference and the second difference.

[0039] When it is determined that the concentration of high-sensitivity troponin I corresponding to the third difference is in the high concentration region, the concentration corresponding to the third difference is determined as the final concentration of high-sensitivity troponin I.

[0040] The advantages of the technical solution of this invention are mainly reflected in:

[0041] This invention utilizes the reusable nature of biosensors to perform multiple fluorescence signal reads. Based on these reads, it selects the most accurate concentration range for determining concentrations in low and medium concentration zones, achieving higher separation in both. Furthermore, it incorporates chemiluminescence detection, employing even higher separation in high concentration regions to determine the final concentration. This fully integrates the advantages of both detection technologies, ultimately achieving better separation across high, medium, and low concentration zones. Therefore, this method can accurately identify sample concentrations across all concentration ranges. It boasts a sensitivity of 0.824 ng / L and a detection range of 0.824 ng / L–100,000 ng / L. The method significantly reduces sample volume, requiring only 20 microliters for accurate detection, thus reducing blood volume and improving blood collection for physical examinations. It also allows for smaller storage containers, promoting miniaturization.

[0042] The method of the present invention involves reacting the biosensor with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody together and then washing it, as well as reacting it with streptavidin-acridone ester and then washing it. This can minimize the time required for separate operations and the storage containers needed, which is beneficial for improving detection efficiency and reducing the size of the test strip. Attached Figure Description

[0043] Figure 1 This is a flowchart of Embodiment 1 of the method of the present invention;

[0044] Figure 2 This is a schematic diagram of the test process of Embodiment 2 of the method of the present invention. Detailed Implementation

[0045] The objectives, advantages, and features of this invention will be illustrated and explained through the following non-limiting description of preferred embodiments. These embodiments are merely typical examples of applying the technical solutions of this invention, and all technical solutions formed by equivalent substitutions or equivalent transformations fall within the scope of protection claimed by this invention.

[0046] In the description of the solution, it should be noted that the terms "center," "upper," "lower," "left," "right," "front," "rear," "vertical," "horizontal," "inner," and "outer," etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings and are only for the convenience and simplification of description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0047] Example 1

[0048] The method for detecting high-sensitivity troponin I disclosed in this invention is described below with reference to the accompanying drawings. Figure 1 As shown, it includes the following steps:

[0049] S1, the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1 is pre-read to obtain the first reading (background signal).

[0050] S2, After reacting the biosensor with a high-sensitivity troponin I sample, the biosensor is cleaned with a cleaning solution.

[0051] S3, the cleaned biosensor is reacted with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain the first fluorescence signal reading.

[0052] S4, the biosensor is reacted with streptavidin-cyanine dye 5 and streptavidin-acridone ester, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain a second fluorescence signal reading.

[0053] S5, the biosensor is reacted sequentially with the pre-excitation solution and the excitation solution, and the chemiluminescence signal generated when the biosensor reacts with the excitation solution is read to obtain the chemiluminescence reading;

[0054] S6, determine the first difference, second difference, and third difference between the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading and the first reading, and determine the concentration of high-sensitivity troponin I corresponding to them;

[0055] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the low concentration region and not in the intersection region between the low concentration region and the medium concentration region, the concentration corresponding to the second difference is determined as the final concentration of high-sensitivity troponin I.

[0056] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the medium concentration region and not in the intersection region of the low concentration region and the medium concentration region, the concentration corresponding to the first difference is determined as the final concentration of high-sensitivity troponin I.

[0057] When it is determined that at least one of the concentrations of high-sensitivity troponin I corresponding to the first difference and the second difference is in the intersection region of the low concentration region and the medium concentration region, the concentration of high-sensitivity troponin I is determined according to the concentrations corresponding to the first difference and the second difference.

[0058] When it is determined that the concentration of high-sensitivity troponin I corresponding to the third difference is in the high concentration region, the concentration corresponding to the third difference is determined as the final concentration of high-sensitivity troponin I.

