CN120084765A

CN120084765A - A method and system for detecting an analyte

Info

Publication number: CN120084765A
Application number: CN202311637207.5A
Authority: CN
Inventors: 李和平; 魏永超; 郭礼江
Original assignee: Leadway HK Ltd
Current assignee: Leadway HK Ltd
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2025-06-03

Abstract

The present invention provides a method and system for detecting an analyte, which involves, after adding a sample to an immunochromatographic test strip, irradiating the T line of the test strip with excitation light emitted by a light source of an optical analyzer, detecting luminescent signals R1 and R2 emitted by the T line at two different time points after the light source is turned off, R2=KR1, comparing R1 with a cutoff value B of the optical analyzer, and when R1≤B, calculating the analyte concentration in the sample using R1; when R1>B, calculating the analyte concentration in the sample using KR2. When the fluorescence signal generated by a high-concentration analyte exceeds the upper detection limit of the optical analyzer, the present invention still accurately and inexpensively obtains the concentration value of the high-concentration analyte, thereby solving the problem of insufficient detection range of conventional optical analyzers, and avoiding the use of a sample dilution method to test samples of high-concentration analytes.

Description

Analyte detection method and detection system

Technical Field

The invention belongs to the technical field of biological detection, and relates to a detection method and a detection system of an analyte.

Background

Fluorescent immunochromatography is one of the common techniques for clinical diagnosis today. Based on the principle of immunochromatography, fluorescent markers are gathered in a test area, light with a wavelength different from that of excitation light of a light source is emitted under the irradiation of the light source with a specific wavelength, and finally the light is captured by a photoelectric sensing device to form a tiny current, and the magnitude of the current is related to the concentration of an analyte in a sample, so that the analyte is qualitatively and/or quantitatively detected.

The current captured by the photoelectric sensing device is very weak, and the current signal can be formed for later result calculation after the current is amplified by the amplifying circuit. Therefore, in designing a light analyzer, it is often necessary to select a reasonable current amplification factor. The current amplification is generally dependent on two aspects:

(1) The lower limit of current amplification is typically 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 3pg/mL, and the lower limit of detection of N-terminal brain natriuretic peptide precursor (NT-proBNP) is 15pg/L, the optical analyzer used should be capable of having better resolution even when the fluorescent signal is extremely low. In other words, the optical analyzer is required to have a large current amplification factor without affecting the signal-to-noise ratio.

(2) The upper limit of current amplification is typically determined by the upper limit of detection of the analyte and the word length of the analog-to-digital converter. The detection range of clinical analytes varies widely, and some analytes have smaller detection ranges, for example, serum Amyloid A (SAA) detection ranges of 5-200 mg/L, upper detection limit/lower detection limit ratio of 40 times, and some analytes have larger detection ranges, for example, IL-6 detection ranges of 3-4000 pg/mL, and upper detection limit/lower detection limit ratio of 1333 times. From the cost point of view, the word length of the current common analog-to-digital converter is 16 bits, and the counting range is 0-65535 (2 ¹⁶ -1). In order to prevent the current amplified by the amplifying circuit from exceeding the upper limit of the count of the analog-to-digital converter, the current amplification factor of the optical analyzer should not be too large, or else the current amplified by the amplifying circuit exceeds the upper limit of the count of the analog-to-digital converter.

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 3pg/mL IL-6 is detected by an optical analyzer is generally regulated to about 160, and theoretically calculated by using the detection upper limit/detection lower limit ratio of IL-6 as 1333.33 times, the upper limit signal can reach 213332.8, and the maximum counting range of an analog-to-digital converter in the optical analyzer is far larger than 65535.

Conventional methods for solving this problem generally include (1) diluting a high-concentration clinical sample with a sample diluent and then retesting, (2) reducing the current amplification factor, and (3) replacing the analog-to-digital converter to expand the count range.

However, the first method is extremely inconvenient for the user, and this also increases 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, the upper limit signal is 53333.2, which accords with the requirement of a hardware chip, and the fluorescence signal generated by the detection lower limit (3 pg/mL) of IL-6 is also reduced from 160 to 40, however, in view of the fact that the noise signal of a conventional optical analyzer is about 20, the ratio of the noise signal is 50% in the signal-to-noise ratio, and the inaccuracy of the test result is easily caused. The third method can effectively enlarge the counting range, but in view of the fact that the mainstream analog-to-digital converter in the current market is 16 bits, the analog-to-digital converter technology with larger word length is still immature, the performance is unstable, and the price is high. Thus, there is an urgent need in the marketplace for a new method for accurately and cost-effectively detecting high concentrations of analytes in samples without dilution.

Disclosure of Invention

Aiming at the defects of the prior art, the invention discovers that the immunoassay method based on the time-resolved luminescent marker can accurately and low-cost acquire the concentration value of the high-concentration analyte in the sample under the condition that the detection of the low-concentration analyte is not affected, when the luminescent signal generated by the high-concentration analyte during the detection exceeds the detection upper limit of the optical analyzer, thereby solving the problem of insufficient detection range of the analyte concentration existing in the conventional optical analyzer, avoiding the use of a sample dilution method to detect the concentration of the high-concentration analyte, saving the test time and the test cost.

The invention provides a detection method of an analyte, which comprises the steps of (1) providing a detection device, wherein the detection device carries a first reagent marked by a first signal marker when in detection, the first reagent can specifically bind to the analyte in a sample, after the sample is added, the first reagent marked by the first signal marker can be captured in a test area of the detection device, and the first signal marker is a time-resolved luminescent marker;

(3) Illuminating a test area of the detection device with excitation light emitted by a light source of the light analyzer, then turning off the light source, capturing luminescence signals R1 and R2 generated by a first signal marker captured in the test area at a first time point T1 and a second time point T2 after the light source is turned off by a detector of the light analyzer, T1< T2;

(4) Comparing the magnitude of the boundary value B of R1 and the light analyzer, when R1 is less than or equal to B, calculating the analyte concentration in the sample by using R1, and when R1 is more than B, calculating the analyte concentration in the sample by using KR2, wherein K is more than 1.

The invention also provides a detection system which comprises a detection device and an optical analyzer, wherein the detection device carries a first reagent marked by a first signal marker when detecting, the first reagent can specifically bind with an analyte in a sample, the first reagent marked by the first signal marker can be captured in a test area of the detection device after the sample is added, the first signal marker is a time resolution luminous marker, the optical analyzer comprises a light source, a detector, an analog-to-digital converter and a processor, the light source emits excitation light to illuminate the test area, the detector captures luminous signals R1 and R2 generated by the first signal marker captured in the test area at a first time point T1 and a second time point T2 after the light source is turned off, T1< T2, the processor can compare the boundary value B of R1 and the optical analyzer, when R1 is less than or equal to B, the processor selects R1 to calculate the concentration of the analyte, when R1 is more than B, the processor selects KR2 to calculate the concentration of the analyte, and K >1. In the invention, the demarcation value B of the optical analyzer is less than or equal to the detection upper limit A of the optical analyzer.

