Analyte detection method and detection system

Abstract

The invention provides a detection method and a detection system of an analyte, and relates to the following steps: after a sample is added into an immunochromatography test strip, exciting light emitted by a light source of a light analyzer is utilized to irradiate a T line of the test strip, light-emitting signals R1 and R2 emitted by the T line are detected at two different time points after the light source is turned off, R2 = KR1, R1 is compared with a boundary value B of the light analyzer, and the detection result is obtained. When R1 is less than or equal to B, calculating the analyte concentration in the sample by using R1; when R1gt; and B, calculating the analyte concentration in the sample by using KR2. When the fluorescence signal generated by the high-concentration analyte exceeds the detection upper limit of the light analyzer, the concentration value of the high-concentration analyte is still accurately obtained at low cost, so that the problem that the detection range of a conventional light analyzer is insufficient is solved, and meanwhile, a sample of the high-concentration analyte is prevented from being tested by using a sample dilution method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a method and a system for detecting an analyte. Background Art

[0002] Fluorescence immunoassay chromatography analysis technology is one of the commonly used technologies in current clinical diagnosis. Based on the principle of immunochromatography, fluorescent labels aggregate in the test area. Under the irradiation of a light source with a specific wavelength, light with a wavelength different from the excitation light of the light source is emitted, and finally captured by a photoelectric induction device to form a tiny current. The magnitude of the current is related to the concentration of the analyte in the sample, so as to qualitatively and / or quantitatively detect the analyte.

[0003] The current captured by the photoelectric induction device is very weak and needs to be amplified by an amplifier circuit before it can form a current signal for subsequent result calculation. Therefore, when designing an optical analyzer, it is usually necessary to select a reasonable current amplification factor. The current amplification factor usually depends on the following two aspects:

[0004] (1) The lower limit of the current amplification factor is usually determined by the lower limit of detection of the analyte. When the lower limit of detection of the analyte is low, for example, the lower limit of detection of interleukin 6 (IL-6) is 3 pg / mL, and the lower limit of detection of N-terminal pro-brain natriuretic peptide (NT-proBNP) is 15 pg / L, the optical analyzer used should be able to have good resolution even when the fluorescence signal is extremely low. In other words, without affecting the signal-to-noise ratio, it is required that the optical analyzer has a large current amplification factor.

[0005] (2) The upper limit of the current amplification factor is usually determined by the upper limit of detection of the analyte and the word length of the analog-to-digital converter. The detection ranges of clinical analytes vary greatly. Some analytes have a small detection range. For example, the detection range of serum amyloid A (SAA) is 5 - 200 mg / L, and the ratio of the upper limit of detection to the lower limit of detection is 40 times. Some analytes have a large detection range. For example, the detection range of IL-6 is 3 - 4000 pg / mL, and the ratio of the upper limit of detection to the lower limit of detection is 1333 times. Considering costs, the current commonly used word length of the analog-to-digital converter is 16 bits, and the counting range is 0 - 65535 (2 16 -1). In order to ensure that the current amplified by the amplifier circuit does not exceed the counting upper limit of the analog-to-digital converter, the current amplification factor of the optical analyzer should not be too large, otherwise it will exceed the counting upper limit of the analog-to-digital converter.

[0006] Obviously, the above two points are contradictory. Taking IL-6 as an example, in order to reduce the influence of circuit noise, the fluorescence signal value generated when detecting 3 pg / mL IL-6 using an optical analyzer is usually adjusted to about 160. Theoretically, calculated with the detection upper limit / detection lower limit ratio of IL-6 being 1333.33 times, the upper limit signal can reach 213332.8, which will be far greater than the maximum counting range of 65535 of the analog-to-digital converter in the optical analyzer.

[0007] The common methods to solve this problem usually include the following: (1) Dilute the high-concentration clinical sample with sample diluent and test again; (2) Reduce the current amplification factor; (3) Replace the analog-to-digital converter to expand the counting range.

[0008] However, the first method is extremely inconvenient for users, and it will also increase the detection cost and time. In the second method, taking IL-6 as an example, if the current amplification factor is reduced to 1 / 4, its upper limit signal is 53333.2, meeting the requirements of the hardware chip. At this time, the fluorescence signal generated during the detection of the detection lower limit (3 pg / mL) of IL-6 also drops from 160 to 40. However, considering that the noise signal of a conventional optical analyzer is about 20, at this time, in the signal-to-noise ratio, the proportion of the noise signal is 50%, which is extremely likely to cause inaccurate test results. The third method can effectively expand the counting range, but considering that the mainstream analog-to-digital converter on the current market is 16-bit, the technology of analog-to-digital converters with a larger word length is not yet mature, the performance is unstable, and in addition, the price is expensive. Therefore, there is an urgent need in the market for a new method that can accurately and low-costly detect high-concentration analytes in samples without dilution. Summary of the Invention

[0009] Aiming at the deficiencies of the prior art, the present invention discovers that by using the time-resolved luminescence immunoassay method, without affecting the detection of low-concentration analytes, when the luminescence signal generated during the detection of high-concentration analytes exceeds the detection upper limit of the optical analyzer, the concentration value of the high-concentration analytes in the sample can still be accurately and low-costly obtained, thereby solving the problem of insufficient detection range of analyte concentration existing in conventional optical analyzers, and at the same time avoiding using the method of sample dilution to detect the concentration of high-concentration analytes, which not only saves the test time but also saves the test cost.

[0010] The present invention provides a method for detecting an analyte, and the steps thereof include: (1) providing a detection device which, during detection, carries a first reagent labeled with a first signal marker, the first reagent being capable of specifically binding to the analyte in the sample. After adding the sample, the first reagent labeled with the first signal marker can be captured in the test area of the detection device, and the first signal marker is a time-resolved luminescence marker; (2) adding the sample to the detection device, and the first reagent labeled with the first signal marker moves with the sample towards the test area and is captured by a second reagent in the test area;

[0011] (3) irradiating the test area of the detection device with excitation light emitted by the light source of the light analyzer, then turning off the light source, and using the detector of the light analyzer to capture the luminescence signals R1 and R2 generated by the first signal marker captured in the test area at the first time point T1 and the second time point T2 after turning off the light source, where T1 < T2;

[0012] (4) comparing the magnitude of R1 with the threshold value B of the light analyzer. When R1 ≤ B, the concentration of the analyte in the sample is calculated using R1; when R1 > B, the concentration of the analyte in the sample is calculated using KR2, where K > 1.

[0013] The present invention also provides a detection system, which includes a detection device and a light analyzer. The detection device carries a first reagent labeled with a first signal marker during detection. The first reagent can specifically bind to the analyte in the sample. After adding the sample, the first reagent labeled with the first signal marker can be captured in the test area of the detection device, and the first signal marker is a time-resolved luminescence marker; the light analyzer includes a light source, a detector, an analog-to-digital converter, and a processor. The light source emits excitation light to irradiate the test area, and the detector captures the luminescence signals R1 and R2 generated by the first signal marker captured in the test area at the first time point T1 and the second time point T2 after turning off the light source, where T1 < T2; the processor can compare the magnitude of R1 with the threshold value B of the light analyzer. When R1 ≤ B, the processor selects R1 to calculate the concentration of the analyte; when R1 > B, the processor selects KR2 to calculate the concentration of the analyte, where K > 1. In the present invention, the threshold value B of the light analyzer ≤ the detection upper limit A of the light analyzer.