[0059] In step S1, the biosensor is fabricated using a gold-plated matrix probe. The gold-plated matrix probe is made from a substrate such as quartz, silicon, ceramic, or plastic, and a gold plating layer is deposited on the substrate. Furthermore, to ensure stable adhesion between the gold plating layer and the substrate, a titanium or chromium layer can be deposited between the gold plating layer and the substrate. The titanium or chromium layer can effectively bond with both the substrate and the gold plating layer, thereby effectively increasing the bonding strength between the substrate and the gold plating layer.

[0060] The gold-plated matrix probe can be directly coated with a layer of fluorescein antibody-coupled polymer (FCCP) on its bottom surface through hydrophobic interactions and gold-sulfur bonds. This is because FCCPs typically incorporate intentionally introduced thiol-containing chemical groups or molecules, with the sulfur atoms in the thiol groups exhibiting a high affinity for the gold surface. When these groups approach the gold surface, they replace other molecules originally adsorbed on the gold surface, forming stable Au-S covalent bonds with the gold atoms. This securely, stably, directionally, and densely fixes the FCCP to the gold-plated surface.

[0061] The fluoresceinized goat anti-human troponin I monoclonal antibody 1 can specifically bind to the fluorescein antibody conjugate polymer, which makes the fluoresceinized goat anti-human troponin I monoclonal antibody 1 firmly and uniformly fixed on the bottom surface of the probe, thus providing a reliable guarantee for subsequent accurate detection.

[0062] Specifically, the biosensor coated with a fluorescein antibody-conjugated polymer and a fluoresceinized goat anti-human troponin I monoclonal antibody 1 is obtained by sequentially immersing the probe in PBS buffer, R1 working solution, blocking buffer including milk powder and HS-PEG, PBST washing solution, R2 working solution, blocking solution, PBST washing solution, and sucrose aqueous solution, followed by drying. Furthermore, in each reaction step, each reaction solution can be loaded into a 96-well plate, which is placed on a shaker or mixer. During the reaction, the rotation speed of the shaker or mixer is maintained between 1000-1200 rpm.

[0063] The amount of PBS buffer used is between 250-300 μL, and the reaction time of the biosensor with the PBS buffer is between 30-50 seconds.

[0064] The R1 working solution is obtained by diluting the fluorescein antibody-conjugated polymer to 30-40 μg / ml with 0.01 M PBS buffer. The volume of the R1 working solution is between 180 and 240 ml, and the reaction time between the R1 working solution and the biosensor is between 500 and 800 seconds. The above concentration range was chosen because excessively low concentrations can lead to uneven or insufficient probe surface coating, affecting subsequent reaction efficiency and signal intensity; while excessively high concentrations can result in reagent waste and may cause performance degradation due to multilayer stacking or steric hindrance. At the above concentration range, a dense and ordered layer of fluorescein antibody-conjugated polymer can be formed on the gold surface, providing a large number of uniform binding sites for subsequent antibody capture.

[0065] The blocking buffer contains 5% milk powder by mass and volume, 0.25% HS-PEG (high molecular weight polyethylene glycol) by mass and volume, and the remainder is PBS buffer. The volume of the blocking buffer is 250-300 uL, and the reaction time between the biosensor and the blocking buffer is between 60-120 s.

[0066] The reason for combining milk powder with HS-PEG is that they can work synergistically, complementing each other to achieve a synergistic effect greater than the sum of its parts. The proteins in milk powder (such as casein) can rapidly and non-specifically adsorb onto any unoccupied hydrophobic or hydrophilic sites on the gold surface or polymer, providing a biocompatible microenvironment for HS-PEG. However, because its blocking is reversible and non-covalent, its stability is slightly weaker. HS-PEG, on the other hand, forms strong, irreversible gold-sulfur bonds with the gold surface. Simultaneously, the PEG chains of HS-PEG are highly hydrophilic and flexible, forming a dense "hydration brush" on the sensor surface, physically blocking the approach of any non-specific molecules and greatly reducing background signal. Together, they form a tight, sealed structure, effectively ensuring the sealing effect.

