WO2020062490A1

WO2020062490A1 - Detection device

Info

Publication number: WO2020062490A1
Application number: PCT/CN2018/115802
Authority: WO
Inventors: 李金峰; 柯跃斌; 何洁; 肖云军; 吕子全
Original assignee: 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所）
Priority date: 2018-09-26
Filing date: 2018-11-16
Publication date: 2020-04-02
Also published as: CN109444413B; CN109444413A

Abstract

A detection device for quantitatively detecting the content of a target substance in a sample. By means of comparing the signal intensity in a detection area (11) and the signal intensity in a reference area (12), target molecules in a sample can be quantitatively detected. The detection device has low production costs, and accurate and reliable detection results.

Description

Specification Invention Name: A detection device

Technical field

[0001] The present invention relates to the field of biological detection, and in particular, to a detection device for detecting components in a sample.

Background technique

[0002] The rapid detection technology is currently developing rapidly, and has been widely used in clinical, food safety, forensic, environmental, military, and many other fields. However, most of these technologies are applied in the field of qualitative detection, and it is difficult to perform quantitative detection. Many factors such as the homogeneity of the marker signal, the reaction time, the reaction temperature and humidity, and the reaction speed will affect the reaction result, thereby increasing the coefficient of variation, restricting the development of rapid and precise detection technology and thus failing to achieve the goal of quantitative detection. Taking the most commonly used immunochromatographic test strips as an example, low temperature will significantly reduce the signal strength of the test strips; too long or too short a reaction time will directly affect the signal ratio between the detection area and the reference area; The chromatographic speed of the sample on the chromatographic membrane will be different, which will also affect the quantitative results; the uniformity of the chromatographic membrane material will also affect the signal ratio between the detection area and the reference area. The inconsistent reaction time between the detection zone and the reference zone is also an important reason for the instability of the quantitative results of immunochromatography.

Summary of invention

technical problem

Problem solution

Technical solutions

[0003] The present invention provides a detection device to make a detection zone and a reference zone react at the same time and under the same conditions, so as to improve the accuracy of quantitative detection.

[0004] The technical problem to be solved by the present invention is achieved by the following technical solutions:

[0005] A detection device is provided with a detection area and a reference area independent of each other, and the detection area and the reference area are located at the same cross section of the sample flow direction.

[0006] The detection device may be an immunochromatographic test strip, or a microfluidic chip, or other device that realizes detection by flowing a sample through a detection area and a reference area, and the detection device measures adjacent locations The ratio of the signal in the detection zone to the reference zone to determine the amount of target molecules in the sample. [0007] The detection device has two or more detection zones and one or more reference zones at the same cross section of the sample flow direction.

[0008] In the present invention, the detection area and the reference area are in accordance with the "detection area, reference area, detection area, reference area ...

... "in the same cross-section in the direction of sample flow.

[0009] In the present invention, the shape of the detection area and / or the reference area is any one of a regular pattern, a line, or other shapes; wherein the regular pattern includes a circle, a square, a rectangle, At least one of ellipse, polygon, and star.

[0010] In the present invention, the concentration of the capture molecules coated in different detection regions at the same cross section may be different.

The beneficial effects of the invention

Beneficial effect

[0011] The present invention has the following beneficial effects:

[0012] The detection device provided by the present invention enables the detection zone and the reference zone to participate in the reaction at the same time, various conditions in the reaction process are infinitely close, and the quantitative result is more accurate. In order to further improve the accuracy of quantification, the present invention also provides a detection device with multiple detection zones and reference zones. In addition to the accurate detection results, the detection scheme provided by the present invention is also easy to produce on a large scale without increasing production costs.

Brief description of the drawings

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a schematic view of the structure of a chromatography membrane component of an immunochromatographic test strip in scheme A and embodiment 6 of Example 1 of the present invention (one detection area, one reference area);

[0014] Note: 11. Detection area; 12. Reference area; 13. Chromatographic membrane; Arrow: direction of liquid flow.