[0014] In some embodiments of the present invention, the detection device includes an immunochromatographic test strip. The first reagent labeled with the first signal marker is located on the test strip during detection. The second reagent coated on the T line in the test area can capture the first reagent labeled with the first signal marker on the T line; (2) adding the sample to the sample addition area of the test strip, and the first reagent labeled with the first signal marker moves with the sample towards the T line and is captured by the second reagent on the T line.

[0015] In some embodiments of the present invention, first, an optical analyzer is used to measure the fluorescence signal R generated by the first signal marker captured in the test area (such as the T line) at time point T1 when the concentration of the analyte is at the lower limit of analyte detection. R is M times (M>1) the noise signal S of the optical analyzer at time point T1. Then, the concentration of the analyte is calculated as the luminescence signal R' that the first signal marker captured in the test area should theoretically generate when the analyte concentration is at the upper limit of analyte detection, where R' = R × upper limit of analyte detection / lower limit of detection. Next, the ratio of R' to A is calculated. K is the ratio of the luminescence signal values of the standard luminescence signal time decay curve of the first signal marker at the selected time points T1 and T2 and is greater than the ratio of R' to A, so as to improve the detection range of the analyte concentration. In addition, after adding the sample, in order to avoid the interference of the background fluorescence signal in the sample, at T1, the background luminescence signal in the sample completely disappears or the interference generated by the background luminescence signal can be ignored in the standard luminescence signal time decay curve of the first signal marker. In some embodiments of the present invention, R≥3×S. In some embodiments of the present invention, B is set to 70% - 95% of the detection upper limit A of the optical analyzer, and A = 2 N -1, and N is the word length of the analog-to-digital converter in the optical analyzer. In some embodiments of the present invention, B is set to 70% - 90% of the detection upper limit A of the optical analyzer. In some embodiments of the present invention, N = 16 and B = 50000.

[0016] In some embodiments of the present invention, the first signal marker is selected from lanthanide elements and their chelates, platinum / palladium porphyrin compounds, upconversion luminescent materials, and time-resolved luminescent microspheres. In some embodiments of the present invention, the first signal marker is a time-resolved fluorescent microsphere internally encapsulated with an europium chelate.

[0017] In some embodiments of the present invention, 200μs≤T1≤600μs and 900μs≤T2≤2000μs. In some embodiments of the present invention, T1 = 300μs and T2 = 1200μs. In some embodiments of the present invention, K>3.26. In some embodiments of the present invention, K≥4.

[0018] In some embodiments of the present invention, the first reagent labeled with the first signal marker is coated on the test strip or added to the test strip during detection.

[0019] In some embodiments of the present invention, when the analyte is an antigen, the first reagent and the second reagent are antibodies that specifically bind to the analyte, or the first reagent is an antibody that specifically binds to the analyte (such as a mouse IgG antibody that specifically binds to the analyte), and the second reagent is a secondary antibody that specifically binds to the first reagent (such as a rabbit anti-mouse IgG antibody that specifically binds to the mouse IgG antibody); when the analyte is an antibody, the first reagent and the second reagent are antigens that specifically bind to the analyte, or one of the first reagent and the second reagent is an antigen that specifically binds to the analyte and the other is a secondary antibody that specifically binds to the analyte, or the first reagent is an antigen that specifically binds to the analyte (such as the receptor-binding domain of the SARS-CoV-2 spike protein), and the second reagent (such as the human ACE protein) competes with the analyte (such as the SARS-CoV-2 neutralizing antibody) for binding to the first reagent; when the analyte is a hapten, the first reagent is an antibody that specifically binds to the analyte, and the second reagent is a conjugate of the analyte or its analog and a carrier protein (such as BSA, KLH, etc.).

[0020] In some embodiments of the present invention, the immunoassay strip includes a sample application pad, and the sample application area is located on the sample application pad. In some embodiments of the present invention, the labeling reagent is coated on the sample application pad. In some embodiments of the present invention, the immunoassay strip includes a labeling pad, the sample application area is located on the labeling pad, and the labeling reagent is coated on the labeling pad. In some embodiments of the present invention, the strip includes a sample application pad and a labeling pad, the labeling pad is located between the sample application pad and the detection pad, the sample application area is located on the sample application pad, and the labeling reagent is coated on the labeling pad.

[0021] In some embodiments of the present invention, a C line is further provided on the detection pad. The reference reagent labeled with the second signal label and the first reagent labeled with the first signal label are coated on the strip or added to the strip during detection. The reference capture reagent coated on the C line can capture the reference reagent labeled with the reference label on the C line. In some embodiments of the present invention, the reference reagent / reference capture reagent is selected from non-human antibodies / anti-non-human antibodies, biotin / streptavidin, DNP-BSA / anti-DNP antibodies, and receptors / ligands. In some embodiments of the present invention, the reference reagent / reference capture reagent is goat anti-rabbit IgG antibody / rabbit IgG antibody. In some embodiments of the present invention, the second signal label is selected from lanthanide elements and their chelates, platinum / palladium porphyrin compounds, time-resolved luminescent microspheres, colored luminescent microspheres, colored colloidal particles, magnetic nanoparticles, and luminescent compounds.

[0022] In some embodiments of the present invention, when the second signal marker is the same as the first signal marker, in step (3), the detector of the optical analyzer is further used to capture the luminescence signal R3 of the second signal marker captured on the C line at T1; in step (4), when R1 ≤ B, R1 is corrected by using R3 to obtain the corrected R1, and then the corrected R1 is used to calculate the concentration of the analyte; when R1 > B, KR2 is corrected by using R3 to obtain the corrected KR2, and then the corrected KR2 is used to calculate the concentration of the analyte. In some embodiments of the present invention, when R1 ≤ B, the correction of R1 is achieved by calculating the ratio of R1 / R3 or R1 / (R1 + R3), and the corrected R1 is the ratio of R1 / R3 or R1 / (R1 + R3); when R1 > B, the correction of KR2 is achieved by calculating the ratio of KR2 / R3 or KR2 / (KR2 + R3), and the corrected KR2 is the ratio of KR2 / R3 or KR2 / (KR2 + R3).

[0023] In the present invention, the standard luminescence signal time decay curve of the first signal marker refers to the values of the luminescence signals generated by a certain number of first signal markers at different time points after the light source is turned off under the condition that the luminescence signals generated after irradiating with the excitation light for a period of time and then turning off the light source do not exceed the detection range of the optical analyzer, and then the standard luminescence signal time decay curve of the first signal marker is plotted according to the different time points and the measured luminescence signal values.