In some embodiments of the invention, the detection device comprises an immunochromatographic strip, a first reagent labeled with a first signal marker is positioned on the strip during detection, a second reagent coated on a T line positioned in a test area can capture the first reagent labeled with the first signal marker on the T line, and (2) a sample is added to a sample adding area of the strip, and the first reagent labeled with the first signal marker moves towards the T line along with the sample and is captured by a second reagent on the T line.

In some embodiments of the present invention, the fluorescence signal R generated by the first signal marker captured in the test area (such as T line) at the time point T1 is measured by the optical analyzer when the concentration of the analyte is the lower limit of analyte detection, where R is M times (M > 1) the noise signal S of the optical analyzer at the time point T1, then the luminescence signal R ' =r×the upper limit of analyte detection that should be theoretically generated by the first signal marker captured in the test area at the upper limit of analyte detection is calculated, and then the ratio of R ' to a is calculated, where 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 that the detection range of the analyte concentration can be improved. In addition, after the sample is added, in order to avoid interference of the background fluorescent signal in the sample, the standard luminescence signal time attenuation curve of the first signal marker is at the time of T1, the background luminescence signal in the sample completely disappears or the interference generated by the background luminescence signal is negligible. In some embodiments of the invention, R.gtoreq.3 XS. In some embodiments of the present invention, B is set to 70% -95% of the detection upper limit a of the optical analyzer, 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 invention, B is set to 70% -90% of the detection upper limit A of the optical analyzer. In some embodiments of the invention, n=16, b=50000.

In some embodiments of the invention, the first signal label is selected from the group consisting of lanthanoids and chelates thereof, platinum/palladium porphyrin compounds, up-conversion luminescent materials, and time-resolved luminescent microspheres. In some embodiments of the invention, the first signal marker is a time-resolved fluorescent microsphere with europium chelate coated inside.

In some embodiments of the present invention, T1 is less than or equal to 200 mu s and less than or equal to 600 mu s, T2 is less than or equal to 900 mu s and less than or equal to 2000 mu s. In some embodiments of the invention, t1=300 μs and t2=1200 μs. In some embodiments of the invention, K >3.26. In some embodiments of the invention, K.gtoreq.4.

In some embodiments of the invention, the first reagent labeled with the first signal marker is coated on the test strip or added to the test strip at the time of detection.

In some embodiments of the invention, the first and second reagents are antibodies that specifically bind to the analyte when the analyte is an antigen, 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), the second reagent is a second antibody that specifically binds to the first reagent (such as a rabbit anti-mouse IgG antibody that specifically binds to a mouse IgG antibody), when the analyte is an antibody, the first and second reagents are antibodies that specifically bind to the analyte, or one of the first and second reagents is an antigen that specifically binds to the analyte, the other is a second antibody that specifically binds to the analyte, or the first reagent is an antigen that specifically binds to the analyte (such as a receptor binding domain of SARS-CoV-2 spike protein), the second reagent (such as a human protein) competes with the analyte (such as a SARS-CoV-2 neutralizing antibody) for binding to the first reagent, and when the analyte is a hapten, the first reagent is an antibody that specifically binds to the analyte, the second reagent is an antibody that specifically binds to the analyte, or a conjugate thereof with an analyte (such as H, an analog of an carrier, or the like).

In some embodiments of the invention, an immunoassay test strip includes a loading pad, and a loading zone is located on the loading pad. In some embodiments of the invention, the labeling reagent is coated on the loading pad. In some embodiments of the invention, the immunoassay test strip includes a label pad, the sample application region is positioned on the label pad, and the labeling reagent is coated on the label pad. In some embodiments of the invention, the test strip includes a loading pad and a labeling pad, the labeling pad is positioned between the loading pad and the detection pad, the loading region is positioned on the loading pad, and the labeling reagent is coated on the labeling pad.

In some embodiments of the present invention, the detection pad is further provided with a C line, and the second signal marker-labeled reference reagent and the first signal marker-labeled first reagent are coated on the test strip, or are added to the test strip during detection, and the reference capture reagent coated on the C line can capture the reference marker-labeled reference reagent on the C line. In some embodiments of the invention, the reference reagent/reference capture reagent is selected from the group consisting of non-human antibody/anti-non-human antibody, biotin/streptomycin, DNP-BSA/anti-DNP antibody, and receptor/ligand. In some embodiments of the invention, the reference reagent/reference capture reagent is a goat anti-rabbit IgG antibody/rabbit IgG antibody. In some embodiments of the invention, the second signal label is selected from the group consisting of lanthanoids and chelates thereof, platinum/palladium porphyrin compounds, time-resolved luminescent microspheres, colored colloid particles, magnetic nanoparticles, and luminescent compounds.

In some embodiments of the invention, when the second signal marker is the same as the first signal marker, the luminescence signal R3 at T1 of the second signal marker captured on the C line is also captured with the detector of the optical analyzer in step (3), corrected R1 is obtained by correcting R1 with R3 when R1. Ltoreq.B, then the concentration of the analyte is calculated with corrected R1, and corrected KR2 is obtained by correcting KR2 with R3 when R1> B, then the concentration of the analyte is calculated with corrected KR2 in step (4). In some embodiments of the invention, when R1 is less than or equal to B, the correction of R1 is achieved by calculating the ratio of R1/R3 or R1/(R1+R3), the corrected ratio of R1 is R1/R3 or R1/(R1+R3), and when R1 is greater than B, the correction of KR2 is achieved by calculating the ratio of KR2/R3 or KR 2/(KR2+R3), and the corrected ratio of KR2 is KR2/R3 or KR 2/(KR2+R3).

In the present invention, the standard luminescence signal time-decay curve graph of the first signal markers refers to a curve of the standard luminescence signal time-decay curve of the first signal markers, which is obtained by measuring the values of the generated luminescence signals at different time points after the light source is turned off under the condition that the luminescence signals generated after the light source is previously irradiated with excitation light for a period of time and then turned off do not exceed the detection range of the light analyzer, and then the standard luminescence signal time-decay curve graph of the first signal markers is drawn according to the different time points and the measured luminescence signal values.