[0067] Meanwhile, a 5% milk powder concentration ensures sufficient saturation while also taking cost into account and avoiding material waste. Milk powder is inexpensive, and its presence helps reduce the concentration of HS-PEG, allowing it to be reduced to 0.25%. This concentration range not only ensures the formation of a dense PEG protective layer with the milk powder, but also avoids excessively high viscosity of the blocking buffer, which could lead to uneven coating, large differences in sensor performance, and the formation of non-specific background signals.

[0068] The amount of PBST cleaning solution used is between 250-300 μL, and the reaction time between the biosensor and the PBST cleaning solution is between 20-40 seconds.

[0069] The R2 working solution is obtained by diluting fluoresceinized goat anti-human troponin I monoclonal antibody 1 to 20-30 μg / ml with 0.01 M PBST solution. The volume of the R2 working solution is between 180 and 240 mL, and the reaction time between the R2 working solution and the biosensor is between 180 and 240 seconds.

[0070] Preferably, the biosensor is prepared according to the process parameters in the table below.

[0071]

[0072]

[0073] The above-mentioned concentration, dosage, and reaction time settings can effectively ensure that a sufficient number of antibody molecules bind to the fluorescein in the R1 layer, forming a high-density capture site to achieve high sensitivity, while avoiding non-specific binding or aggregation caused by excessive antibody, thus achieving monolayer antibody coverage with optimal binding activity.

[0074] The blocking solution contains 5% milk powder by mass and volume, with the remainder being PBS buffer. The amount of blocking solution used is between 250-300 μL, and the reaction time between the biosensor and the blocking solution is between 30-50 seconds.

[0075] The sucrose aqueous solution contains 15% by mass and volume, and the amount of the sucrose aqueous solution used is between 250-300 μL. The reaction time of the biosensor with the sucrose aqueous solution is between 30-50 seconds.

[0076] The biosensor after the final reaction was placed in a constant temperature drying oven for drying at a temperature between 37-40℃ for 3.5-5 hours.

[0077] Furthermore, in S1, the first reading of the biosensor is read using a known fluorescence reading method. The specific reading method is a known technique and will not be described in detail here.

[0078] In step S2, the amount of the high-sensitivity troponin I sample is 20 μL, and the reaction time between the biosensor and the high-sensitivity troponin I sample is 180 seconds. During the reaction, the shaker or mixer rotates at 1200 rpm. Simultaneously, the washing solution is PBST washing solution, and the washing is performed three times, with each washing session lasting 7 seconds. During the washing process, the shaker or mixer rotates at 1200 rpm.

[0079] In step S3, the biotin-labeled mouse anti-human troponin I monoclonal antibody is obtained by biotin labeling of Hs-cTnI mouse anti-human troponin I monoclonal antibody obtained from Hytest (Hytest Biotechnology) using a standard method. The specific labeling method is a known technique and not an innovation of this invention, so it will not be elaborated here. Typically, each antibody contains approximately four biotin molecules. The concentration of mouse anti-human troponin I monoclonal antibody in the biotin-labeled mouse anti-human troponin I monoclonal antibody solution is 2-3 μg / ml.

[0080] The anthocyanin-labeled mouse anti-human troponin I monoclonal antibody was prepared according to the following procedure:

[0081] 90 μL of a 5 mg / mL DMF solution of Cy5-NHS (anthocyanin 5-succinimide ester, purchased from GE Healthcare) was reacted with 30 μL of antibody solution at room temperature for 60 minutes. After the reaction, the mixture was purified by passing it through a PD10 column (Pharmacia) to remove unconjugated Cy5 and obtain a purified anthocyanin-labeled mouse anti-human troponin I monoclonal antibody solution. The antibody solution was obtained by dissolving troponin I monoclonal antibody (Hytest) in sodium carbonate buffer at pH 7.0 with a molar concentration of 0.12 M, and the concentration of troponin I monoclonal antibody in the antibody solution was 1.5 mg / mL.

[0082] The concentration of mouse anti-human troponin I monoclonal antibody in the anthocyanin-labeled mouse anti-human troponin I monoclonal antibody solution is between 0.5 and 1 ug / ml.