[0024] Advantages: (1) When the luminescence signal R1 generated by detecting a high-concentration analyte in the sample at the time point T1 exceeds the detection upper limit of the analog-to-digital converter in the optical analyzer, based on the characteristic that the time-resolved luminescence signal generated after excitation by the time-resolved luminescence marker continuously decays over time, the luminescence signal R2 measured at the time point T2 after T1 is less than R1, and the farther the distance between T2 and T1, the smaller R2 is compared with R1. When R1 exceeds the detection upper limit of the analog-to-digital converter in the optical analyzer, R1 is indirectly measured by measuring R2 at this time. In this way, without reducing the current amplification factor of the optical analyzer and without replacing the analog-to-digital converter with a larger word length, the concentration of the high-concentration analyte in the sample can still be accurately and low-costly detected by using the existing optical analyzer, solving the problem of insufficient detection range of the analyte concentration in the conventional method; or when a larger detection range of the analyte concentration is required, a high-performance analog-to-digital converter needs to be replaced, thus increasing the cost; (2) When the analyte concentration in the sample is too high, it is often solved by diluting the sample. However, by using the detection method of the present invention, the concentration of the high-concentration analyte in the sample can be detected without diluting the sample, which not only saves the test time but also saves the test cost; (3) In addition, while accurately detecting the high-concentration analyte in the sample, the present invention does not affect the detection of the low-concentration analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 .A three-dimensional schematic diagram of an analyzer used in the present invention.

[0026] Figure 2 .A schematic diagram of the internal circuit of an analyzer used in the present invention.

[0027] Figure 3 . Schematic diagram of the decomposition of the test card used in the present invention.

[0028] Figure 4 . Schematic diagram of the immunoassay strip in the test card used in the present invention.

[0029] Figure 5 .Time decay curve of standard fluorescence signal of europium chelate after being excited by excitation light (wavelength 365nm), showing two measurement time periods: the first measurement time period (200~600μs) and the second measurement time period (900~2000μs). DETAILED DESCRIPTION

[0030] like Figure 1 and Figure 2 As shown, the optical analyzer 100 used in the present invention includes an analyzer housing 1, a display 2 and a test card insertion port 3. The test card 5 is connected to the optical analyzer 100 through the test card insertion port 3. After the clinical sample is added to the test card 5, it is inserted into the optical analyzer 100 for detection after a reaction time outside the optical analyzer 100, or the clinical sample is immediately inserted into the optical analyzer 100 after the test card 5 is added, and then the optical analyzer 100 reacts for a period of time before detection. The optical analyzer 100 also includes an optical system, an amplifier circuit 8, an analog-to-digital conversion chip (ADC) 9, a main control unit 10, a digital-to-analog conversion chip (DAC) 11, a light source drive circuit 12 and a feedback circuit 13 located inside the analyzer housing 1. The optical system includes a light source 4, an optical path structure 6 and a detector 7. The optical system can also be equipped with some optical devices such as narrowband filters or gratings as needed. The light source 4 can be selected from a light emitting diode (LED), a flash lamp and other appropriate light sources, such as an LED. The illumination of the light source 4 can be continuous or pulsed. The detector 7 can be selected from a photomultiplier tube, a photodiode, a charge coupled device, a charge injection detector or a CMOS photosensitive element, for example, a photodiode (PD), such as a silicon photodiode. A plurality of detectors 7 can be provided as required. Of course, the optical analyzer used in the present invention can also be selected from commercially available analyzers, such as the FIAflex of Acon Biotechnology (Hangzhou) Co., Ltd. TM Fluorescent Immunoassay Analyzer (iFIA-100).

[0031] The main control unit 10 includes a microprocessor. The control signal applied by the microprocessor is converted by the digital-to-analog conversion chip 11 and then provides a light source current control signal to the light source drive circuit 12. After being fed back by the feedback circuit 13, the light source 4 operates in a constant current state. During the scanning of the test card 5 by the optical system, the light emitted by the light source 4 irradiates the test card 5 with the sample to be tested added through an optical path structure (such as an optical fiber) 6. After the sample reacts for a period of time, the time-resolved luminescent markers captured on the T line and C line of the test card 5 generate optical signals after irradiation. The generated optical signals are output to the detector 7 through the optical path structure 6 (such as an optical fiber, different from the optical fiber through which the excitation light emitted by the light source 4 passes), and are converted into current signals by the detector 7. The generated current signals are modulated and converted into a suitable voltage range by the amplifier circuit 8 and then converted by the analog-to-digital conversion chip 9 and transmitted to the main control unit 10. The microprocessor in the main control unit 10 calculates the received analog-to-digital converted electrical signals to determine the presence or concentration of the analyte in the sample to be tested.

[0032] As Figure 3 and Figure 4 shown, the test card 5 includes a test strip 15, a card cover 14, and a card seat 16. The test strip 15 used is an immunochromatographic test strip, which includes a sample application pad 51, a labeling pad 52, a detection pad 53, and an absorbent pad 54 that are sequentially overlapped. The detection pad 53 is made of materials such as nitrocellulose, glass fiber, polyethersulfone, or nylon. Therefore, in some cases, the detection pad 53 is a nitrocellulose membrane. A detection line (also called the T line) 56 and a control line (also called the C line) 57 are provided on the detection pad 53. The sample application pad 51 is made of a water-absorbing material and can be selected from glass fiber or non-woven fabric. The labeling pad 52 is also made of a water-absorbing material and can be selected from polyester film, glass fiber, or non-woven fabric.

[0033] The number of detection lines provided on the detection pad 53 can be adjusted according to actual needs. For example, when detecting one analyte, only one detection line needs to be set. When detecting two or more analytes, the corresponding number of detection lines should be set.

[0034] The test strip 15 further includes a bottom support layer 55, which is made of a common hydrophobic material such as polyvinyl chloride. The sample application pad 51, the labeling pad 52, the detection pad 53, and the absorbent pad 54 are provided on the bottom support layer. One end of the sample application pad 51 partially overlaps with the labeling pad 52; one end of the labeling pad 52 partially overlaps with the sample application pad 51, and the other end of the labeling pad 52 partially overlaps with the detection pad 53; the absorbent pad 54 is made of a hydrophilic material; one end of the absorbent pad 54 partially overlaps with the detection pad 53. In addition, in some cases, the overlapping area between any two adjacent pads is 0.5 to 5 millimeters.

[0035] The test strip 15 is located within a housing, which is composed of a snap cap 14 and a card holder 16. A test strip slot 60 is provided in the middle of the card holder 16 for placing the test strip 15.

[0036] The snap cap 14 is also provided with a sample inlet 58 and an observation window 59. When a clinical sample is added through the sample inlet 58, the sample enters the sampling pad 51 located below the sample inlet 58. Under capillary action, the sample migrates along the length direction of the test strip 15 towards the absorbent pad 54. The observation window 59 is arranged above the test line 56 and the control line 57 of the detection pad 53. The light emitted by the light source 4 can irradiate the test line 56 and the control line 57 of the test strip 15 through the transparent or semi-transparent observation window 59. After being irradiated by the excitation light from the light source 4, the optical signals generated by the test line 56 and / or the control line 57 are output to the detector 7 through the optical path structure 6.

