Assembled 96-channel immunochromatography test paper array sample adding device and high-throughput detection method

Abstract

The invention provides an assembled 96-channel immunochromatography test paper array sample adding device and a high-throughput detection method, and belongs to the technical field of immunochromatography. According to the present invention, the 96-well matched immunochromatography test paper array sample adding device is adopted to achieve the simultaneous sample injection of the 96-well sample, the whole plate scanning detection method is adopted to achieve the high-flux immunochromatography test paper detection, and the method is provided for the instant detection requiring the ultra-high flux.

Description

Technical Field

[0001] The present invention provides an ultra-high throughput lateral flow immunochromatography detection method, device and application, belonging to the technical field of high throughput detection. Background Art

[0002] Lateral flow chromatography is a simple, fast, easy-to-use, low-cost rapid detection technology that can obtain results on-site in a short time (5-15 minutes). It is widely used in clinical diagnosis, health inspection, epidemic prevention inspection, and military inspection. With the development of this technology and the continuous expansion of application scenarios, when faced with the demand for huge sample testing, traditional multi-channel detection technology is completely unable to meet the needs of high-throughput and high-efficiency detection, such as the screening and detection of a large number of samples during the epidemic.

[0003] There are some throughput detection technologies in the market, which are generally single-sample, multi-indicator combined detection. One solution is to coat multiple detection reagents on the test paper to increase the detection items, but the anti-antibody load is limited, generally 3-5, such as patents CN111089956A, CN118655309A, and there is interference between reagents. The technology is difficult to implement and is less used.

[0004] Another technology is to increase the number of test paper channels. One sample addition hole corresponds to multiple channels for multi-indicator joint detection. Generally, the number of channels is 5-10. If there are more channels, the size will be very large, such as patents CN216900570U, CN1768267A, and CN209117696U.

[0005] Another is the single-channel test card, which adds samples independently, tests simultaneously, and uses a multi-channel detector for analysis. The commonly used throughput is 8-10, such as patents CN215339892U, CN110568203A, and CN209656713U, but adding samples is also time-consuming.

[0006] When faced with testing a large number of samples simultaneously, such as up to 100 samples, the detection efficiency of these technologies will be very low, requiring repeated testing, which is time-consuming.

[0007] In some high-throughput screening experiments, 96-well plate detection is a routine experimental operation. Sometimes multiple 96 plates need to be detected simultaneously. Immunoassay technologies such as ELISA can perform 96-well parallel operations at the same time. The entire process needs to be operated under the support of laboratory equipment, which is time-consuming and labor-intensive and does not meet the needs of instant detection. Summary of the invention

[0008] The purpose of the present invention is to solve the problem that the existing lateral flow detection technology cannot meet the requirements of ultra-high throughput detection. Based on the 96-well plate commonly used in the detection field, an ultra-high throughput lateral flow chromatography array loading device and a high-throughput detection method are invented.

[0009] An assembled 96-channel immunochromatographic test strip array sample loading device and a high-throughput detection method, the specific scheme of the present invention is as follows: The detection method and steps include: Assembled 96-channel immunochromatographic test strip array loading device: includes a 96-well plate fixing bracket and a test strip support plate; the immunochromatographic test strips are arranged and fixed by the positioning and limiting blocking on the test strip support plate, and the test strip loading area protrudes from the test strip support plate, and multiple support plates can be inserted into the card slots of the 96-well fixing bracket and fixed to form a 96-channel immunochromatographic test strip array loading device.