[0083] The biosensor reacts with the biotin-labeled mouse anti-human troponin I monoclonal antibody and the anthocyanin-labeled mouse anti-human troponin I monoclonal antibody for 60 seconds. The amount of the biotin-labeled mouse anti-human troponin I monoclonal antibody used is between 40-45 μL, and the amount of the anthocyanin-labeled mouse anti-human troponin I monoclonal antibody used is between 15-20 μL. During the reaction, the shaking incubator or mixer rotates at 1200 rpm.

[0084] In step S3, the cleaning solution is also PBST cleaning solution, and the cleaning is performed 3 times, with each cleaning lasting 7 seconds. During the cleaning, the speed of the shaker or mixer is 1200 rpm.

[0085] In step S4, the streptavidin-cyanine dye 5 is prepared according to the following process:

[0086] 32 μL of a 5 mg / mL Cy5-NHS (GE Healthcare) DMF solution was reacted with 1 mL of streptavidin (SA) solution at 30 °C for 40 min. After the reaction, the mixture was purified by passing it through a PD10 column (Pharmacia) to remove unconjugated Cy5, resulting in a purified streptavidin-cyanine dye 5 solution. The concentration of streptavidin in the streptavidin-cyanine dye 5 solution was between 5-8 μg / mL.

[0087] The streptavidin solution was obtained by dissolving streptavidin (ScrIppsLabs) in a sodium carbonate buffer solution at pH 9.5 with a molar concentration of 0.1 M, and the concentration of streptavidin in the streptavidin solution was 2.4 mg / mL.

[0088] The streptavidin-acridone ester is prepared according to the following process:

[0089] A 4.5 μL solution of 10 mg / mL acridine inner salt (synonyms NSP-SA-NHS, Biosynth Carbosynth) in DMF was reacted with 3.0 mL of streptavidin-crosslinked ficolyl solution at room temperature for 1 hour. The resulting mixture was purified by passing it through a PD10 column to remove unlinked acridine esters (AEs) and obtain a purified streptavidin-crosslinked ficolyl solution. The streptavidin-crosslinked ficolyl solution was prepared by mixing streptavidin-crosslinked ficolyl with PBS buffer at pH 7.4, wherein the concentration of streptavidin-crosslinked ficolyl was 0.3 mg / mL. The concentration of streptavidin in the streptavidin-crosslinked ficolyl solution was between 2-4 μg / mL.

[0090] In step S4, the biosensor reacts with streptavidin-anthocyanin dye 5 and streptavidin acridine ester for 30 seconds. The amount of streptavidin-anthocyanin dye 5 is between 40-45 μL, and the amount of streptavidin acridine ester is between 15-20 μL. During the reaction, the shaker or mixer speed is 1200 rpm. After the reaction, the washing solution is also PBST washing solution, and the washing is performed 3 times, with each washing lasting 7 seconds. During washing, the shaker or mixer speed is 1200 rpm.

[0091] In step S5, the pre-excitation solution is a hydrogen peroxide solution, the chemiluminescence excitation solution is a sodium hydroxide solution, and the amount of both the pre-excitation solution and the excitation solution is 30 μL.

[0092] In step S6, determining the concentration of high-sensitivity troponin I based on the concentrations corresponding to the first difference and the second difference involves taking the average of the concentrations corresponding to the first difference and the second difference. Specifically, this is determined based on their respective standard curves. How to determine the standard curves will be described below and will not be elaborated here.

[0093] In practice, the method involves loading a biosensor and various reaction solutions onto a reagent strip. Then, an automated detection device performs various operations on the reagent strip. For example, the biosensor on the reagent strip is sequentially immersed into the reaction solutions in each well according to the described procedure. The biosensor is then transferred to a reading well for fluorescence signal reading and chemiluminescence signal reading. The specific structure of the automated detection device is known and not innovative, therefore it will not be described in detail here. Furthermore, the specific methods for reading fluorescence and chemiluminescence signals are known technologies and not innovative, therefore they will not be described in detail here.