[0037] According to the differences in the analyte to be measured (such as antigen, antibody or hapten) and the immunoassay principle (double antigen sandwich method, double antibody sandwich method, competitive method, indirect method, capture method), the substances coated on the labeling pad 52 and the test line 56 will vary. Here, taking the analyte to be measured as IL-6, the detection principle as the double antibody sandwich method, and the time-resolved luminescence label as europium chelate as an example for illustration. The labeling pad 52 is coated with a first reagent labeled with europium chelate (the first anti-IL-6 antibody) and a reference reagent labeled with europium chelate. The test line 56 is coated with a second reagent (the second anti-IL-6 antibody). The first reagent and the second reagent specifically bind to IL-6 in the clinical sample. Thus, when a clinical sample is added to the sampling pad 51 through the sample inlet 58, the clinical sample flows along the length direction of the test strip 15. When the clinical sample reaches the labeling pad 52, the first reagent labeled with europium chelate specifically binds to IL-6 (if present) in the clinical sample. The formed europium chelate - first reagent - IL-6 complex continues to flow and specifically binds to the second reagent on the test line 56, thereby capturing the europium chelate - first reagent - IL-6 complex on the test line 56. After being excited by the excitation light from the light source, a detection signal related to the concentration of IL-6 can be generated. Meanwhile, the reference reagent labeled with europium chelate continues to flow. When it reaches the control line 57, the reference capture reagent coated on the control line 57 can capture the reference reagent labeled with europium chelate or the complex formed by the specific binding of the reference reagent labeled with europium chelate and a non-analyte on the control line 57. After being irradiated by the light from the light source, the europium chelate captured on the control line 57 generates a control signal.

[0038] In some cases, the reference capture reagent coated on the control line 57 directly captures the europium chelate-labeled reference reagent on the control line 57. At this time, the reference reagent and the reference capture reagent can be selected from any one of the following combinations: non-human antibody / anti-non-human antibody (for example, rabbit IgG antibody / goat anti-rabbit IgG antibody, chicken IgY antibody / goat anti-chicken IgY antibody), biotin / streptavidin, DNP-BSA / anti-DNP antibody, receptor / ligand, etc.

[0039] The non-analyte mentioned in the present invention refers to a substance that does not affect the detection of the analyte in the sample. It can be present in the sample or not. If it is not present in the sample, it can be added to the sample or the test strip in advance. In some cases, the non-analyte is a substance that is not present in the sample or is present in a negligible amount, so that the calibration signal is not affected by the clinical sample. Of course, the non-analyte can also be a substance present at a relatively high level in the sample. For example, when the analyte to be detected is IL-6, the non-analyte in the present invention can be human IgG present in the clinical sample. At this time, the reference reagent is a rabbit anti-human IgG antibody that specifically binds to human IgG, and the reference capture reagent coated on the control line 57 is a goat anti-rabbit IgG antibody that specifically binds to the rabbit anti-human IgG antibody.

[0040] The time-resolved luminescence marker in the present invention has the characteristic of luminescence delay, that is, when the excitation light emitted by the light source is turned off, they can still continuously generate emission light for a certain period of time. The wavelength of the emission light can be greater than or less than the wavelength of the excitation light. This luminescence delay characteristic makes the emission light generated by the time-resolved luminescence marker after excitation have a long lifetime. Thus, in actual detection, after the excitation light from the light source excites the time-resolved luminescence marker, the light source can be turned off and waited for a period of time, and then the emission light signal emitted by the time-resolved luminescence marker is detected, thereby eliminating the interference of the background light signal with a short lifetime and the scattered excitation light.

[0041] The time-resolved luminescence marker can exist in molecular form, called time-resolved luminescence molecule, and can be selected from lanthanide elements such as samarium (Sm(III)), dysprosium (Dy(III)), europium (Eu(III)) and terbium (Tb(III)) and their chelates, as well as upconversion luminescent materials; platinum / palladium porphyrin compounds that can emit phosphorescence after excitation. The delay time of the europium chelate is about 2 ms, and the emitted fluorescence signal gradually decays with time within 2 ms. In view of the fact that some proteins in clinical samples can also produce fluorescence under the excitation of the light source but do not have the delay characteristic, the fluorescence produced by most proteins completely disappears within 200 μs after the light source is turned off. Therefore, the time-resolved fluorescence detection of the europium chelate usually selects to collect the fluorescence signal at a time of 200 - 400 μs after the light source is turned off. In some cases, the standard fluorescence signal time decay curve of the europium chelate after excitation by the excitation light is as Figure 5As shown. A suitable europium chelate is N-(p-isothiocyanatophenyl)-diethylenetriaminepentaacetic acid-Eu +3 .

[0042] Time-resolved luminescence markers can also exist in another form: time-resolved luminescent microspheres, that is, time-resolved luminescent molecules are encapsulated inside or on the surface of natural or synthetic microspheres or microbeads. Each time-resolved luminescent microsphere can encapsulate tens of thousands of time-resolved luminescent molecules, effectively improving the detection sensitivity. Time-resolved luminescent microspheres that produce fluorescence after excitation are called time-resolved fluorescent microspheres.

[0043] Upconversion luminescent materials refer to materials that are excited by low-energy light and emit high-energy light. The optical signals generated by upconversion luminescent materials after excitation have strong stability, long lifetime, and high sensitivity, and are also suitable for time-resolved immunoassays. Common upconversion luminescent materials can be selected from Y 2 O 3 、Y 2 O 2 S, LaF 3 、NaYF 4 、NaGdF 4 、NaYF 4 :Yb 3+ / Nd 3+ / Ho 3+ 、NaGdF 4 :Yb 3+ / Nd 3+ / Ho 3+ 、Y 2 O 3 :Er, Yb, etc.

[0044] The C line of the present invention is not necessary. When the C line is provided, the reference signal C generated by the C line in the present invention can be used not only to indicate whether the added clinical sample has flowed to the absorbent pad 54, but also to correct the detection signal T generated by the time-resolved luminescent label on the T line. The corrected T signal can be used to calculate the analyte concentration in the sample. This correction can be achieved by calculating the ratio of T / C or T / (T + C), and the corrected T signal is T / C or T / (T + C). In addition, the reference signal C generated by the C line in the present invention can also be only used to indicate whether the added clinical sample has flowed to the absorbent pad 54 and is not used to correct the detection signal T. Therefore, the reference signal can also be a color signal, such as a colored aggregate generated after colored colloidal particles aggregate on the reference line 57. When a color signal appears on the reference line 57, it can be determined whether the added clinical sample has flowed to the absorbent pad 54. Thus, there is no need to collect optical signals such as reflected light, fluorescence, and phosphorescence generated by the C line after light source irradiation. Therefore, the second signal label can be selected from time-resolved luminescent labels, colored luminescent microspheres, colored colloidal particles, magnetic nanoparticles, and luminescent compounds. Colored luminescent microspheres refer to microspheres or microbeads with luminescent compounds such as quantum dots and fluorescent dyes wrapped on the surface or inside, and can generate optical signals without luminescence delay characteristics after being irradiated with excitation light of an appropriate wavelength, and can be selected from green fluorescent microspheres, blue fluorescent microspheres, red fluorescent microspheres, yellow fluorescent microspheres, and colored fluorescent microspheres (emitting multiple specific colors of fluorescence). Colored colloidal particles refer to colloidal particles that generate colored aggregates after aggregating on the T line and / or C line during the immunochromatographic reaction, and can be selected from latex, colloidal gold, colloidal carbon, and colloidal selenium. Luminescent compounds refer to those that can generate light rays without luminescence delay characteristics after being irradiated with excitation light of an appropriate wavelength, and can be selected from quantum dots; fluorescein and its derivatives, such as fluorescein isothiocyanate (FITC); fluorescent proteins and their improved variant forms that can emit fluorescence after excitation, such as green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, etc.; chemiluminescent labels, and can be selected from luminol, isoluminol and its derivatives, 1,2-dioxetane derivatives (commonly including AMPPD, CSPD, CDP, and CDP-Star, as well as PPD, Lumi-Phos, and Lumi-Plus of Lumigen company), and acridinium esters or acridinium sulfonamides.