[0010] Loading: The sample is in a 96-well plate. The assembleable 96-channel immunochromatographic test strip array loading device is aligned with the 96 wells of the 96-well plate and installed vertically, so that each test strip loading area is inserted into the well and reaches below the liquid surface. Immune response: After contacting the liquid surface, the siphon effect of the test paper is used to load samples in 96 channels at the same time. The sample crawls upward, passes through the binding pad of the immunochromatographic test paper and combines with the labeled antibody, continues to pass through the detection T line, and is bound by the capture antibody or antigen on the T line. The excess labeled antibody continues to crawl and combines with the control C line, and the excess liquid is absorbed by the absorbent pad to complete the entire immunochromatographic test; Scanning detection: After the reaction is completed, a test strip support plate is placed in a high-dose immunochromatographic test strip detection device, which includes three mutually isolated photoelectric sensor detection modules, and the three photoelectric sensor detectors correspond to the T line, C line and blank area b between TC of the immunochromatographic test strip; the scanning detection of the immunochromatographic test strips on all the support plates is completed in sequence, and the T line signal, C line signal and background signal of the blank area b between TC are obtained; the signal curve of each test strip on each support plate is recorded and displayed; Result calculation: According to the overall curve of each test strip, calculate the peak height or area under the curve of the T-line peak and C-line peak signal, deduct the background signal of the blank area b between the C line and TC, calculate the (Tb) / (Cb) value, which is linearly related to the concentration, and calculate with the pre-stored calibration curve to obtain the concentration of the substance to be tested in each sample. The result data of 96 samples are displayed and saved at the same time. Quantitative calculation: The light signal of the T-line peak and C-line peak of each test strip minus the light signal of the blank area between the T line and the C line is the T-line peak and the C-line peak. Calculate the peak height or area under the curve of the T-line peak and the C-line peak signal. The T / C value is linearly related to the concentration. Calculate with the pre-stored calibration curve to obtain the concentration of the substance to be tested in each sample. The result data of 96 samples are displayed and saved at the same time.

[0011] The present invention has at least one of the following beneficial effects: The detection throughput is close to 100, which is 10 times higher than the previous maximum detection throughput of 10. In the same time, the efficiency is nearly 10 times higher. The depth of the sample adding area of ​​the test strip into the sample can control the sample loading amount and keep the sample loading amount consistent. There is no need to add the sample by dripping, which reduces the workload. The combination configuration can flexibly adjust the detection throughput, so that multiple indicators can be measured with the same sample, or multiple samples can be measured with the same indicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic structural diagram of the immunochromatographic test paper array sample loading device of the present invention; Figure 2 It is a top view of the immunochromatographic test paper array sample loading device of the present invention; Figure 3 This is a side view of the immunochromatographic test paper array sample loading device of the present invention; Figure 4 It is a schematic diagram of high-throughput detection of the present invention; Figure 5 It is a high-throughput detection signal curve diagram of the present invention. In the figure, 1. 96-well plate fixing bracket, 2. test strip support plate, 3. immunochromatographic test strip, 4. test strip support plate clamping position, 5. test strip support plate limit blocking, 6. test strip sample adding area, 7. 96-well fixed bracket slot, 8. 96-well plate, 9. 3 isolated photoelectric sensor detection modules, 10. T line signal, 11. C line signal, 12. TC blank area optical signal. DETAILED DESCRIPTION

[0013] In order to more clearly illustrate the embodiments of the present invention, the specific implementation methods of the present invention will be described below with reference to the accompanying drawings. Obviously, for those skilled in the art, other drawings and other implementation methods can be obtained based on these drawings without creative work. Example 1

[0014] Reference Figure 1 to Figure 5 The present invention proposes an assembled high-throughput immunochromatographic test paper array loading device, comprising a 96-well plate fixing bracket and 12 test paper strip support plates, 8 immunochromatographic test papers are arranged and fixed on the test paper strip support plate through the test paper strip support plate clamping and limiting blocking, and the 8 test paper strip loading areas protrude 10 mm from the test paper strip support plate, and the 12 support plates are inserted into the card slots of the 96-well fixing bracket side by side to form a 96-channel immunochromatographic test paper array loading device; The assembled array loading device is installed on a 96-well plate, and the loading areas of 96 test strips simultaneously contact the samples in the 96 wells of the 96-well plate, and high-throughput sample loading detection is completed through the siphon effect of the test strips. In this embodiment, the immunochromatographic test strip is a standard 8cm long strip, the loading area is about 3.5mm deep into the sample liquid surface, and the loading volume is about 100μL; the reaction time is 15min; The detection marker is time-resolved fluorescent nanoparticles, the detection excitation wavelength is 365nm, and the detection emission wavelength is 610nm; in the dark box, there are 3 mutually isolated photoelectric sensing detection modules, the LED wavelength in the photoelectric sensing detection module is 365, the fluorescence detection channel has a 610nm narrow bandpass filter, PD is a sensor, and the 3 photoelectric sensing detection modules correspond to the T line, C line, and the blank area between TC respectively. The linear slide rail drives the 3 mutually isolated photoelectric sensing detection modules to move, and scans and obtains the signal curves of the T line, C line, and the blank area between TC of each test paper; By calculating the value of (Tb) / (Cb), which is correlated with the concentration of the detection target, the concentration of each test paper test sample is obtained by comparing it with the standard series concentration curve. Example 2