[0015] FIG. 2 is a schematic structural diagram of a chromatographic membrane component of a scheme B immunochromatographic test strip in Example 1 of the present invention (one detection area, one reference area);

[0016] Note: 14. Detection area; 15. Reference reference area; 13. Chromatographic membrane; Arrow: direction of liquid flow.

[0017] FIG. 3 is a schematic structural diagram of a chromatography membrane component of a scheme A immunochromatographic test strip in Embodiment 2 of the present invention (multiple detection zones, multiple reference zones);

[0018] Note: 21. Detection area; 22. Reference area; 23. Chromatographic membrane; Arrow: direction of liquid flow.

[0019] FIG. 4 is a schematic structural diagram of a chromatography membrane component of a protocol B immunochromatographic test strip in Example 2 of the present invention; [0020] Note: 24. Detection area; 25. Reference area reference area; 23. Chromatography membrane; arrow: direction of liquid flow. [0021] FIG. 5 is a schematic diagram of pipelines related to a detection area and a reference area of a solution A microfluidic chip in Embodiment 3 of the present invention;

[0022] Note: 31. Detection area; 32. Reference area; Arrow: direction of liquid flow.

Invention Examples

Embodiments of the invention

[0023] The present invention is described in detail below with reference to the drawings and embodiments.

Example 1 Quantitative detection of alpha-fetoprotein (AFP) in serum by immunochromatographic test strips

[0025] A. The solution of this embodiment:

[0026] 1.

Nanoparticle-labeled detection antibody (nanoparticle-detection antibody conjugate): Take rare earth fluorescent nanoparticles (particle size: 300nm) with HXVL concentration of 10mg / mL at 90 (^ L

In MES (2- (N-morpholine) ethanesulfonic acid, 50mM, PH6.1), vortex and mix; add 20mg NHS (N-hydroxysuccinimide), 20mg EDC (1- (3-dimethylformaldehyde) Aminopropyl) -3 -ethylcarbodiimide), vortex and mix; react at room temperature for 30min; centrifuge at 1000rpm for 10min, discard the supernatant; vortex and resuspend the particles; add AF P detection antibody 20, react at room temperature for 2 ~ 4h Add casein to a final concentration of 0.1% and continue the reaction for 2 to 4 h; centrifuge at 1000 rpm for 10 min and discard the supernatant; wash the particles three times with PBST (phosphate Tween buffer); add 2 mL of particle resuspension (10 mM Tris -HCl buffer, 1% BSA (bovine serum albumin)

, 5% trehalose) Resuspend the pellet and set aside.

[0027] 2.

Nanoparticle-labeled reference antibody (nanoparticle-reference antibody conjugate): Take 10 (VL concentration 10mg / mL rare earth fluorescent nanoparticles (particle size: 300nm)) in 900pL MES (50mM, PH6.1), Vortex to mix; add 20mg NHS, 20mg

EDC, vortex and mix; react at room temperature for 30 min; centrifuge at 1000 rpm for 10 min and discard the supernatant; vortex and resuspend the particles; add melamine monoclonal antibody 40 and react at room temperature for 2 to 4 h; add casein to make the final concentration of 0.1%, Continue the reaction for 2 ~ 4h; centrifuge at 1000rpm for 10min, discard the supernatant; wash the particles three times with PBST; add 2mL of particle resuspension (10mM Tris-HCl, 1% BSA, 5% trehalose) and resuspend the particles for later use.

[0028] 3. Preparation of binding pad: The prepared nanoparticle-detection antibody conjugate and nanoparticle-reference antibody conjugate were mixed in equal volumes, and then sprayed onto the glass cellulose membrane at 3 pL / cm. At 37 ° C Oven dry and set aside.

[0029] 4. Assembly of test strip: Paste the nitrocellulose membrane (chromatographic membrane 13) to the middle position of the PVC bottom plate. Then, affix the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the bonding pad, the chromatography membrane 13, and the absorbent paper are successively overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. Then cut into 5.5mm wide immunochromatographic test strips.

[0030] 5.