[0045] In time-resolved luminescent microspheres and colored luminescent microspheres, the polymers forming the microspheres or microbeads can be selected from polystyrene, butadiene styrene, styrene acrylic acid-ethylene terpolymer, polymethyl methacrylate, polyethyl methacrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, polydivinylbenzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or their aldehyde group, carboxyl group, amino group, hydroxyl group, hydrazide derivatives, or mixtures thereof. In addition, the surface of the microspheres or microbeads usually carries groups such as hydroxyl group, carboxyl group, amino group, aldehyde group, sulfo group, etc., and can be coupled and bound together with antibodies or antigens or hapten-carrier protein conjugates through conventional chemical coupling reagents. In some cases, the particle size of the time-resolved luminescent microspheres is 20 nm to 100 μm.

[0046] When detecting one or more analytes in a sample, according to the different immunodetection principles, the intensity of the detection signal generated by the time-resolved luminescent label on the T line is positively or negatively correlated with the concentration of the analyte. Among them, except for the competitive method, the intensity of the detection signal generated by the time-resolved luminescent label on the T line is positively correlated with the concentration of the analyte.

[0047] The clinical samples in the present invention can be selected from serum, plasma, whole blood, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, nasopharyngeal swab, sputum, feces, skin lesion samples (including swabs of rash / pustule exudates; pustule fluid; scabs, etc.). According to the different types of clinical samples and analytes, some clinical samples need to be pretreated (such as lysed with lysis buffer to release the analytes to be detected) before being added to the test card 5 for detection.

[0048] The analytes that can be detected by the present invention include antigens or antibodies, and even haptens, such as inflammatory markers, heart failure markers, tumor markers, bone metabolism series markers, sex markers, thyroid function markers; infectious disease markers, diabetes markers, liver fibrosis markers, allergy markers, intestinal health series markers, etc.

[0049] Example 1: Preparation of the first type of test strip and test card of the present invention

[0050] A method for preparing a test strip and a test card for quantitatively detecting IL-6 in a sample includes the following steps:

[0051] A. Antibody preparation: Animals such as mice, rats, and rabbits can be immunized with antigens, or hybridoma cells can be counted and screened to obtain paired monoclonal or polyclonal antibodies for detecting IL-6. Commercially available paired IL-6 antibodies (one is an IL-6 capture antibody and the other is an IL-6 detection antibody) can also be selected. In this example, commercially available paired IL-6 antibodies (purchased from Phoenix Biotech Co., Ltd., product numbers IL6-MAB-F1-001 and IL6-MAB-F1-002) are used as an example for illustration. Rabbit IgG antibody and goat anti-rabbit IgG antibody are prepared in-house or purchased from the market.

[0052] B. Detection pad spotting solution

[0053] The IL-6 capture antibody is added to 0.02 M phosphate buffer (pH 7.2) to obtain the T-line solution, and the concentration of the IL-6 capture antibody in the T-line solution is 1 mg / ml. The goat anti-rabbit IgG antibody is added to 0.02 M phosphate buffer (pH 7.2) to obtain the C-line solution, and the concentration of the goat anti-rabbit IgG antibody in the C-line solution is 0.3 mg / ml. Then, a quantitative spotting device (AUTOKUN continuous membrane coating machine spotter HGS101 of Hangzhou Fenghang Technology Co., Ltd.) is used to coat the T-line solution and the C-line solution on a 2.5-cm-wide nitrocellulose membrane (purchased from Satorious, model 1UN95ER100025NT) used as the detection pad at a rate of 1 μl / cm with an interval of 0.8 cm, thereby forming the T-line and the C-line respectively. It is dried at 45°C for 18 hours and then sealed with a desiccant for standby.

[0054] C. Preparation of fluorescent microspheres:

[0055] Fluorescent microsphere selection: Time-resolved fluorescent microspheres (Merk, product number F1-XC010) with europium chelate encapsulated inside, having an excitation wavelength of 365 nm and an emission wavelength of 615 nm.

[0056] Preparation of MES buffer: Morpholineethanesulfonic acid sodium salt is added to pure water and mixed evenly to make the concentration of morpholineethanesulfonic acid sodium salt 1.0% (w / v).

[0057] Preparation of storage buffer: 50 mM (pH 8.0) Tris-HCl buffer.

[0058] Preparation of Time-Resolved Fluorescent Microsphere-Labeled IL-6 Detection Antibody: Wash the fluorescent microspheres with MES buffer, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to make their final concentrations 0.4 mg / ml and 0.1 mg / ml respectively, and react at room temperature for 20 minutes to activate the time-resolved fluorescent microspheres. After thoroughly washing the activated microspheres with MES buffer, add the IL-6 detection antibody at a ratio of 1 mg:0.1 mg (mass ratio), react at room temperature for 1.5 hours, and after thoroughly washing with MES buffer, add 0.05 M pH 8.0 Tris-HCl buffer containing 10% BSA (w / v), and block at room temperature for 1 hour. Wash the time-resolved fluorescent microspheres with MES buffer, redissolve the detected time-resolved fluorescent microspheres with the storage buffer to make the final concentration of the detected time-resolved fluorescent microspheres 5 mg / ml, and store at 4°C for later use; prepare the time-resolved fluorescent microsphere-labeled rabbit IgG antibody in the same way as the preparation of the time-resolved fluorescent microsphere-labeled IL-6 detection antibody, and use the storage buffer to make the final concentration of the time-resolved fluorescent microsphere-labeled rabbit IgG antibody 5 mg / ml, and store at 4°C for later use.

[0059] Finally, thoroughly mix the time-resolved fluorescent microsphere-labeled IL-6 detection antibody, the time-resolved fluorescent microsphere-labeled rabbit IgG antibody and the fluorescent label diluent in a volume ratio of 6:1:13 to obtain a fluorescent microsphere mixture, where the fluorescent label diluent is 50 mM (pH 8.0) Tris-HCl buffer containing 15% sucrose (w / v), 5% trehalose (w / v), 2% Tween-20 (v / v), 0.5% PVP (w / v), and 0.5% BSA (w / v).

[0060] D. Spraying and Drying of Fluorescent Microspheres

[0061] Use the special liquid-drawing head of the AUTOKUN continuous membrane-drawing machine dotting instrument HGS101 to evenly spray the prepared fluorescent microsphere mixture on a 1.0 cm-wide marking pad (glass fiber) at a rate of 2 μl / cm, dry at 45°C for 18 hours, and add a desiccant for storage.