[0094] In specific applications and the following tests, the preferred amounts of each reagent are as follows:

[0095] The amount of the high-sensitivity troponin I sample used was 20 μL; the amount of biotinylated mouse anti-human troponin I monoclonal antibody used was 45 μL; the amount of anthocyanin-labeled mouse anti-human troponin I monoclonal antibody used was 15 μL; the amount of streptavidin-anthocyanin dye 5 used was 40 μL; the amount of streptavidin-acridone ester used was 20 μL; and the amount of washing solution used in each step was 90 μL.

[0096] In order to accurately determine the concentration of high-sensitivity troponin I based on the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading, it is necessary to establish their respective standard curves.

[0097] In practice, different concentrations of high-sensitivity troponin I antigen are used to perform the reaction according to the method described above, and the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading are recorded. During the reaction...

[0098] The concentrations of the high-sensitivity troponin I antigen were 100,000 ng / L, 33,333 ng / L, 11,111 ng / L, 3,704 ng / L, 1,235 ng / L, 412 ng / L, 137 ng / L, 45.7 ng / L, 15.2 ng / L, 5.08 ng / L, 1.69 ng / L, and 0 ng / L, respectively. The results are shown in Table 1 below.

[0099] Table 1: Test results of the standard curve.

[0100]

[0101]

[0102] Since the main difference between fluorescence detection systems and chemiluminescence detection systems lies in the resolution between standard concentration points (resolution is calculated as the ratio of a signal of one concentration to its immediately adjacent lower concentration), the above standard concentrations are diluted in a ratio, usually 3-fold. Therefore, the closer the resolution is to 3-fold, the better the resolution, and the higher the resolution, the more accurate the detection results and the better the linearity.

[0103] Therefore, with a resolution > 2.5 as the acceptance limit for resolution, it can be seen from Table 1 that when the concentration is greater than 0 and less than 45.7 ng / L, the resolution corresponding to the second fluorescence signal reading is close to 3. Therefore, the concentration range of greater than 0 and less than 45.7 ng / L is defined as the low concentration region. Thus, when the concentrations corresponding to the first fluorescence signal reading and the second fluorescence signal reading are determined to be in the above-mentioned low concentration region, the concentration of high-sensitivity troponin I in the measured sample can be determined based on the second fluorescence signal reading.

[0104] When the concentration is greater than or equal to 15.2 ng / L and less than 1235 ng / L, the resolution corresponding to the first fluorescence signal reading is close to 3. Therefore, the concentration range of greater than or equal to 15.2 ng / L and less than 1235 ng / L is defined as the medium concentration region. So when the concentrations corresponding to the first fluorescence signal reading and the second fluorescence signal reading are in the above-mentioned medium concentration region, the concentration of high-sensitivity troponin I in the measured sample can be determined based on the first fluorescence signal reading.

[0105] Furthermore, when the concentration is greater than or equal to 1235 ng / L and less than or equal to 100,000 ng / L, the resolution corresponding to the chemiluminescence signal is closer to 3. Therefore, the concentration range greater than or equal to 1235 ng / L is defined as the high concentration region. Thus, when the concentration region corresponding to the chemiluminescence reading is the aforementioned high concentration region, the concentration of high-sensitivity troponin I in the measured sample can be directly determined based on the chemiluminescence reading.

[0106] Finally, since the low-concentration region and the medium-concentration region have an overlap area (15.2-45.7 ng / L), in order to avoid interference, when at least one of the concentrations corresponding to the first fluorescence signal reading and the concentrations corresponding to the second fluorescence signal reading falls within the overlap area, the average of the concentrations corresponding to the first difference and the concentrations corresponding to the second difference is taken as the concentration of high-sensitivity troponin I in the final sample.

[0107] By fitting a linear equation based on the above results, we can obtain the linear equations corresponding to the concentration values ​​for the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading, and thus obtain their respective standard curves.

[0108] Therefore, in actual testing, after reading the first fluorescence signal, the second fluorescence signal, and the chemiluminescence reading, they can be substituted into their respective linear equations to obtain their respective concentration values. Based on their corresponding concentration values, their corresponding concentration ranges can be determined. Then, based on the determined concentration ranges and the above method, the final concentration of high-sensitivity troponin I in the sample can be determined.