The invention has the advantages that (1) when a high-concentration analyte in a sample exceeds the detection upper limit of an analog-to-digital converter in an optical analyzer at a time point T1, according to the characteristic that a time-resolved luminescence signal generated by a time-resolved luminescence marker after excitation continuously fails along with the time, the farther the luminescence signal R2 measured at a time point T2 after T1 is smaller than R1, the farther the T2 is away from T1, the smaller the R2 is, when the R1 exceeds the detection upper limit of the analog-to-digital converter in the optical analyzer, the R1 is indirectly measured by measuring the R2, so that the problem that the existing optical analyzer is insufficient in the detection range of the analyte is still solved under the condition that the existing optical analyzer accurately and low-cost detects the concentration of the high-concentration analyte in the sample, or in order to obtain a larger detection range of the analyte, the cost is often required to be replaced, and therefore, when the R1 exceeds the detection upper limit of the analog-digital converter in the optical analyzer, the high-concentration analyte is not required to be diluted by the sample, the invention can be accurately detected by measuring the high-concentration analyte in the optical analyzer, and the sample is not required to be diluted by the high-concentration sample, and the high-concentration analyte is not required to be accurately detected in the sample.

Drawings

FIG. 1 is a schematic perspective view of an analyzer used in the present invention.

Fig. 2 is a schematic diagram of the internal circuitry of an analyzer used in the present invention.

FIG. 3 is an exploded view of a test card used in the present invention.

FIG. 4 is a schematic diagram of an immunoassay strip in a test card used in the present invention.

FIG. 5 shows a graph of time decay of standard fluorescent signal after excitation of europium chelate with excitation light (wavelength 365 nm), showing two measurement periods, a first measurement period (200-600. Mu.s) and a second measurement period (900-2000. Mu.s).

Detailed Description

As shown in fig. 1 and 2, an 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 slot 3. The clinical sample is inserted into the optical analyzer 100 after being added to the test card 5 for a reaction time outside the optical analyzer 100 for detection, or is inserted into the optical analyzer 100 immediately after the clinical sample is added to the test card 5 and then reacted in the optical analyzer 100 for a reaction time for detection. The optical analyzer 100 further includes an optical system, an amplifying 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 driving circuit 12, and a feedback circuit 13, which are located inside the analyzer housing 1. The optical system comprises a light source 4, an optical path structure 6 and a detector 7. The optical system can be provided with some optical devices such as a narrow-band filter or a grating according to requirements. The light source 4 may be selected from Light Emitting Diodes (LEDs), flash lamps and other suitable light sources, such as LEDs. The illumination of the light source 4 may be continuous or pulsed. The detector 7 may be selected from a photomultiplier tube, a photodiode, a charge coupled device, a charge injection detector, or a CMOS photosensitive element, etc., such as a Photodiode (PD), such as a silicon photodiode. The detector 7 may be provided in plural as necessary. Of course, the light analyzer used in the present invention may be a commercially available analyzer such as FIAflex ^TM Fluorescent Immunoassay Analyzer (iFIA-100) available from Aikang Biotechnology (Hangzhou).

The main control unit 10 comprises a microprocessor, wherein a control signal applied by the microprocessor is subjected to digital-to-analog conversion through the digital-to-analog conversion chip 11, then a light source current control signal is provided for the light source driving circuit 12, and the light source 4 is enabled to work in a constant current state after feedback through the feedback circuit 13. During the scanning of the test card 5 by the optical system, the light emitted from the light source 4 irradiates the test card 5 to which the sample to be tested has been added through the optical path structure (such as an optical fiber) 6. After a period of time of reaction with the added sample, the time-resolved luminescent markers captured on the T-line and C-line of the test card 5 are irradiated to generate a light signal. The generated optical signal is output to the detector 7 through an optical path structure 6 (such as an optical fiber, which is different from the optical fiber through which the excitation light emitted from the light source 4 passes), and is converted into a current signal by the detector 7. The generated current signal is modulated and converted into a proper voltage interval by an amplifying circuit 8, and then is subjected to analog-digital conversion by an analog-digital conversion chip 9 and is transmitted to a main control unit 10. The microprocessor in the main control unit 10 calculates the received analog-to-digital converted electrical signal to determine the presence or concentration of the analyte in the sample to be measured.

As shown in fig. 3 and 4, the test card 5 includes a test strip 15, a card cover 14, and a card holder 16. The test strip 15 used was an immunochromatographic test strip comprising a sample addition pad 51, a label pad 52, a detection pad 53 and a water absorption pad 54 which are sequentially overlapped. The detection pad 53 is made of nitrocellulose, glass fiber, polyethersulfone, nylon, or the like, and thus, in some cases, the detection pad 53 is a nitrocellulose membrane. A detection line (also called T line) 56 and a control line (also called C line) 57 are provided on the detection pad 53. The sample addition pad 51 is made of a water-absorbent material, and glass fiber or nonwoven fabric is selected. The marking pad 52 is also made of a water absorbent material, and may be a polyester film, fiberglass, or a nonwoven fabric.

The number of detection lines provided on the detection pad 53 may be adjusted according to actual needs, for example, when detecting one analyte, only one detection line is required, and when detecting two or more analytes, a corresponding number of detection lines is required.

The test strip 15 also includes a bottom support layer 55. The bottom support layer 55 is made of a hydrophobic material such as polyvinyl chloride, which is commonly used. The loading pad 51, the marking pad 52, the detection pad 53 and the absorbent pad 54 are provided on the bottom support layer. One end of the sample addition pad 51 is partially overlapped with the label pad 52, one end of the label pad 52 is partially overlapped with the sample addition pad 51, the other end of the label pad 52 is partially overlapped with the detection pad 53, the water absorption pad 54 is made of hydrophilic material, and one end of the water absorption pad 54 is partially overlapped with the detection pad 53. In addition, in some cases, the overlap area between any two adjacent pads is 0.5 to 5 millimeters long.

The test strip 15 is located in a housing that is comprised of a cartridge 14 and a cartridge 16. A test strip slot 60 is provided in the middle of the cartridge 16 for receiving the test strip 15.

The card cover 14 is also provided with a sample inlet 58 and a viewing window 59. When a clinical sample is added through the sample addition port 58, the sample enters the addition pad 51 located below the sample addition port 58, and migrates along the length of the test strip 15 toward the absorbent pad 54 by capillary action. The observation window 59 is provided above the detection line 56 and the control line 57 of the detection pad 53. The light from the light source 4 may be directed through a transparent or translucent viewing window 59 to the detection line 56 and the control line 57 of the test strip 15. After being irradiated with excitation light from the light source 4, the light signals generated by the detection line 56 and/or the control line 57 are output to the detector 7 through the light path structure 6.