[0062] E. Treatment of Sample Pad

[0063] Soak a 2.5 cm-wide sample pad in the sample pad treatment solution for 1 hour, then take it out and dry it at 37°C overnight (12 - 24 h). The sample pad treatment solution is a buffer containing 50 mM (pH 8.0) Tris-HCl buffer, 1% BSA (w / v), and 0.5% Tween-20 (v / v).

[0064] E. Assembly and Cutting of Test Strip Plate

[0065] Assembly of the test strip plate: Manually or by machine, a 2.5-cm-wide sample pad, a 1.0-cm-wide marker pad, a 2.5-cm-wide nitrocellulose membrane, and a 2.5-cm-wide absorbent paper (as the absorbent pad) are adhesively pasted onto an 8-cm-long plastic base plate (as the bottom support layer) such that the 2.5-cm-wide sample pad, the 1.0-cm-wide marker pad, the 2.5-cm-wide nitrocellulose membrane, and the 2.5-cm-wide absorbent paper overlap each other alternately by 2.0 mm in sequence to assemble the test strip plate.

[0066] Cutting of the test strip plate: The assembled test strip plate is cut into 4-mm-wide single-person test strips using an AUTOKUN HGS201 strip cutter.

[0067] F. Assembly of the test card

[0068] Place the cut single-person test strip into the card slot on the plastic card holder, cover the card cover, and use a card pressing machine or manually press the card holder and the card cover tightly to ensure that the entire immunoassay test strip is in a taut state. Add a desiccant and seal it at room temperature for standby.

[0069] After the test card is assembled, it can be placed into a kit containing a desiccant and an instruction manual. In addition, according to needs, the kit can also contain a sampling tube, a test tube containing lysis solution, a test tube containing sample diluent, a sampling swab, etc.

[0070] Example 2: Preparation of the second type of test strip and test card of the present invention

[0071] The differences between this example and the first example are as follows: (1) The paired antibodies for detecting N-terminal pro-brain natriuretic peptide (NT-proBNP) --- NT-proBNP detection antibody and NT-proBNP capture antibody (purchased from GenScript Biotech Corporation, product numbers V00706 and V00711) are used to replace the IL-6 detection antibody and IL-6 capture antibody in Example 1 respectively; (2) The fluorescent label diluent is a 50 mM (pH 8.0) Tris-HCl buffer containing 15% sucrose (w / v), 5% trehalose (w / v), 2% Tween-20 (v / v), 0.5% PVP (w / v), and 0.5% Casien (w / v). This example can prepare test strips and test cards for detecting NT-proBNP in samples.

[0072] Example 3: Preparation of standard test strips and standard test cards

[0073] The preparation methods of the standard test strip and the standard test card are as follows: Add time-resolved fluorescent microspheres (Merk product number F1-XC 010) into Tris-HCl (pH 8.0) buffer solution containing 0.5% NaCl (w / v) to make the concentration of the time-resolved fluorescent microspheres 1 mg / ml. Then, directly coat the obtained time-resolved fluorescent microsphere solution onto the T line of the nitrocellulose membrane (purchased from Satorious, model 1UN95ER100025NT) in an amount of 1 μl / cm by means of scribing, and dry it. Paste the nitrocellulose membrane coated with time-resolved fluorescent microspheres onto the plastic base plate with adhesive tape, and then cut it into standard test strips with a width of 4 mm. Place the cut single-person test strip into the card slot on the plastic card holder, cover the card cover, and use a card pressing machine or manually press the card holder and the card cover tightly to form a standard test card that can generate a stable fluorescent signal. Additionally, when preparing the standard test strip and the test card, do not add time-resolved fluorescent microspheres (Merk product number F1-XC 010), thereby blank test strips and blank test cards can be prepared for measuring the background fluorescent signal.

[0074] Example 4: Detection method

[0075] Use the test card prepared in Example 1 or Example 2 to detect the analyte (such as IL-6, NT-proBNP, etc.) in the sample. The specific process is as follows: Add 100 μl of the sample (here, the whole blood sample is taken as an example for illustration) into the sample inlet of the test card. After incubating for 15 min, insert the test card into the optical analyzer for detection. The word length of the analog-to-digital converter in the optical analyzer determines the detection upper limit of the optical analyzer. When the word length of the analog-to-digital converter is N and the counting range is 0 to 2 N -1. The word length of the analog-to-digital converter of the used optical analyzer is 16 bits, and the counting range is 0 to 65535 (2 16 -1), that is, the detection upper limit A of the optical analyzer is 65535. When the concentration of the analyte in the sample is very high, the fluorescent signal emitted by the time-resolved fluorescent microspheres captured on the T line may exceed the detection upper limit A of the optical analyzer. The common practice is to dilute the sample and then measure the diluted sample, which will increase the test time and cost.

[0076] After inserting the test card into the optical analyzer, the present invention uses an optical system to scan the test card. During the scanning, first, the light source emits excitation light with a wavelength of 365 nm to irradiate the time-resolved fluorescence microspheres captured on the T line and the C line respectively, and each line is irradiated for 2 - 8 ms. Then, the light source is turned off, and the time-resolved fluorescence microspheres containing europium chelate captured on the T line and the C line emit fluorescence signals with a wavelength of 615 nm. Utilizing the characteristic that the fluorescence signal of the europium chelate gradually decays, at the first time point T1 (200 - 600 μs) and the second time point T2 (900 - 2000 μs) after the light source is turned off, the detector of the optical analyzer is used to capture and count, and the fluorescence signals R1 and R2 emitted from the T line at T1 and T2 and (if necessary) the fluorescence signal R3 emitted from the C line at T1 are obtained respectively.

[0077] In view of the situation that sometimes occurs: when the measured fluorescence signal is near the detection upper limit A of the optical analyzer, due to the measurement error existing in the optical analyzer itself, it is not clear at this time whether the measured fluorescence signal is actually less than or equal to the detection upper limit A of the optical analyzer or greater than the detection upper limit A of the optical analyzer. To avoid this situation, a demarcation value B is set, and the size of B is 70% - 90% of the detection upper limit A. For example, when A = 65535, B can be selected as 50000, and at this time B is 76.3% of A.

[0078] After determining the demarcation value B of the optical analyzer, compare the magnitudes of R1 and B. When R1 ≤ B, at this time, select R1 to calculate the analyte concentration in the sample; when R1 > B, select KR2 to calculate the analyte concentration in the sample. This calculation can be carried out in two ways: (1) When there is no C line or the signal emitted by the C line does not participate in the correction, only use R1 (when R1 ≤ B) or KR2 (when R1 > B) to calculate the analyte concentration in the sample; (2) When the signal R3 emitted by the C line participates in the correction, use R3 to correct R1 (when R1 ≤ B) or KR2 (when R1 > B) to obtain the corrected R1 (when R1 ≤ B) or KR2 (when R1 > B), and then use the corrected R1 (when R1 ≤ B) or the corrected KR2 (when R1 > B) to calculate the concentration of the analyte. In some cases, when R1 ≤ B, the correction of R1 is achieved by calculating the ratio of R1 / R3 or R1 / (R1 + R3), and the corrected R1 is the ratio of R1 / R3 or R1 / (R1 + R3); when R1 > B, the correction of KR2 is achieved by calculating the ratio of KR2 / R3 or KR2 / (KR2 + R3), and the corrected KR2 is the ratio of KR2 / R3 or KR2 / (KR2 + R3).