[0109] To test the sensitivity of the method of the present invention, the detection sample buffer (blank sample) was tested 20 times according to the above reaction steps. The detection sample buffer included PBS, Tween-20, BSA, mIgG, and NaN3, wherein the molar concentration of MPBS was 12 mM (millimoles per liter), the mass-volume percentage of Tween-20 was 0.05%, the concentration of BSA was 5 mg / mL, the concentration of mIgG was 0.2 mg / mL, and the mass-volume percentage of NaN3 was 0.05%. The results are shown in Table 2 below.

[0110] Table 2: Sensitivity Detection Results: (The detection result is the concentration corresponding to the second difference determined by the second fluorescence signal reading, unit: ng / L)

[0111]

[0112] The concentration corresponding to the signal with mean +2*SD was used as the sensitivity of this test. It can be seen that the fluorescence sensitivity is 0.824ng / L, which reaches the level of ultra-trace analysis and belongs to the high-performance level.

[0113] Furthermore, to verify the precision of the method of the present invention, clinical samples with high, medium and low concentrations were selected and tested on a clinical instrument (Beckman Access2 analyzer) with concentrations of approximately 15000 ng / L, 500 ng / L and 5 ng / L, respectively. According to the above reaction steps, the high, medium and low concentration samples were measured to evaluate the precision of the method of the present invention. Each sample was measured 10 times. The specific test results are shown in Tables 3-5 below.

[0114] Table 3: Precision detection results for low-concentration samples (unit: ng / L)

[0115]

[0116] Table 4: Precision detection results for medium-concentration samples:

[0117]

[0118]

[0119] Table 5: Precision detection results for high-concentration samples:

[0120]

[0121]

[0122] Precision is determined based on the CV value; the smaller the CV value, the higher the precision. Table 3 shows that low-concentration samples have better CV values ​​and higher accuracy when calculated at the second fluorescence reading. Table 4 shows that medium-concentration samples have better CV values ​​and higher accuracy when calculated at the first fluorescence reading. Table 5 shows that high-concentration samples have better CV values ​​and higher accuracy when calculated at the chemiluminescence reading.

[0123] Select a high-concentration sample of high-sensitivity troponin I with a known concentration, and then perform a 3-fold serial dilution with a negative sample. Each sample is measured twice, and the mean value is taken as the measured value. The percentage between the measured value and the theoretical value is calculated as the recovery rate.

[0124] Recovery rate test

[0125] A clinical sample with a known concentration of high-sensitivity troponin I was selected, and its concentration was tested using a clinical instrument (Beckman Access 2 analyzer). The sample was then serially diluted 3-fold using a negative sample. Each sample was measured 3 times according to the method of this invention, and the mean value was taken as the measured value. The percentage between the measured value and the theoretical value was calculated as the recovery rate. The specific results are shown in Tables 6-9.

[0126] Table 6: Recovery test results corresponding to the first fluorescence reading: (unit: ng / L)

[0127]

[0128]

[0129] Table 7: Recovery test results corresponding to the second fluorescence reading:

[0130]

[0131] Table 8: Recovery rate test results corresponding to chemiluminescence readings:

[0132]

[0133]

[0134] Table 9: Recovery results of integrating the concentrations corresponding to the differences determined by two fluorescence readings and chemiluminescence readings.

[0135] As can be seen from Table 9, the method of the present invention combines the advantages of high recovery rates of the first and second fluorescence readings in the medium and low concentration regions and high recovery rates of the chemiluminescence readings in the high concentration region, thereby achieving a recovery rate that remains within the range of 90%-110% across the entire linear range.

[0136] Real-time Example 2

[0137] This embodiment discloses another highly sensitive method for detecting troponin I. Unlike Embodiment 1, this embodiment does not involve co-reacting the biosensor with streptavidin-anthocyanin dye 5 and streptavidin-acridone ester; instead, the reaction is performed stepwise, as detailed in the attached diagram. Figure 2 As shown, the high-sensitivity troponin I detection method includes the following steps:

[0138] S1, the first reading is obtained by pre-reading the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1.