Treatment of test strips: 2mg / ml AFP capture antibody and 2mg / ml melamine-BSA (bovine serum albumin) conjugate spray points at the middle position of the chromatography membrane 13 (30mm away from the end of the sample pad), respectively As the detection area 11 and the reference area 12, the detection area 11 and the reference area 12 are arranged side by side on a horizontal line perpendicular to the long side of the test strip, so that the detection area 11 and the reference area 12 are simultaneously with the sample to be measured Contact and reaction. Dry at 37 ° C, then store at 2-8 ° C in a dry, dark place.

[0031] 6. Quantitative detection of AFP in serum: Placing a test strip on the surface of a water platform, drop 18 (VL serum on a sample pad, leave it for 15 minutes, and use a quantitative detection device for quantitative detection.

[0032] B. Comparative scheme: 5 see an immunochromatographic test strip

[0033] 1.

Preparation of Nanoparticle-labeled Detection Antibody (Nanoparticle-Detection Antibody Conjugate): Refer to "A. Scheme of this embodiment" in this example.

[0034] 2. Preparation of nanoparticle-labeled reference antibody (nanoparticle-reference antibody conjugate): See this example

"A. Scheme of this embodiment".

[0035] 3. Preparation of the bonding pad: See the "A. Scheme of this embodiment" in this embodiment.

[0036] 4. Preparation of chromatographic membrane 13: AFP capture antibody (detection area 14) with a concentration of 2 mg / mL was sprayed on the chromatographic membrane 13 in the order of 0.8 pL / cm along the chromatographic direction, 2 mg / mL The melamine-BSA (bovine serum albumin) conjugate (reference zone 15) was dried at 37 ° C for 2h and used.

[0037] 5. Assembly of test strip: Paste the nitrocellulose membrane (chromatographic membrane 13) to the middle position of the PVC bottom plate

(The detection area 14 is near the end of the sample pad, and the reference area 15 is near the end of the absorbent paper). Then, attach the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the binding pad chromatography membrane 13, and the absorbent paper are sequentially overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. . Finally, a 5.5mm wide immunochromatographic test strip was cut and stored at 2-8 ° C, in a dry and dark place. [0038] 6. Quantitative detection of AFP in serum: Placing a test strip flat on a water platform, drop 18 (VL serum on a sample pad, leave it for 15 minutes, and use a quantitative detection device for quantitative detection.

[0039] Evaluation of results: Compared with the prior art (scheme B), the immunochromatographic test strip (scheme A) has significantly improved detection stability, and the experimental data is shown in Table 1 (the same concentration is repeated 10 times with two schemes) Statistical Analysis) .

[0040] Table 1 Comparison of the AFP detection scheme (Scheme A) and the existing immunochromatographic test strip (Scheme B) in this embodiment

[0041] Note: The coefficient of variation, also known as the coefficient of dispersion or relative deviation (rsd), is the ratio of the standard deviation sd to the mean mean, expressed as a percentage, and the calculation formula is: cv = sd / mean xl00%. The larger the coefficient of variation, the more unstable it is.

[0042]

Example 2 Quantitative detection of galectin -3 by immunochromatographic test strips

[0044] A. The solution _of this embodiment:

[0045] 1.

Nanoparticle-labeled detection antibody (nanoparticle-detection antibody conjugate): Take rare earth fluorescent nanoparticles (particle size: 300nm) with HXVL concentration of 10mg / mL in 900pL MES (50mM, PH6.1), and mix by vortexing ; Add 20mg NHS, 20mg

EDC, vortex and mix; react at room temperature for 30min; centrifuge at 1000rpm for 10min, discard the supernatant; vortex and resuspend the particles; add galectin-3 detection antibody 5 (Vg, react at room temperature for 2 ~ 4h; add casein to make The final concentration is 0.1%, and the reaction is continued for 2 ~ 4h; centrifuge at 1000rpm for 10min, discard the supernatant; wash the particles three times with PBST; add 2mL particle resuspension (10mM Tris-HCl, 1% BSA, 5% trehalose) and resuspend the particles Spare.