[0079] Example 5: Determination of the K value

[0080] The excitation light (wavelength 365 nm) emitted by the light source of the optical analyzer is used to irradiate the T line of the standard test card prepared in Test Example 3. Then, the light source is turned off, and the fluorescence signal emitted from the T line is read at different delay times (all the measured fluorescence signals are within the measurement range of the optical analyzer). The results are shown in the following table, and the resulting standard fluorescence signal time decay curve is as shown in Figure 5 shown. The background fluorescence signal can be measured by irradiating the blank test card prepared in Example 3 with the excitation light (wavelength 365 nm) emitted by the light source of the optical analyzer. It can be seen that the background fluorescence signal completely disappears 200 μs after the light source is turned off.

[0081]

[0082] The calculation method of the K value is to divide the average value of the fluorescence signal R1 at the delay time T1 by the average value of the fluorescence signal R2 at the delay time T2. For example, when the average value of R1 at T1 of 200, 300, 400, 600 μs is divided by the average value of R2 at T2 of 900 μs, the K values can be calculated as 3.02, 2.64, 2.19, and 1.58 respectively; when the average value of R1 at T1 of 200, 300, 400, 600 μs is divided by the average value of R2 at T2 of 1200 μs, the K values can be calculated as 5.15, 4.51, 3.73, and 2.70 respectively, and so on.

[0083] In addition, when calculating the K value, it can also be obtained by calculating the ratio of the single measurement of R1 at T1 to the single measurement of R2 at T2, or by calculating the ratio of the average value of at least two parallel measurements of R1 at T1 to the average value of at least two parallel measurements of R2 at T2.

[0084] For IL-6, the detection range of IL-6 is 3 - 4000 pg / mL, and the ratio of the upper detection limit to the lower detection limit is 1333.33 times; for NT-proBNP, the detection range of NT-proBNP is 15 - 20000 pg / L, and the ratio of the upper detection limit to the lower detection limit is also 1333.33 times. The noise signal of the optical analyzer at time point T1 is 30, and the signal-to-noise ratio of the fluorescence signal R generated on the T line when detecting IL-6 with a concentration of the lower detection limit (3 pg / mL) or NT-proBNP with a concentration of the lower detection limit (15 pg / L) at time point T1 is ≥ 3. Here, it is selected as 160. Therefore, when the concentration of IL-6 is the upper detection limit (4000 pg / mL) or the concentration of NT-proBNP is the upper detection limit (20000 pg / L), the fluorescence signal R' generated on the T line at time point T1 should theoretically reach 213332.8 (160 × 1333.33), which is 3.26 times the maximum count value 65535 of the commonly used 16-bit analog-to-digital converter in the optical analyzer. Therefore, K > 3.26 should be selected. After determining K > 3.26, the selection of time points T1 and T2 can be determined. From the above table and Figure 5 As can be seen, when T1 = 200 μs and T2 = 1200 μs, K = 5.15 > 3.26; when T1 = 300 μs and T2 = 1200 μs, K = 4.51 > 3.26; when T1 = 400 μs and T2 = 1200 μs, K = 3.73 > 3.26.

[0085] In view of the characteristic that the fluorescence signal generated after the excitation of the europium chelate continuously decays over time, the fluorescence signal R2 measured at time point T2 after T1 is less than R1. The farther T2 is from T1, the smaller R2 is compared to R1, and the larger K is. Even when the IL-6 concentration in the sample is greater than the upper detection limit, as long as T2 selected is farther from T1, R1 can still be indirectly measured by measuring KR2. Therefore, using the characteristic of the fluorescence signal generated by the europium chelate decaying over time to obtain the signal values at different time points can solve the problem of insufficient detection range of analyte concentration existing in conventional optical analyzers.

[0086] Example 6: IL-6 Detection Results

[0087] The test strip and test card for quantitatively detecting IL-6 in serum were prepared using the preparation method in Example 1.

[0088] A series of IL-6 reference standards with concentrations of 3.21 pg / ml, 30.85 pg / ml, 310.5 pg / ml, 989.2 pg / ml, 1985 pg / ml, and 3942 pg / ml were prepared. 100 μl of each IL-6 reference standard was added to the IL-6 test card, and the fluorescence signals were read using the following three methods. Each concentration was tested 5 times, and the coefficient of variation (CV) of the fluorescence signal values at each concentration was calculated.

[0089] Method 1: With the normal current amplification factor, the fluorescence signal was captured once every 300 μs. The measured fluorescence signals are shown in the following table:

[0090]

[0091]

[0092] Obviously, the fluorescence signals of the high-concentration IL-6 reference standards (1985 pg / mL and 3942 pg / mL) had no gradient and could not be distinguished.

[0093] Using the average fluorescence signal (T line) as the X-axis and the IL-6 reference standard concentration as the Y-axis, a standard curve was made and a piecewise linear regression equation was established. The test results are calculated as shown in the following table:

[0094]

[0095] As can be seen from the above table, the accuracy and repeatability of the test results for the high-concentration IL-6 reference standards were both poor.

[0096] Method 2: The current amplification factor was reduced to 1 / 4, and the fluorescence signal was captured once every 300 μs. The measured fluorescence signals are shown in the following table.

[0097]

[0098]

[0099] For the low-concentration IL-6 reference standard (3.21 pg / mL), due to the too low fluorescence signal, the consistency was very poor and the test results were inaccurate.

[0100] Using the average fluorescence signal as the X-axis and the IL-6 reference standard concentration as the Y-axis, a standard curve was made and a piecewise linear regression equation was established. The test results are calculated as shown in the following table:

[0101]

[0102] Compared with the test results of Method 1, Method 2 had great progress, but for the low-concentration IL-6 reference standard (3.21 pg / mL), due to the influence of the noise of the light analyzer, the repeatability was poor.

[0103] Method 3: Normal current amplification factor, capture the fluorescence signal once at 300 μs and once at 1200 μs

[0104] During the measurement, the K value was 4.51 and B was 50000. Compare the magnitude of the fluorescence signal R1 captured at 300 μs with B. When R1 ≤ B, select R1 as the fluorescence signal and use it to calculate the concentration of IL-6; when R1 > B, select KR2 as the fluorescence signal and use it to calculate the concentration of IL-6. The measured fluorescence signals are shown in the following table:

[0105]

[0106] The test results of Method 3 show good repeatability for the test results of the low-concentration IL-6 reference product and good gradient for the high-concentration IL-6 reference product.

[0107] Using the average fluorescence signal as the X-axis and the concentration of the IL-6 reference product as the Y-axis, make a standard curve and establish a piecewise linear regression equation. The calculated test results are shown in the following table:

[0108]

[0109]

[0110] The test results of Method 3 are both good in repeatability and accuracy.