[0139] S2, After reacting the biosensor with a high-sensitivity troponin I sample, the biosensor is cleaned with a cleaning solution.

[0140] S3, the cleaned biosensor is reacted with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain the first fluorescence signal reading.

[0141] S4, after reacting the biosensor with streptavidin-cyanine dye 5, it is cleaned with a cleaning solution, and the fluorescence signal of the biosensor is read after cleaning to obtain a second fluorescence signal reading;

[0142] S5, after reacting the biosensor with streptavidin-acridone ester and cleaning it, it is reacted sequentially with pre-excitation solution and excitation solution, and the chemiluminescence signal generated by the biosensor when reacting with the excitation solution is read to obtain the chemiluminescence reading;

[0143] S6, determine the first difference, second difference, and third difference between the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading and the first reading, and determine the concentration of high-sensitivity troponin I corresponding to them;

[0144] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the low concentration region and not in the intersection region between the low concentration region and the medium concentration region, the concentration corresponding to the second difference is determined as the final concentration of high-sensitivity troponin I.

[0145] When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the medium concentration region and not in the intersection region of the low concentration region and the medium concentration region, the concentration corresponding to the first difference is determined as the final concentration of high-sensitivity troponin I.

[0146] When it is determined that at least one of the concentrations of high-sensitivity troponin I corresponding to the first difference and the second difference is in the intersection region of the low concentration region and the medium concentration region, the concentration of high-sensitivity troponin I is determined according to the concentrations corresponding to the first difference and the second difference.

[0147] When it is determined that the concentration of high-sensitivity troponin I corresponding to the third difference is in the high concentration region, the concentration corresponding to the third difference is determined as the final concentration of high-sensitivity troponin I.

[0148] This invention has many other embodiments, and all technical solutions formed by equivalent transformation or equivalent transformation fall within the protection scope of this invention.

Claims

1. A method for high sensitivity troponin I detection characterized in that, Includes the following steps: S1, the first reading is obtained by pre-reading the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1. S2, After reacting the biosensor with a high-sensitivity troponin I sample, the biosensor is cleaned with a cleaning solution. S3, the cleaned biosensor is reacted with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain the first fluorescence signal reading. S4, the biosensor is reacted with streptavidin-cyanine dye 5 and streptavidin-acridone ester, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain a second fluorescence signal reading. S5, the biosensor is reacted sequentially with the pre-excitation solution and the excitation solution, and the chemiluminescence signal generated when the biosensor reacts with the excitation solution is read to obtain the chemiluminescence reading; S6, determine the first difference, second difference, and third difference between the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading and the first reading, and determine the concentration of high-sensitivity troponin I corresponding to them; When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the low concentration region and not in the intersection region between the low concentration region and the medium concentration region, the concentration corresponding to the second difference is determined as the final concentration of high-sensitivity troponin I. When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the medium concentration region and not in the intersection region of the low concentration region and the medium concentration region, the concentration corresponding to the first difference is determined as the final concentration of high-sensitivity troponin I. When it is determined that at least one of the concentrations of high-sensitivity troponin I corresponding to the first difference and the second difference is in the intersection region of the low concentration region and the medium concentration region, the concentration of high-sensitivity troponin I is determined according to the concentrations corresponding to the first difference and the second difference. When it is determined that the concentration of high-sensitivity troponin I corresponding to the third difference is in the high concentration region, the concentration corresponding to the third difference is determined as the final concentration of high-sensitivity troponin I.

2. The high-sensitivity troponin I detection method according to claim 1, characterized in that: The biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1 is obtained by sequentially immersing the probe in PBS buffer, R1 working solution, blocking buffer including milk powder and HS-PEG, PBST washing solution, R2 working solution, blocking solution, PBST washing solution and sucrose aqueous solution, and then drying it.