[0046] 2.

[0047] 3. Preparation of binding pad: The prepared nanoparticle-detection antibody conjugate and nanoparticle-reference antibody conjugate were mixed in equal volumes, and then sprayed onto the glass cellulose membrane at 3 pL / cm. , Dry in 37 ° C oven, set aside.

[0048] 4. Assembly of test strips: Paste the nitrocellulose membrane (chromatographic membrane 23) to the middle position of the PVC bottom plate

. Then, affix the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the binding pad chromatography membrane 23, and the absorbent paper are sequentially overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. . Finally cut into 6.0mm immunochromatographic test strips.

[0049] 5. Processing of test strips: As shown in FIG. 3, at the middle position of the chromatography membrane 23 (30 mm away from the end of the sample pad), 2 mg / ml galectin-3 capture antibody and 2 mg / ml were used, respectively. The melamine-BSA conjugate spray point is used as the detection area 21 and the reference area 22 (in the order of "detection area 21, reference area 22, detection area 21, reference area 22, detection area 21, and reference area 22"). (A) a horizontal line arranged side by side perpendicular to the long side of the test strip is convenient for the detection area 21 and the reference area 22 to contact and react with the sample to be tested at the same time). Dry at 37 ° C, then store in a dry place.

[0050] 6.

Quantitative detection of galectin-3 in serum: Place the test strip on the flat surface of the water, drop 18 (VL serum on the sample pad, and let it stand for 15 min. Use the quantitative detection equipment for quantitative detection.

[0051]

[0052] B _· comparison scheme: [0053] 1.

[0054] 2. Preparation of nanoparticle-labeled reference antibody (nanoparticle-reference antibody conjugate): See this example

"A. Scheme of this embodiment".

[0055] 3. Preparation of the bonding pad: See the "A. Scheme of this embodiment" in this embodiment.

[0056] 4. Preparation of chromatographic membrane 23: As shown in FIG. 4, galectin-3 was sprayed on the chromatographic membrane 23 at a concentration of 2 mg / mL in the order of 0.8 pL / cm along the chromatographic direction. Capture antibody (detection zone 24), 2mg / mL melamine-BSA (bovine serum albumin) conjugate (reference zone 25), dry at 37 ° C for 2h, and reserve

[0057] 5. Assembly of test strips: Paste a nitrocellulose membrane (chromatographic membrane 23) to the middle position of the PVC base plate

(The detection area 24 is near the end of the sample pad, and the reference area 25 is near the end of the absorbent paper). Then, affix the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the binding pad chromatography membrane 23, and the absorbent paper are sequentially overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. . Finally, 5.5mm wide immunoassay strips were cut and stored at 2-8 ° C in a dry and dark place.

[0058] 6.

Quantitative detection of galectin in serum -3: Place the test strip on the flat surface of the water, drop 18 (VL serum on the sample pad, and let it stand for 15 minutes, and use the quantitative detection equipment for quantitative detection.

[0059] Evaluation of results: Compared with the prior art (scheme B), the immunochromatographic test strip (scheme A) has significantly improved detection stability. The experimental data are shown in Table 2 (the same concentration is repeated 10 times with two schemes) Statistical Analysis) .

[0060]

Table 2 Comparison of the scheme (scheme A) of this embodiment and the existing immunochromatographic test strip (scheme B) for the detection of galectin-3

[]

Example 3 Quantitative Detection of Insulin-Like Growth Factor Binding Protein (IGFBP-7) with a Microfluidic Chip

[0063] The reference region 32 and the detection region 31 on the microfluidic chip are located at the same cross section of the liquid flow, or in different reaction regions through which the liquid flows simultaneously. Although the detection area 31 and the reaction area are located in different reaction areas of the chip, the liquid to be detected flows through the two areas at the same time to ensure the synchronization of the reactions.