Depending on the analyte to be detected (e.g., antigen, antibody or hapten) and the principle of immunodetection (double antigen sandwich, double antibody sandwich, competition, indirect, capture), the substance coated on the label pad 52 and the detection line 56 will vary. The detection principle is a double antibody sandwich method, and the time-resolved luminescent marker is europium chelate. The labeling pad 52 is coated with a europium chelate-labeled first reagent (first anti-IL-6 antibody) and a europium chelate-labeled reference reagent, and the detection line 56 is coated with a second reagent (second anti-IL-6 antibody), which specifically binds to IL-6 in the clinical sample, so that the clinical sample flows along the length direction of the test strip 15 after the clinical sample is added to the sample addition pad 51 through the sample addition port 58. When the clinical sample reaches the label pad 52, the europium chelate-labeled first reagent specifically binds to IL-6 (if present) in the clinical sample, and the europium chelate-first reagent-IL-6 complex formed continues to flow and specifically binds to the second reagent on the detection line 56, thereby capturing the europium chelate-first reagent-IL-6 complex on the detection line 56, which upon excitation by excitation light from a light source can generate a detection signal related to the concentration of IL-6. At the same time, the europium chelate-labeled reference reagent continues to flow, and when flowing onto the control line 57, the europium chelate-labeled reference reagent or a complex formed by the europium chelate-labeled reference reagent specifically binding to the non-analyte is captured on the control line 57 by the coated reference capture reagent on the control line 57, and after being irradiated with light from the light source, the europium chelate captured on the control line 57 generates a control signal.

In some cases, the coated reference capture reagent on control line 57 directly captures the europium chelate-labeled reference reagent on control line 57. In this case, the reference reagent and the reference capture reagent may be selected from any one of a combination of non-human antibody/anti-non-human antibody (e.g., rabbit IgG antibody/sheep anti-rabbit IgG antibody, chicken IgY antibody/sheep anti-chicken IgY antibody), biotin/streptomycin, DNP-BSA/anti-DNP antibody, receptor/ligand, and the like.

Reference herein to a non-analyte means a substance that does not affect the detection of the analyte in the sample, either in the sample or not, if not present in the sample, which may be added to the sample or to the test strip in advance. In some cases, the non-analyte is a substance that is not present or is present in a negligible amount in the sample, such that the correction signal is not affected by the clinical sample. Of course, the non-analyte may also be a substance present at a higher level in the sample, e.g., when the analyte to be detected is IL-6, the non-analyte of the present invention may be human IgG present in the clinical sample, where the reference reagent is a rabbit anti-human IgG antibody that specifically binds human IgG and the reference capture reagent coated on the control line 57 is a sheep anti-rabbit IgG antibody that specifically binds rabbit anti-human IgG antibody.

The time-resolved luminescent markers of the present invention have the property of luminescence delay, i.e. they can still continuously produce emitted light for a certain period of time after the excitation light emitted by the light source is turned off. The wavelength of the emitted light may be greater than or less than the wavelength of the excitation light. The characteristic of the luminescence delay enables the emitted light generated after the time-resolved luminescence marker is excited to have longer service life, so that when in actual detection, the light source can be turned off for a period of time after the excitation light from the light source excites the time-resolved luminescence marker, and then the emitted light signal emitted by the time-resolved luminescence marker is detected, thereby eliminating the interference of the background light signal and scattered excitation light with shorter service life.

The time-resolved luminescent marker may exist in a molecular form, called a time-resolved luminescent molecule, and may be selected from lanthanoids such as samarium (Sm (III)), dysprosium (Dy (III)), europium (Eu (III)), and terbium (Tb (III)), chelates thereof, and up-conversion luminescent materials, and platinum/palladium porphyrin compounds that can emit phosphorescence upon excitation. The delay time of the europium chelate is about 2ms, and the emitted fluorescent signal gradually decays with time within 2 ms. Whereas some proteins in clinical samples can also fluoresce under excitation of the light source, without a time-lapse feature, most proteins produce fluorescence that completely disappears 200 μs after the light source is turned off. Therefore, the time-resolved fluorescence detection of europium chelates usually selects to collect fluorescence signals 200-400 μs after the light source is turned off. In some cases, the time decay profile of the standard fluorescence signal after excitation of the europium chelate by excitation light is shown in FIG. 5. One suitable europium chelate is N- (p-isothiocyanatobenzene) -diethylenetriamine tetraacetic acid-Eu ⁺³.

The time-resolved luminescent markers may also be present in another form, time-resolved luminescent microspheres, i.e. time-resolved luminescent molecules are encapsulated within or on the surface of natural or synthetic microspheres or microbeads. Each time-resolved luminescent microsphere can wrap thousands of time-resolved luminescent molecules, thereby effectively improving the detection sensitivity. The time-resolved luminescent microspheres that produce fluorescence upon excitation are referred to as time-resolved fluorescent microspheres.

Up-converting luminescent materials refer to materials that are excited by light of low energy and emit light of high energy. The light signal generated by the up-conversion luminescent material after excitation has stronger stability, longer service life and higher sensitivity, and can also be suitable for time-resolved immunodetection. The usual up-conversion luminescent material may be selected from Y₂O₃、Y₂O₂S、LaF₃、NaYF₄、NaGdF₄、NaYF₄:Yb³⁺/Nd³⁺/Ho³⁺、NaGdF₄:Yb³⁺/Nd³⁺/Ho³⁺、Y₂O₃:Er,Yb and the like.