3. The high-sensitivity troponin I detection method according to claim 2, characterized in that: The R1 working solution was obtained by diluting the fluorescein antibody-conjugated polymer to 30-40 ug / ml with 0.01M PBS buffer. The R2 working solution was obtained by diluting fluoresceinized goat anti-human troponin I monoclonal antibody 1 to 20-30 ug / ml with 0.01 M PBST solution. The amounts of R1 and R2 working solutions are between 180 and 240 ml, the reaction time of R1 working solution with the biosensor is between 500 and 800 seconds, and the reaction time of R2 working solution with the biosensor is between 180 and 240 seconds.

4. The high-sensitivity troponin I detection method according to claim 1, characterized in that: The amount of the high-sensitivity troponin I sample used was 20 μL.

5. The high-sensitivity troponin I detection method according to claim 1, characterized in that: The concentration of the mouse anti-human troponin I monoclonal antibody labeled with cyanine dye is between 0.5-1 ug / ml.

6. The high-sensitivity troponin I detection method according to claim 1, characterized in that: The concentration of streptavidin in the streptavidin-cyanine dye 5 is between 5-8 ug / ml.

7. The high-sensitivity troponin I detection method according to claim 1, characterized in that: The concentration of streptavidin in the streptavidin-acridone ester is between 2-4 μg / mL.

8. The high-sensitivity troponin I detection method according to claim 1, characterized in that: The concentration in the low concentration region is less than or equal to 45.7 ng / L, the concentration in the medium concentration region is greater than or equal to 15.2 ng / L and less than 1235 ng / L, and the concentration in the high concentration region is greater than or equal to 1235 ng / L.

9. The method for detecting high-sensitivity troponin I according to any one of claims 1-8, characterized in that: The determination of the concentration of high-sensitivity troponin I based on the concentrations corresponding to the first difference and the second difference is to take the average of the concentrations corresponding to the first difference and the second difference.

10. A high-sensitivity method for detecting troponin I, characterized in that, Includes the following steps: S1, the first reading is obtained by pre-reading the biosensor coated with fluorescein antibody-conjugated polymer and fluoresceinized goat anti-human troponin I monoclonal antibody 1. S2, After reacting the biosensor with a high-sensitivity troponin I sample, the biosensor is cleaned with a cleaning solution. S3, the cleaned biosensor is reacted with biotin-labeled mouse anti-human troponin I monoclonal antibody and anthocyanin-labeled mouse anti-human troponin I monoclonal antibody, and then cleaned with a cleaning solution. After cleaning, the fluorescence signal of the biosensor is read to obtain the first fluorescence signal reading. S4, after reacting the biosensor with streptavidin-cyanine dye 5, it is cleaned with a cleaning solution, and the fluorescence signal of the biosensor is read after cleaning to obtain a second fluorescence signal reading; S5, after reacting the biosensor with streptavidin-acridone ester and cleaning it, it is reacted sequentially with pre-excitation solution and excitation solution, and the chemiluminescence signal generated by the biosensor when reacting with the excitation solution is read to obtain the chemiluminescence reading; S6, determine the first difference, second difference, and third difference between the first fluorescence signal reading, the second fluorescence signal reading, and the chemiluminescence reading and the first reading, and determine the concentration of high-sensitivity troponin I corresponding to them; When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the low concentration region and not in the intersection region between the low concentration region and the medium concentration region, the concentration corresponding to the second difference is determined as the final concentration of high-sensitivity troponin I. When it is determined that the concentration of high-sensitivity troponin I corresponding to the first difference and the second difference is in the medium concentration region and not in the intersection region of the low concentration region and the medium concentration region, the concentration corresponding to the first difference is determined as the final concentration of high-sensitivity troponin I. When it is determined that at least one of the concentrations of high-sensitivity troponin I corresponding to the first difference and the second difference is in the intersection region of the low concentration region and the medium concentration region, the concentration of high-sensitivity troponin I is determined according to the concentrations corresponding to the first difference and the second difference. When it is determined that the concentration of high-sensitivity troponin I corresponding to the third difference is in the high concentration region, the concentration corresponding to the third difference is determined as the final concentration of high-sensitivity troponin I.

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