[0064] Evaluation of the results: Table 3 shows a microfluidic chip (scheme A, shown in FIG. 5) using the reference region 32 and the detection region 31 at the same cross-section of the liquid flow direction and the prior art (reference region And the detection zone 31 is located at a cross section of different liquid flow direction, scheme B) Experimental results of detecting IGFBP-7 in serum. It can be seen that the detection stability of scheme A has been significantly improved (the same concentration was repeatedly measured 10 times for statistical analysis with two schemes).

Table 3 Solution (A) and Existing Solution (B) of this Example

Example 4 Quantitative detection of alpha-fetoprotein (AFP) in serum by immunochromatographic detection [0067] For specific solutions, see A and B in Example 1.

[0068] This embodiment differs from Example 1 in that: The processing of the test strip of step 5 in the scheme A is changed to: In the middle position of the chromatography membrane (30 mm away from the end of the sample pad), respectively, 2 mg / ml, 1.2 mg / ml, 0.6 mg / ml AFP capture antibody, and 2 mg / ml melamine-BSA (bovine serum albumin) conjugate spray point as the detection area and reference area (according to the "detection area (2 mg / ml

AFP capture antibody), reference area (2mg / ml melamine-BSA), detection area (1.2mg / ml AFP capture antibody), reference area (2mg / ml melamine-BSA), detection area (0.6mg / ml AFP capture Antibodies) "sequence is placed side by side on a horizontal line perpendicular to the long side of the test strip, so that the detection area and the reference area can contact and react with the sample to be tested at the same time). Dry at 37 ° C, then place Store at 2-8 ° C in a dry and dark place.

[0069] Evaluation of results: Compared with the prior art (scheme B), the immunochromatographic test strip (scheme A) has significantly improved detection stability, and compared with the scheme A in Example 1, the coefficient of variation is also significantly reduced. The experimental data are shown in Table 4 (repeated determination of the same concentration with two protocols 10 times for statistical analysis).

Table 4 Comparison of the AFP detection scheme of this embodiment (scheme A) and the existing immunochromatographic test strip (scheme B)

[]

Example 5 Quantitative detection of chloramphenicol by immunochromatographic test strips

[0072] A. The solution of this embodiment:

[0073]

Nanoparticle-labeled detection antibody (nanoparticle-detection antibody conjugate): Take rare earth fluorescent nanoparticles (particle size: 300nm) with HXVL concentration of 10mg / mL in 900pL MES (50mM, PH6.1) and vortex Well; add 20mg NHS, 20mg

EDC, vortex and mix; react at room temperature for 30min; centrifuge at 1000rpm for 10min, discard the supernatant; vortex and resuspend the particles; add 15pg of chloramphenicol detection antibody, react at room temperature for 2 ~ 4h; add casein to make the final concentration of 0.1% Continue to react for 2 ~ 4h; Centrifuge at 1000rpm for 10min, discard the supernatant; wash the particles three times with PBST; add 2mL of particle resuspension (10mM Tris-HCl, 1% BSA, 5% trehalose) and resuspend the particles for later use.

[0074] 2.

EDC, vortex and mix; react at room temperature for 30min; centrifuge at 1000rpm for 10min, discard the supernatant; vortex and resuspend the particles; add melamine monoclonal antibody 15 and react at room temperature for 2 ~ 4h; add casein to make the final concentration of 0.1%, Continue the reaction for 2 ~ 4h; centrifuge at 1000rpm for 10min, discard the supernatant; wash the particles three times with PBST; add 2mL of particle resuspension (10mM Tris-HCl, 1% BSA, 5% trehalose) and resuspend the particles for later use.

[0075] 3. Preparation of binding pad: The prepared nanoparticle-detection antibody conjugate and nanoparticle-reference antibody conjugate were mixed in equal volumes, and then sprayed onto a glass cellulose membrane at 2 pL / cm ( Width: 6mm), put it in an oven at 37 ° C for drying, and set aside.

[0076] 4. Assembly of test strips: Paste a nitrocellulose membrane (chromatographic membrane) to the middle position of the PVC bottom plate.