The C-line of the present invention is not required. When line C is set, the control signal C generated by line C of the present invention can be used to correct the detection signal T generated by the time-resolved luminescent marker on line T, in addition to the effect of indicating whether the added clinical sample is flowing to the absorbent pad 54, and the corrected T signal can be used to calculate the analyte concentration in the sample. This correction can be achieved by calculating the T/C or T/(T+C) ratio, the corrected T signal being T/C or T/(T+C). In addition, the control signal C generated by line C in the present invention may be used only to indicate whether the added clinical sample flows to the absorbent pad 54 or not, and is not used to correct the detection signal T, so that the control signal may also be a color signal, such as a colored aggregate generated after colored colloidal particles are aggregated on the control line 57, and when the color signal appears on the control line 57 is observed, it may be judged whether the added clinical sample flows to the absorbent pad 54, so that there is no need to collect the reflected light, fluorescence, phosphorescence, etc. generated by line C after the light source is irradiated, so that the second signal marker may be selected from the group consisting of time-resolved luminescent markers, colored luminescent microspheres, colored colloidal particles, magnetic nanoparticles, and luminescent compounds. The colored luminescent microsphere is a microsphere or a microsphere with luminescent compounds such as quantum dots, fluorescent dyes and the like coated on the surface or inside, and can generate a light signal without luminescence delay characteristic by irradiation of excitation light with proper wavelength, and can be selected from green fluorescent microsphere, blue fluorescent microsphere, red fluorescent microsphere, yellow fluorescent microsphere and color fluorescent microsphere (emitting fluorescence of various specific colors). Colored colloidal particles refer to colloidal particles that, upon aggregation on the T-line and/or C-line during an immunochromatographic reaction, produce colored aggregates, and can be selected from the group consisting of latex, colloidal gold, colloidal carbon, and colloidal selenium. The luminescent compound is selected from quantum dots, fluorescein and its derivatives such as Fluorescein Isothiocyanate (FITC), fluorescent protein capable of emitting fluorescence after excitation and its modified variants such as green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein, etc., and chemiluminescent marker selected from luminol, isoluminol and its derivatives, 1, 2-dioxyethane derivatives (commonly known as AMPPD, CSPD, CDP and CDP-Star and PPD, lumi-Phos and Lumi-Plus of Lumigen company, etc.), and acridine ester or acridine sulfonamide.

In the time-resolved luminescent microspheres and the colored luminescent microspheres, the polymer forming the microspheres or microbeads may be selected from polystyrene, butadiene styrene, styrene acrylic-ethylene terpolymer, polymethyl methacrylate, styrene-maleic anhydride copolymer, polyvinyl acetate, polyvinyl pyridine, polydivinylbenzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or aldehyde, carboxyl, amino, hydroxyl, hydrazide derivatives thereof, or mixtures thereof. In addition, the surface of the microsphere or microbead typically carries hydroxyl, carboxyl, amino, aldehyde, sulfo, etc. groups, which can be coupled to the antibody or antigen or hapten-carrier protein conjugates by conventional chemical coupling reagents. In some cases, the time-resolved luminescent microspheres have a particle size of 20nm to 100 μm.

When detecting one or more analytes in a sample, the intensity of the detection signal produced by the time-resolved luminescent marker on the T-line is positively or negatively correlated with the concentration of the analyte, depending on the principle of immunodetection, wherein the intensity of the detection signal produced by the time-resolved luminescent marker on the T-line is positively correlated with the concentration of the analyte in addition to the competition method.

Clinical samples in the present invention may be selected from serum, plasma, whole blood, cerebrospinal fluid, urine, bronchoalveolar lavage, nasopharyngeal swab, sputum, stool, dermatological lesions samples (including swabs of rash/wheal exudates; wheal fluid; wheal, etc.), and the like. Depending on the type of clinical sample and the analyte, some clinical samples may be added to the test card 5 for testing after pretreatment (e.g., cleavage with a lysate to release the analyte to be tested) prior to testing.

Analytes detectable by the present invention include antigens or antibodies, even haptens, such as inflammatory markers, heart failure markers, tumor markers, bone metabolism markers, sex markers, thyroid function markers, infectious disease markers, diabetes markers, liver fibrosis markers, allergy markers, intestinal health markers, and the like.

Example 1 preparation of a first type of test strip and test card according to the invention

A preparation method of a test strip and a test card for quantitatively detecting IL-6 in a sample comprises the following steps:

A. The antibody preparation may be carried out by using antigen-immunized mice, rats, rabbits and other animals or hybridoma cell counts to screen out paired monoclonal or polyclonal antibodies for detecting IL-6, or alternatively, commercially available IL-6 paired antibodies (one is an IL-6 capturing antibody and the other is an IL-6 detecting antibody), and in this example, commercially available IL-6 paired antibodies (available from Fei Peng Bio Inc., under the designations IL6-MAB-F1-001 and IL 6-MAB-F1-002) are used as examples. Rabbit IgG antibodies and goat anti-rabbit IgG antibodies are self-produced or commercially available.

B. liquid for detecting pad spots

IL-6 capture antibody was added to 0.02M phosphate buffer (pH 7.2) to obtain a T-line solution, IL-6 capture antibody concentration in the T-line solution was 1mg/ml, goat anti-rabbit IgG antibody was added to 0.02M phosphate buffer (pH 7.2) to obtain a C-line solution, goat anti-rabbit IgG antibody concentration in the C-line solution was 0.3mg/ml, and then the T-line solution and the C-line solution were coated on a 2.5cm wide nitrocellulose membrane (model No. 1UN95ER100025NT from Satorious) serving as a detection pad at an interval of 0.8cm using a quantitative spot solution apparatus (Hangzhou Peak-air technologies, inc. AUTOKUN continuous laminator spot film machine HGS 101) at an amount of 1. Mu.l/cm, thereby forming T-line and C-line, respectively, oven-drying at 45℃for 18 hours, and sealing with a desiccant for use.

C. Preparation of fluorescent microspheres:

The fluorescent microsphere is a time-resolved fluorescent microsphere (Merk Co., product number F1-XC 010) with europium chelate coated inside, and has excitation wavelength of 365nm and emission wavelength of 615nm.

And (3) preparing the MES buffer solution, namely adding the morpholine ethanesulfonic acid sodium salt into pure water, and uniformly mixing to ensure that the concentration of the morpholine ethanesulfonic acid sodium salt is 1.0% (w/v).

Preparation of preservation buffer 50mM (pH 8.0) Tris-HCl buffer.

The preparation of the time-resolved fluorescence microsphere-labeled IL-6 detection antibody comprises the steps of washing the fluorescence microsphere by using MES buffer solution, adding carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to enable the final concentration of the fluorescence microsphere to be 0.4mg/ml and 0.1mg/ml respectively, reacting for 20 minutes at room temperature, fully washing the activated microsphere by using the MES buffer solution, adding the IL-6 detection antibody in the ratio of 1mg to 0.1mg (mass ratio), reacting for 1.5 hours at room temperature, fully washing by using the MES buffer solution, adding 0.05M Tris-HCl buffer solution with pH of 8.0 containing 10% BSA (w/v), sealing for 1 hour at room temperature, washing the time-resolved fluorescence microsphere by using the MES buffer solution, and using a preservation buffer solution to re-measure the time-resolved fluorescence microsphere so that the final concentration of the time-resolved fluorescence microsphere is 5mg/ml and preserving for standby, preparing the time-resolved fluorescence microsphere-labeled IL-6 detection antibody in the same manner as the preparation of the time-resolved fluorescence microsphere-labeled IL-6 detection antibody, preparing the time-resolved fluorescence microsphere, and preserving the rabbit antibody by using the final concentration of the IgG-labeled IgG and preserving the rabbit antibody at the final concentration of 4mg and preserving the time-resolved microsphere at 4 ℃ after the time-resolved IgG.