[0111] Example 7: NT-proBNP test results

[0112] Using the preparation method in Example 2, prepare test strips and test cards for quantitatively detecting NT-proBNP in serum. Configure a series of NT-proBNP reference products with concentrations of 16.5 pg / ml, 151.2 pg / ml, 1188 pg / ml, 3965 pg / ml, 9827 pg / ml, and 19287 pg / ml respectively. Take 100 μl of each NT-proBNP reference product, add it to the NT-proBNP test card, and read the fluorescence signal using the following three methods respectively. Each concentration is tested 5 times repeatedly, and calculate the coefficient of variation CV of the fluorescence signal values at each concentration.

[0113] Method 1: Normal current amplification factor, capture the fluorescence signal once at 300 μs. The measured fluorescence signals are shown in the following table:

[0114]

[0115] Obviously, there is no gradient for the high-concentration NT-proBNP reference products (9827 pg / mL and 19287 pg / mL).

[0116] Taking the average fluorescence signal as the X-axis and the NT-proBNP reference concentration as the Y-axis, a standard curve was made and a piecewise linear regression equation was established. The test results are calculated as follows:

[0117]

[0118] As can be seen from the above table, the accuracy and repeatability of the test results for high-concentration NT-proBNP reference are both poor.

[0119] Method 2: Reduce the current amplification factor to 1 / 4 and capture the fluorescence signal once every 300 μs. The measured fluorescence signals are shown in the following table:

[0120]

[0121] Due to the too low time-resolved signal, the consistency of the low-concentration NT-proBNP reference (16.5 pg / mL) is very poor and the test results are inaccurate.

[0122] Taking the average fluorescence signal as the X-axis and the NT-proBNP reference concentration as the Y-axis, a standard curve was made and a piecewise linear regression equation was established. The test results are calculated as follows:

[0123]

[0124] Compared with the test results of Method 1, Method 2 has made great progress, but for the low-concentration NT-proBNP reference (16.5 pg / mL), due to the influence of the analyzer noise on fluorescence, the repeatability is poor.

[0125] Method 3: Normal current amplification factor, capture the fluorescence signal once at 300 μs and once at 1200 μs respectively

[0126] During the measurement, the value of K is 4.51 and B is 50000. Compare the magnitude of the fluorescence signal R1 captured at 300 μs with B. When R1 ≤ B, select R1 as the fluorescence signal and use it to calculate the concentration of NT-proBNP; when R1 > B, select KR2 as the fluorescence signal and use it to calculate the concentration of NT-proBNP. The measured fluorescence signals are shown in the following table:

[0127]

[0128] For Method 3, the test results show good repeatability for the low-concentration NT-proBNP reference and good fluorescence signal gradient for the high-concentration NT-proBNP reference.

[0129] Taking the average fluorescence signal as the X-axis and the NT-proBNP reference concentration as the Y-axis, a standard curve was made and a piecewise linear regression equation was established. The test results are calculated as follows:

[0130]

[0131] The repeatability and accuracy of the test results of Method 3 are both good.

Claims

1. A method for detecting an analyte, the steps comprising: (1) Providing a detection device which, during detection, carries a first reagent labeled with a first signal marker. The first reagent can specifically bind to the analyte in the sample. After adding the sample, the first reagent labeled with the first signal marker can be captured in the test area of the detection device. The first signal marker is a time-resolved luminescence marker; (2) Adding the sample to the detection device, and the first reagent labeled with the first signal marker moves with the sample towards the test area and is captured by a second reagent in the test area; (3) Using the excitation light emitted by the light source of the light analyzer to irradiate the test area of the detection device, then turning off the light source, and using the detector of the light analyzer to capture the luminescence signals R1 and R2 generated by the first signal marker captured in the test area at the first time point T1 and the second time point T2 after turning off the light source, where T1 < T2; (4) Comparing the magnitude of R1 with the demarcation value B of the light analyzer. When R1 ≤ B, calculating the concentration of the analyte in the sample using R1. When R1 > B, calculating the concentration of the analyte in the sample using KR2, where K > 1.

2. The method according to claim 1, wherein, the detection device comprises an immunochromatographic test strip. The first reagent labeled with the first signal marker is located on the test strip during detection. The second reagent coated on the T line in the test area can capture the first reagent labeled with the first signal marker on the T line; (2) Adding the sample to the sample application area of the test strip, and the first reagent labeled with the first signal marker moves with the sample towards the T line and is captured by the second reagent on the T line.

3. The method according to claim 1, wherein, using the light analyzer to measure that the luminescence signal R generated by the first signal marker captured in the test area of the detection device at T1 when the concentration of the analyte is the lower limit of analyte detection is M times the noise signal S of the light analyzer at T1, where M > 1. Then calculating the luminescence signal R' that should theoretically be generated by the first signal marker captured in the test area of the detection device when the concentration of the analyte is the upper limit of analyte detection, R' = R × upper limit of analyte detection / lower limit of analyte detection. Then calculating the ratio of R' to the detection upper limit A of the light analyzer. K is the ratio of the luminescence signal values of the standard luminescence signal time decay curve of the first signal marker at T1 and T2 and is greater than the ratio of R' to A.

4. The method according to claim 3, wherein, R ≥ 3 × S.

5. The method according to claim 1, wherein, B is set to 70% - 95% of the detection upper limit A of the optical analyzer, and A = 2 N -1, where N is the word length of the analog-to-digital converter in the optical analyzer.

6. The method according to claim 1, wherein, the first signal marker is selected from lanthanide elements and their chelates, platinum / palladium porphyrin compounds, upconversion luminescent materials, and time-resolved luminescent microspheres.

7. The method according to claim 6, wherein, the first signal marker is a time-resolved fluorescence microsphere internally encapsulated with europium chelate.

8. A detection system, the detection system comprising a detection device and a light analyzer, wherein, The detection device carries a first reagent labeled with a first signal marker during detection. The first reagent can specifically bind to the analyte in the sample. After adding the sample, the first reagent labeled with the first signal marker can be captured in the test area of the detection device. The first signal marker is a time-resolved luminescence marker; The optical analyzer includes a light source, a detector, an analog-to-digital converter, and a processor. The light source emits excitation light to irradiate the test area. The detector captures the luminescence signals R1 and R2 generated by the first signal marker captured in the test area at the first time point T1 and the second time point T2 after turning off the light source, where T1 < T2; The processor can compare the magnitudes of R1 and the threshold value B of the optical analyzer. When R1 ≤ B, the processor selects R1 to calculate the concentration of the analyte. When R1 > B, the processor selects KR2 to calculate the concentration of the analyte, where K > 1.

9. The method according to claim 8, characterized in that, When using the optical analyzer to measure that the concentration of the analyte is the lower limit of analyte detection, the luminescence signal R generated by the first signal marker captured in the test area of the detection device is M times the noise signal S of the optical analyzer at the time point T1, where M > 1. Preferably, R ≥ 3×S. Then calculate the luminescence signal R' that the first signal marker captured in the test area should theoretically generate when the concentration of the analyte is at the upper limit of analyte detection: R × upper limit of analyte detection / lower limit of detection. Then calculate the ratio of R' to A. K is the ratio of the luminescence signal values of the standard luminescence signal time decay curve of the first signal marker at T1 and T2 and is greater than the ratio of R' to A.

10. The method according to claim 8, characterized in that, B is set to be 70% to 95% of the detection upper limit A of the optical analyzer, and A = 2 N -1, where N is the word length of the analog-to-digital converter.

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