Then, attach the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the binding pad chromatography membrane, and the absorbent paper are sequentially overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. Finally cut into 6.0mm immunochromatographic test strips.

[0077] 5. Treatment of test strips: As shown in FIG. 1, at the middle position of the chromatography membrane (30 mm away from the end of the sample pad), 2 mg / ml chloramphenicol-BSA conjugate and 2 mg / ml melamine were used, respectively. -BSA conjugate spray line, as detection area and reference area. The two lines are independent of each other, and there is no overlap, and the detection area and the reference area are arranged side by side on a horizontal line perpendicular to the long side of the test strip, which is convenient for the detection area and the reference area to contact and react with the test sample at the same time. . Dry at 37 ° C and store in a dry place.

[0078] 6. Quantitative detection of chloramphenicol in milk: Place a test strip flat on the surface of a water platform, drop 18 (VL milk on a sample pad, and let stand for 5 minutes, using a quantitative detection device for quantitative detection. [0079]

[0080] B _· comparison scheme:

[0081] 1.

[0082] 2. Preparation of nanoparticle-labeled reference antibody (nanoparticle-reference antibody conjugate): see this example

"A. Scheme of this embodiment".

[0083] 3. Preparation of the bonding pad: See the "A. Scheme of this embodiment" in this embodiment.

[0084] 4. Preparation of chromatography membrane: As shown in FIG. 2, a chloramphenicol-BSA conjugate with a concentration of 2 mg / mL was sprayed on the chromatography membrane in the order of 0.8 pL / cm along the chromatography direction. (Detection area), 2mg / mL melamine-BS A (bovine serum albumin) conjugate (reference area), dried at 37 ° C for 2h, ready for use.

[0085] 5. Assembly of test strips: Paste a nitrocellulose membrane (chromatographic membrane) to the middle position of the PVC bottom plate (the detection area is near the end of the sample pad and the reference area is near the end of the absorbent paper). Then, attach the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the binding pad chromatography membrane, and the absorbent paper are sequentially overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. Finally, 5.5mm wide immunochromatographic test strips were cut and stored at 2-8 ° C, in a dry and dark place.

[0086] 6. Quantitative detection of chloramphenicol residues in milk: Place the test strip on the surface of the water platform, drop 18 (VL milk on the sample pad, leave it for 5 minutes, and use the quantitative detection equipment for quantitative detection.

[0087] Evaluation of results: Compared with the prior art (scheme B), the detection stability of immunochromatographic test strips (scheme A) is significantly improved. The experimental data are shown in Table 5 (the same concentration is repeated 10 times with two schemes) Statistical Analysis) .

Table 5 Comparison of the scheme (scheme A) of this embodiment and the existing immunochromatographic test strip (scheme B) for the detection of chloramphenicol

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Effect of Example 6 Carbomer 940 on Improving Detection Stability

[0090] 1.

In MES (2- (N-morpholine) ethanesulfonic acid, 50mM, PH6.1), vortex and mix; add 20mg NHS (N-hydroxysuccinimide), 20mg EDC (1- (3-dimethylformaldehyde) Aminopropyl) -3 -ethylcarbodiimide), vortex and mix; react at room temperature for 30min; centrifuge at 1000rpm for 10min, discard the supernatant; vortex and resuspend the particles; add C-reactive protein (CRP) detection antibody 20, room temperature Reaction for 2 ~ 4h; Add casein to make final concentration 0.1%, continue reaction for 2 ~ 4h; centrifuge at 1000rpm for 10min, discard supernatant; wash particles with PBST (phosphate Tween buffer) three times; add 2mL particles to resuspend Solution (10 mM Tris-HCl buffer, 1% BSA (bovine serum albumin), 5% trehalose). Resuspend the pellet and set aside.

[0091] 2.