Finally, fully mixing the IL-6 detection antibody marked by the time-resolved fluorescence microsphere, the rabbit IgG antibody marked by the time-resolved fluorescence microsphere and a fluorescence marking diluent according to the volume ratio of 6:1:13 to obtain a fluorescence microsphere mixture, wherein the fluorescence marking diluent is 50mM (pH 8.0) Tris-HCl buffer solution 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).

D. spraying and drying of fluorescent microspheres

The prepared fluorescent microsphere mixture is uniformly sprayed on a marking pad (glass fiber) with the width of 1.0cm in an amount of 2 mu 1/cm by using a special liquid-drawing head of a AUTOKUN continuous film drawing machine point film drawing instrument HGS101, dried for 18 hours at 45 ℃, added with a drying agent and sealed for standby.

E. sample pad handling

And placing the sample pad with the width of 2.5cm into the sample pad treatment solution for soaking treatment for 1 hour, taking out the sample pad, and drying at 37 ℃ overnight (12-24 hours). The sample pad treatment was a buffer containing 50mM (pH 8.0) Tris-HCl buffer, 1% BSA (w/v) and 0.5% Tween-20 (v/v).

E. Assembling and cutting of test strips

Assembling the test strip plate, namely adhering a sample pad with the width of 2.5cm, a marking pad with the width of 1.0cm, a nitrocellulose membrane with the width of 2.5cm and absorbent paper with the width of 2.5cm (serving as an absorbent pad) to a plastic bottom plate with the length of 8cm (serving as a bottom supporting layer) manually or by a machine, so that the sample pad with the width of 2.5cm, the marking pad with the width of 1.0cm, the nitrocellulose membrane with the width of 2.5cm and the absorbent paper with the width of 2.5cm are sequentially overlapped with each other in a staggered manner by 2.0mm, and assembling the test strip plate.

Cutting of the test strips the assembled test strips were cut into 4mm wide single piece test strips using a AUTOKUN HGS model slitter.

F. assembly of test cards

And placing the cut single test strip in a clamping groove on a plastic clamping seat, covering a clamping cover, and tightly pressing the clamping seat and the clamping cover by using a card pressing machine or manually to ensure that the whole immunoassay test strip is in a tight state. Adding a drying agent, and sealing at room temperature for standby.

After the test card is assembled, it can be packaged in a kit with a desiccant and instructions for use. In addition, the kit may be filled with a sampling tube, a test tube containing a lysate, a test tube containing a sample diluent, a sampling swab, and the like, as required.

Example 2 preparation of a second type of test strip and test card according to the invention

This example differs from the first example in that (1) the paired antibodies for detecting N-terminal brain natriuretic peptide precursor (NT-proBNP), NT-proBNP detection antibody and NT-proBNP capture antibody (available from Kirschner technologies Co., ltd., product No. V00706 and V00111), were used in place of the IL-6 detection antibody and IL-6 capture antibody in example 1, respectively, (2) the fluorescent labeling dilution was 50mM (pH8.0) 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). The present example allows the preparation of test strips and cards for the detection of NT-proBNP in a sample.

Example 3 preparation of Standard test strips and Standard test cards

The standard test strip and the standard test card were prepared by adding time-resolved fluorescent beads (Merk. RTM. No. F1-XC 010) to Tris-HCl (pH 8.0) buffer containing 0.5% NaCl (w/v) so that the concentration of the time-resolved fluorescent beads became 1mg/ml, and then directly coating the obtained time-resolved fluorescent bead solution onto a nitrocellulose membrane (model 1UN95ER100025NT, available from Satorious Co.) in the form of a streak in an amount of 1. Mu.l/cm, followed by drying. The nitrocellulose membrane of the fluorescent microsphere with time resolution in coating is stuck on a plastic bottom plate by using self-adhesive, and then cut into standard test strips with the width of 4 mm. And placing the cut single test strip in a clamping groove on the plastic clamping seat, covering a clamping cover, and pressing the clamping seat and the clamping cover by using a card pressing machine or manually to form the standard test card capable of generating stable fluorescent signals. In addition, time-resolved fluorescent microspheres (Merk goods numbers F1-XC 010) are not added when standard test strips and test cards are prepared, so that blank test strips and blank test cards can be prepared and used for measuring background fluorescent signals.

Example 4 detection method

The test card prepared in example 1 or example 2 is used to detect an analyte (e.g., IL-6, NT-proBNP, etc.) in a sample by adding 100. Mu.l of the sample (here, a whole blood sample is illustrated) to the sample addition port of the test card. After incubation for 15min, the test card was inserted into the light analyzer for detection. The word length of the analog-digital converter in the optical analyzer determines the detection upper limit of the optical analyzer, and when the word length of the analog-digital converter is N, the counting range is 0-2 ^N -1. The word length of the analog-digital converter of the optical analyzer is 16 bits, the counting range is 0-65535 (2 ¹⁶ -1), namely 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 microsphere captured on the T-line may exceed the upper detection limit a of the optical analyzer, which is a common practice to dilute the sample and then measure the diluted sample, which increases the test time and cost.

After the test card is inserted into the optical analyzer, the test card is scanned by utilizing an optical system, when scanning is performed, excitation light with the wavelength of 365nm is emitted by a light source to respectively irradiate time-resolved fluorescent microspheres captured on a T line and a C line, each line is irradiated for 2-8 ms, then the light source is turned off, and the time-resolved fluorescent microspheres of europium-containing chelate captured on the T line and the C line emit fluorescent signals with the wavelength of 615 nm. By utilizing the characteristic that fluorescence signals of europium chelates are gradually attenuated, after capturing by using a detector of an optical analyzer at a first time point T1 (200-600 mu s) and a second time point T2 (900-2000 mu s) after turning off a light source, fluorescence signals R1 and R2 emitted on a T line and a fluorescence signal R3 emitted on a C line when the light source is respectively obtained at the T1 and the T2, and when the light source is required.