[0015] The present invention provides an assembled high-throughput immunochromatographic test paper array loading device, comprising a 96-well plate fixing bracket and 8 test paper strip support plates, 12 immunochromatographic test papers are arranged and fixed on the test paper strip support plate through the test paper strip support plate clamping and limit blocking; and the 12 test paper strip loading areas protrude 5mm from the test paper strip support plate, and the 8 support plates are inserted into the card slots of the 96-well fixing bracket side by side and fixed, so as to form a 96-channel immunochromatographic test paper array loading device; The assembled array loading device is installed on a 96-well plate, and the loading areas of 96 test strips simultaneously contact the samples in the 96 wells of the 96-well plate, completing high-throughput sample loading detection through the siphon effect of the test strips.

[0016] In this embodiment, the immunochromatographic test strip is a standard 6 cm short strip, the sample addition area is about 2 mm deep into the sample liquid surface, the sample addition volume is about 56 μL, the detection marker is colloidal gold, and the reaction time is 15 min; After the reaction is finished, the T line and C line appear dark red, and the blank area between TC is light white. Through natural light and camera photography, the image of the entire test paper support plate is obtained; Through image analysis, the image grayscale values ​​of the T line, C line, and blank area b between TC of each test paper are obtained, and the value of (Tb) / (Cb) is calculated. This value is correlated with the concentration of the detection target. By comparing with the standard series concentration curve, the concentration of each test paper detection sample is obtained.

[0017] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An assembled 96-channel immunochromatographic test strip array sample loading device and a high-throughput detection method, characterized in that: The assembled 96-channel immunochromatographic test strip array loading device described in S0 comprises a 96-well plate fixing bracket (1) and a test strip support plate (2); the immunochromatographic test strips (3) are arranged and fixed by means of a clamping position (4) and a limit stop (5) on the test strip support plate (2), and the test strip loading area (6) protrudes from the test strip support plate (2); a plurality of support plates can be inserted into the clamping slot (7) of the 96-well fixing bracket (1) and fixed, thereby forming a 96-channel immunochromatographic test strip array loading device.

2. According to claim 1, the high-throughput detection method comprises: S1 Loading: The sample is placed in a 96-well plate. The 96-channel immunochromatographic test strip array loading device is installed vertically corresponding to the 96 wells of the 96-well plate, so that the loading area of ​​the test strip is inserted into the well and reaches below the liquid surface; S2 reaction: 96 channels are loaded vertically at the same time. Using the siphon effect of the test paper, the sample crawls upward and the sample liquid level drops. When the sample liquid level is disconnected from the test paper, the sample loading is stopped. When the absorbed sample passes through the binding pad of the immunochromatographic test paper and combines with the labeled antibody, it continues to pass through the detection T line and combines with the capture antibody. The excess labeled antibody continues to crawl and combines with the control C line. The excess liquid is absorbed by the absorbent pad, completing the entire immunochromatographic test. S3 Scanning and detection: After the reaction is completed, a test strip support plate is placed in a high-dose immunochromatographic test strip detection device, which includes three mutually isolated photoelectric sensor detection modules (9), and the three photoelectric sensor detectors correspond to the T line, C line and the blank area b between TC of the immunochromatographic test strip; the scanning and detection of the immunochromatographic test strips on all the support plates are completed in sequence, and the curve graphs of the T line signal (10), the C line signal (11) and the background signal (12) of the blank area b between TC are obtained; the signal curve of each test strip on each support plate is recorded and displayed; S4 Result calculation: According to the overall curve of each test strip, calculate the peak height or area under the curve of the T line peak and C line peak signal, deduct the background signal of the blank area b between the C line and TC, calculate the (Tb) / (Cb) value and the concentration is linearly related, calculate with the pre-stored calibration curve, and obtain the concentration of the substance to be tested in each sample. The result data of 96 samples are displayed and saved at the same time.