EDC, vortex and mix; react at room temperature for 30min; centrifuge at 1000rpm for 10min, discard the supernatant; vortex and resuspend the particles; add melamine monoclonal antibody 40 and react at room temperature for 2 ~ 4h; add casein to make the final concentration of 0.1%, Continue the reaction for 2 ~ 4h; centrifuge at 1000rpm for 10min, discard the supernatant; wash the particles three times with PBST; add 2mL of particle resuspension (10mM Tris-HCl, 1% BSA, 5% trehalose) and resuspend the particles. use.

[0092] 3. Preparation of binding pad: Put the binding pad (glass fiber) in carbomer 940 gel of different concentrations in advance for 5 minutes, take it out and dry it at 65 ° C, and prepare the prepared nanoparticles-detection The antibody conjugate and the nanoparticle-reference antibody conjugate were mixed in equal volumes, then sprayed onto the binding pad at 3 pL / cm, dried in an oven at 37 ° C, and set aside.

[0093] 4. Assembly of test strips: Paste a nitrocellulose membrane (chromatographic membrane) to the middle position of the PVC bottom plate.

Then, attach the bonding pad, sample pad, and absorbent paper at the corresponding positions, so that the sample pad, the bonding pad, the chromatography membrane, and the absorbent paper are sequentially overlapped and pasted on the PVC base plate, and there is an overlap of about 1 mm between the two. ; Finally cut into 5.5mm wide immunochromatographic test strips.

[0094] 5.

Treatment of test strips: In the middle position of the chromatography membrane (30mm away from the end of the sample pad), use 2mg / ml CRP capture antibody and 2mg / ml melamine-BSA (bovine serum albumin) conjugate spray points, respectively, as The detection area and the reference area are arranged side by side on a horizontal line perpendicular to the long side of the test strip, which is convenient for the detection area and the reference area to contact and react with the test sample at the same time (see the figure) 1) . Dry at 37 ° C, then store at 2-8 ° C in a dry, dark place.

[0095] 6. Quantitative detection of CRP in serum: Place the test strip flat on the surface of the water platform, drop 18 (VL serum on the sample pad, leave it for 15 minutes, and use the quantitative detection equipment for quantitative detection.

[0096] 7. The prepared test strips were placed at 37 ° C for different times, and a CRP sample of the same concentration was taken out for measurement, and the determination was repeated five times, and the average value was taken. The results are shown in Table 6. It can be seen that adding an appropriate amount (0.1% -0.2%) of Carbom 940 can improve the stability of the test strip detection.

[0097]

Table 6 Effect of different concentrations of Carbomer 940 on detection stability

[] [Table 1]

[0099] Note: Deviation value = (measured average value-actual value) / actual value x 100%)

[0100]

[0101] The above-mentioned embodiment only expresses the implementation manner of the present invention, and its description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent for the utility model. The obtained technical solutions should all fall within the protection scope of the utility model.

[0102]

Claims

Claim

[Claim 1] A detection device, wherein the detection device is provided with a detection area and a reference area independent of each other, and the detection area and the reference area are located at the same cross section of the sample flow direction.

[Claim 2] The detection device according to claim i, wherein the detection device is an immunochromatographic test strip or a microfluidic chip, or other detection is achieved by passing a sample through a detection area and a reference area. The device determines the content of the target molecule in the sample by measuring the signal ratio between the detection area and the reference area at adjacent positions.

[Claim 3] The detection device according to claim i, wherein two or more detection regions and one or more reference regions are provided at the same cross section of the sample flow direction.

[Claim 4] The detection device according to claim 3, wherein the detection area and the reference area are in accordance with the "detection area, reference area, detection area, reference area ..." The sequences are side by side at the same cross section in the direction of sample flow.

[Claim 5] The detection device according to claim 4, wherein the concentration of the capture molecules in different detection regions at the same cross section is different.

[Claim 6] The detection device according to claim 1, wherein the shape of the detection area and / or the reference area is any one of a regular pattern and a line.

[Claim 7] The detection device according to claim 1, wherein the regular pattern includes at least one of a circle, a square, a rectangle, an ellipse, a polygon, and a star.

[Claim 8] The detection device according to claim 1, wherein the concentration of the capture molecules coated in different detection regions at the same cross section may be different.