In view of the fact that there is sometimes a case where, when the measured fluorescence signal is located near the upper detection limit a of the optical analyzer, it is unclear whether the measured fluorescence signal is actually smaller than or equal to the upper detection limit a of the optical analyzer or larger than the upper detection limit a of the optical analyzer, because of the measurement error of the optical analyzer itself. In order to avoid this, a demarcation value B is set, the size of B being 70% -90% of the upper detection limit A. For example, when a=65535, B may be selected to be 50000, where B is 76.3% of a.

After determining the demarcation value B of the optical analyzer, comparing the sizes of R1 and B, when R1 is less than or equal to B, selecting R1 to calculate the analyte concentration in the sample, and when R1 is more than B, selecting KR2 to calculate the analyte concentration in the sample. This calculation can be performed in two ways (1) when no C line or signal from the C line is not involved in the correction, the analyte concentration in the sample is calculated using only R1 (when R1. Ltoreq.B) or KR2 (when R1> B), and (2) when signal R3 from the C line is involved in the correction, R1 (when R1. Ltoreq.B) or KR2 (when R1> B) is corrected using R3 to obtain corrected R1 (when R1. Ltoreq.B) or KR2 (when R1> B), and then the analyte concentration is calculated using corrected R1 (when R1. Ltoreq.B) or corrected KR2 (when R1> B). In some cases, when R1. Ltoreq.B, the correction for R1 is achieved by calculating the ratio of R1/R3 or R1/(R1+R3), the corrected R1 is the ratio of R1/R3 or R1/(R1+R3), and when R1> B, the correction for KR2 is achieved by calculating the ratio of KR2/R3 or KR 2/(KR2+R3), the corrected KR2 is the ratio of KR2/R3 or KR 2/(KR2+R3).

Example 5:K value determination

The T line of the standard test card prepared in test example 3 was irradiated with excitation light (wavelength 365 nm) from the light source of the optical analyzer, then the light source was turned off, and fluorescent signals emitted on the T line were read at different time delays (the measured fluorescent signals were all within the measurement range of the optical analyzer), and the results were shown in the following table, and the resulting time-decay graph of the standard fluorescent signals is shown in fig. 5. The background fluorescence signal was measured by irradiating the blank test card prepared in example 3 with excitation light (wavelength 365 nm) emitted from the light source of the optical analyzer, and it was found that the background fluorescence signal completely disappeared after turning off the light source for 200 μs.

The K value is calculated by dividing the average value of the fluorescent signal R1 when the delay time is T1 by the average value of the fluorescent signal R2 when the delay time is T2. For example, the average R1 value at 200, 300, 400, 600 μs divided by the average R2 value at 900 μs T2 can calculate K values of 3.02, 2.64, 2.19 and 1.58, respectively, the average R1 value at 200, 300, 400, 600 μs divided by the average R2 value at 1200 μs T2 can calculate K values of 5.15, 4.51, 3.73 and 2.70, respectively, and so on.

In addition, when calculating the K value, the R1 and R2 ratio can be obtained by calculating the R1 and R2 ratio, and the R1 average value and the R2 average value can be obtained by calculating the R1 average value and the R2 average value.

IL-6 has a detection range of 3 to 4000pg/mL, an upper limit/lower limit ratio of 1333.33 times, and NT-proBNP has a detection range of 15 to 20000pg/L, and an upper limit/lower limit ratio of 1333.33 times. The noise signal of the light analyzer at the time point T1 is 30, the signal-to-noise ratio of the fluorescent signal R generated on the T line of IL-6 with the concentration of the detection lower limit (3 pg/mL) or NT-proBNP with the concentration of the detection lower limit (15 pg/L) at the time point T1 is not less than 3, and is selected to be 160 here, so that the fluorescent signal R' generated on the T line of IL-6 with the concentration of the detection upper limit (4000 pg/mL) or NT-proBNP with the concentration of the detection upper limit (20000 pg/L) at the time point T1 should theoretically reach 213332.8 (160X 1333.33) which is 3.26 times the maximum count value 65535 of the 16-bit analog-digital converter commonly used in the light analyzer, and K >3.26 should be selected accordingly. When K >3.26 is determined, the choice of T1 and T2 time points can be determined, as can be seen from the above table and fig. 5, k=5.15 >3.26 when t1=200 μs and t2=1200 μs, k=4.51 >3.26 when t1=300 μs and t2=1200 μs, and k=3.73 >3.26 when t1=400 μs and t2=1200 μs.

In view of the characteristic that the fluorescence signal generated by the europium chelate after excitation is continuously depleted over time, the fluorescence signal R2 measured at a time point T2 after T1 is smaller than R1, and the further away from T1 the T2 is, the smaller R1 is, and the larger K is. Even if the IL-6 concentration of the sample is greater than the upper limit of detection, R1 can be indirectly measured by measuring KR2 as long as T2 is selected farther from T1. Therefore, the problem of insufficient detection range of the analyte concentration in the conventional optical analyzer can be solved by taking the signal values of different time points by utilizing the characteristic of the decay of the fluorescent signal generated by the europium chelate with time.

EXAMPLE 6 IL-6 assay results

Test strips and cards for quantitative detection of IL-6 in serum were prepared using the preparation method in example 1.

A series of IL-6 references were prepared at concentrations of 3.21pg/ml, 30.85pg/ml, 310.5pg/ml, 989.2pg/ml, 1985pg/ml and 3942pg/ml, respectively. 100 μl of each IL-6 reference was added to the IL-6 test card, and fluorescent signals were read by the following three methods, and each concentration was repeatedly tested 5 times, to calculate the coefficient of variation CV of the fluorescent signal values of each concentration.

Method 1 normal current amplification, capture fluorescent signal once at 300 μs, measured fluorescent signal as shown in the following table:

it is apparent that the fluorescence signals of the high concentration IL-6 references (1985 pg/mL and 3942 pg/mL) have no gradient and cannot be distinguished.

Taking the average value (T line) of fluorescent signals as an X axis and the concentration of IL-6 reference substances as a Y axis, preparing a standard curve, establishing a piecewise linear regression equation, and calculating test results as shown in the following table:

as can be seen from the above table, the high concentration IL-6 reference test results are not good in accuracy and reproducibility.

Method 2. Reduce the current magnification to 1/4 and capture the fluorescent signal once at 300 mus, the measured fluorescent signal is shown in the table below.

The low concentration IL-6 reference (3.21 pg/mL) had very poor consistency due to the too low fluorescence signal and inaccurate test results.

Taking the average value of fluorescent signals as an X axis and the concentration of IL-6 reference substances as a Y axis, preparing a standard curve, establishing a piecewise linear regression equation, and calculating test results as shown in the following table:

method 2 is a major improvement over the test results of method 1, but the low concentration IL-6 reference (3.21 pg/mL) is less reproducible due to the noise of the light-sensitive analyzer.