3. according to claim 1, an assembled 96-channel immunochromatographic test paper array loading device and a high-throughput detection method, the assembled 96-channel immunochromatographic test paper array loading device is used in combination with a standard 96-well plate, and its immunochromatographic test paper strip support plate has two assembly methods according to the arrangement of the 96-well plate array, the first method is that one support plate can fix 8 immunochromatographic test strips, and 12 support plates are required; the second method is that 1 support plate fixes 12 immunochromatographic test papers, and 8 support plates are required; the support plate is inserted into a 96-well plate fixing bracket to form 96 immunochromatographic test paper array loading devices; the entire loading device is installed on the 96-well plate, and the immunochromatographic test paper strip loading area is exposed 0.5-1cm outside the supporting plate, and the loading area of ​​each test strip corresponds to a plate hole that is just inserted into the hole and contacts the sample, and 96 channels can be loaded and tested simultaneously.

4. According to claim 1, an assembled 96-channel immunochromatographic test strip array loading device and a high-throughput detection method, the assembled 96-channel immunochromatographic test strip array loading device, the number of support plates can be increased or decreased during assembly, and the number of detection channels can be flexibly changed according to the number of samples in the 96-well plate; similarly, several 96-well plates can be used in combination to increase the number of channels, or a matching device with a 384-well plate can be designed to perform 384-channel detection.

5. According to claim 1, an assembled 96-channel immunochromatographic test paper array loading device and a high-throughput detection method, the assembled 96-channel immunochromatographic test paper array loading device, the immunochromatographic test paper fixed on the support plate protrudes from the support plate and penetrates into the sample of the 96-well plate, the support plate is adjustable up and down, so that the depth of the interface of the test paper strip penetrating into the sample can be adjusted, when the absorbed sample liquid level continues to decline and breaks contact with the test paper, the loading is completed, the sample is absorbed less when the depth is shallow, and the sample is absorbed more when the depth is deep, which can ensure the sufficiency of the sample loading amount and the consistency of the loading amount of all test papers.

6. According to claim 1, an assembled 96-channel immunochromatographic test strip array loading device and a high-throughput detection method, wherein the linear slide rail is used as a mobile platform for the test strip support plate, the detection component has three parallel photoelectric detection modules, the optical paths of the three detection modules are completely separated and do not interfere with each other, the photoelectric detection module has a detection light source and a photoelectric sensor, and the optical paths of the detection light source and the sensor are coaxial or at an angle of 3 degrees to 60 degrees. The distance resolution of the three detection units corresponds to the position of the T line, C line and the blank area b (background) between the T line and the C line on the test strip, and the scanning mode is horizontal scanning, that is, the linear slide rail sends the test strip support plate to the detection position, that is, the three photoelectric detection modules correspond to the T line, C line, and the blank area between the T line and the C line on an immunochromatographic test strip, respectively, and scan from the first immunochromatographic test strip to the last test strip, completing the detection of 8 or 12 chromatographic test strips once, and completing the detection of 96 chromatographic test strips multiple times.

7. According to claim 1, an assembled 96-channel immunochromatographic test strip array loading device and a high-throughput detection method, wherein another working principle of the high-throughput detection method is to obtain an image of the entire test strip support plate in a dark box by photographing. If it is color detection, uniform white light is used as the illumination source. If it is a fluorescence detection mode, an LED with a wavelength required for fluorescence excitation is used as the illumination light source. After taking pictures, the T line and C line on each immunochromatographic test strip are identified by image analysis to complete the detection of 96 chromatographic test strips.

8. According to claim 1, an assembled 96-channel immunochromatographic test strip array sample loading device and a high-throughput detection method, wherein the assembled 96-channel multi-channel is not only capable of performing simultaneous detection of multiple samples, but is also applicable to the combined detection of multiple indicators of a single sample.

9. According to claim 1, an assembled 96-channel immunochromatographic test paper array loading device and a high-throughput detection method, wherein the immunochromatographic test paper is also applicable to lateral flow chromatography test paper for nucleic acid molecule detection in principle.

10. According to claim 1, an assembled 96-channel immunochromatographic test strip array loading device and a high-throughput detection method have flexible channel number configuration, high detection throughput, short detection time and high efficiency, and can be used in clinical testing, health testing, epidemic prevention testing, scientific research or high-throughput drug screening and detection, etc., which require a large number of samples for real-time detection. At the same time, it can also be placed in a 96-well automated mobile detection platform to meet the needs of ultra-high-throughput sample automated detection.

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