Method 3 Normal current amplification, capturing fluorescent signals once for 300 μs and 1200 μs each

When measured, K is 4.51, B is 50000, the magnitudes of the fluorescent signals R1 and B captured at 300 μs are compared, R1 is selected as the fluorescent signal and used to calculate the concentration of IL-6 when R1 is less than or equal to B, and KR2 is selected as the fluorescent signal and used to calculate the concentration of IL-6 when R1 is greater than B. The measured fluorescence signals are shown in the following table:

the test results of method 3 showed that the reproducibility of the test results for the low concentration IL-6 reference and the gradient for the high concentration IL-6 reference were both good.

The test result of the method 3 has better repeatability and accuracy.

EXAMPLE 7 NT-proBNP assay

A test strip and a test card for quantitative determination of NT-proBNP in serum were prepared by the preparation method in example 2. A series of NT-proBNP references were prepared at concentrations of 16.5pg/ml, 151.2pg/ml, 1188pg/ml, 3965pg/ml, 9827pg/ml and 19287pg/ml, respectively. 100 μl of each NT-proBNP reference sample was added to the NT-proBNP test card, and the fluorescent signals were read by the following three methods, respectively, and each concentration was repeatedly tested 5 times, to calculate the coefficient of variation CV of the fluorescent signal values of each concentration.

It is apparent that the high concentration NT-proBNP references (9827 pg/mL and 19287 pg/mL) have no gradient.

Taking the average value of fluorescent signals as an X axis and the concentration of the NT-proBNP reference substance as a Y axis, preparing a standard curve and establishing a piecewise linear regression equation, and calculating the test result as follows:

as can be seen from the above table, the high concentration NT-proBNP reference has poor accuracy and reproducibility of the test results.

Method 2, decreasing the current magnification to 1/4, capturing the fluorescent signal once at 300 μs, the measured fluorescent signal is shown in the following table:

the low concentration NT-proBNP reference (16.5 pg/mL) had very poor consistency due to the too low time-resolved signal and inaccurate test results.

method 2 is a major improvement over the test results of method 1, but the low concentration NT-proBNP reference (16.5 pg/mL) is less reproducible due to the influence of the analyzer noise on fluorescence.

Method 3 Normal current amplification, capturing fluorescent signals once at 300 and 1200 μs respectively

When measured, K is 4.51, B is 50000, the magnitudes of the fluorescent signals R1 and B captured at 300 μs are compared, R1 is selected as the fluorescent signal and used to calculate the concentration of NT-proBNP when R1 is less than or equal to B, and KR2 is selected as the fluorescent signal and used to calculate the concentration of NT-proBNP when R1 is greater than B. The measured fluorescence signals are shown in the following table:

test results of method 3 the reproducibility of the test results of the low concentration NT-proBNP reference and the fluorescence signal gradient of the high concentration NT-proBNP reference are both good.

the repeatability and the accuracy of the test result of the method 3 are good.

Claims

1. A method for detecting an analyte, comprising (1) providing a detection device which carries a first reagent labeled with a first signal marker when detected, the first reagent being capable of specifically binding to the analyte in a sample, the first reagent labeled with the first signal marker being captured in a test zone of the detection device after the sample is added, the first signal marker being a time-resolved luminescent marker;

2. The method of claim 1, wherein the test device comprises an immunochromatographic strip, the first signal-labeled first reagent is disposed on the strip during the detection, the second reagent disposed on the T-line of the test zone captures the first signal-labeled first reagent on the T-line, and (2) the sample is added to the sample-adding zone of the strip, and the first signal-labeled first reagent moves with the sample toward the T-line and is captured by the second reagent on the T-line.

3. The method according to claim 1, wherein the luminescence signal R generated by the first signal marker captured in the test area of the detection apparatus at T1 when the concentration of the analyte is the analyte detection lower limit is M times, M >1 of the noise signal S of the optical analyzer at T1 is measured by the optical analyzer, and then the luminescence signal R ' =r×the analyte detection upper/lower limit which should theoretically be generated by the first signal marker captured in the test area of the detection apparatus when the concentration of the analyte is the analyte detection upper limit is calculated, and then the ratio of R ' to the detection upper limit a of the optical analyzer is calculated, and K is the ratio of the luminescence signal values of the standard luminescence signal time decay profile of the first signal marker at T1 and T2 and is larger than the ratio of R ' to a.

4. The method of claim 3, wherein R.gtoreq.3 XS.

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

6. The method of claim 1, wherein the first signal label is selected from the group consisting of lanthanoids and chelates thereof, platinum/palladium porphyrin compounds, up-conversion luminescent materials, and time-resolved luminescent microspheres.

7. The method of claim 6, wherein the first signal label is a time-resolved fluorescent microsphere with europium chelate coated therein.

8. A detection system comprising a detection device and an optical analyzer, wherein the detection device carries a first reagent labeled with a first signal marker when detecting, the first reagent being capable of specifically binding to an analyte in a sample, the first reagent labeled with the first signal marker being capable of being captured in a test zone of the detection device after the sample is added, the first signal marker being a time-resolved luminescent marker;

The light analyzer comprises a light source, a detector, an analog-to-digital converter and a processor, wherein the light source emits excitation light to irradiate a test area, and the detector captures luminescent signals R1 and R2 generated by a first signal marker captured in the test area at a first time point T1 and a second time point T2 after the light source is turned off, wherein T1 is smaller than T2;

The processor may compare the magnitude of the demarcation value B of R1 with the light analyzer, where R1 is selected by the processor to calculate the concentration of the analyte when R1 is less than or equal to B, and KR2 is selected by the processor to calculate the concentration of the analyte when R1> B, where K >1.

9. The method according to claim 8, wherein the luminescence signal R generated by the first signal marker captured in the test area of the detection device when the concentration of the analyte is the analyte detection lower limit is M times the noise signal S of the optical analyzer at the time point T1, M >1, preferably R.gtoreq.3×S, is measured by the optical analyzer, and then the luminescence signal R ' which should theoretically be generated by the first signal marker captured in the analyte detection upper limit test area when the concentration of the analyte is the analyte detection upper limit is calculated by R×the analyte detection upper limit/detection lower limit, and then the ratio of R ' to A is calculated, K being 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 being larger than the ratio of R ' to A.

10. The method of claim 8, wherein B is set to 70% -95% of the upper detection limit a of the optical analyzer, a = 2 ^N -1, and n is the word length of the analog-to-digital converter.