An immunochromatographic test strip for glycated hemoglobin

Abstract

The present invention discloses an immunochromatographic test strip for glycated hemoglobin, which comprises a base plate, a sample pad, a conjugation pad, a reaction membrane, and an absorbent paper pad; the conjugation pad contains an immunoprobe; the immunoprobe is an HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres. The immunochromatographic test strip for glycated hemoglobin of the present invention utilizes the rapid and simple advantages of immunochromatographic technology coupled with the easy excitation, strong luminescence, and strong photostability of quantum dots. Through an evaporation swelling method and innovative reaction principles and synthesis steps, novel quantum dot fluorescent microspheres are prepared. Furthermore, the novel quantum dot fluorescent microspheres are uniquely used as markers to prepare immunoprobes through the EDC method. On this basis, an HbA1c chromatography test strip is constructed, and the purpose of quantifying HbA1c is achieved by detecting the fluorescent signal of the test strip.

Description

Technical Field

[0001] The present invention particularly relates to an immunochromatographic test strip for glycated hemoglobin. Background Art

[0002] Glycated hemoglobin A1c (HbA1c) is a product of the binding of hemoglobin within red blood cells (RBCs) to blood glucose. It is directly proportional to blood glucose concentration. Measuring HbA1c levels can be used to monitor a patient's overall blood glucose control over the past 120 days. It is the recognized gold standard for blood glucose control in diabetes management and an effective indicator for evaluating diabetes treatment plans. Currently, the World Health Organization (WHO) and diabetes associations in many countries have adopted HbA1c as an independent diagnostic indicator for diabetes.

[0003] Immunochromatography is a point-of-care testing technology that integrates immunochromatography, immunolabeling, and monoclonal antibodies to enable rapid, on-site testing of HbA1c. Traditional immunochromatographic techniques, such as colloidal gold, can only provide qualitative or semi-quantitative results, making interpretation subjective and sensitive. Currently, the primary strategy for improving immunochromatographic sensitivity and achieving quantitative testing is the use of highly sensitive fluorescent markers.

[0004] Quantum dots (QDs) are a new type of fluorescent nanocrystals with properties such as easy excitation, strong luminescence, and strong photostability. They are also not affected by solvents and are ideal fluorescent markers. Based on the preparation of high-performance fluorescent markers, fluorescent markers are encapsulated in nanospheres, or they are modified on the surface of nanospheres, or fluorescent materials are embedded in the nanospheres during the synthesis process to prepare fluorescent microspheres. Generally speaking, fluorescent microspheres have better fluorescence intensity and physical and chemical stability than single fluorescent materials, and the surface is also easier to functionalize. However, due to the limited maximum photoluminescence of a single fluorescent substance in quantum dots, in particular, the prepared fluorescent microspheres have poor stability, are prone to fluorescence quenching, and are highly dependent on specific instruments. Therefore, there are currently no reports on the use of quantum dots in immunochromatographic test strips to achieve quantitative detection of glycated hemoglobin. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides an immunochromatographic test strip for glycated hemoglobin, which is composed of a base plate, a sample pad, a conjugation pad, a reaction membrane and an absorbent paper pad;

[0006] The reaction membrane is marked with a T line and a C line; the T line is fixed with an anti-HbA1c monoclonal antibody; the C line is fixed with a goat anti-mouse IgG;

[0007] The conjugate pad contains an immune probe; the immune probe is an HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres.

[0008] Furthermore, the bottom plate is a PVC bottom plate; the sample pad, conjugate pad, reaction membrane and absorbent paper pad are sequentially adhered to the PVC bottom plate from left to right; the T line in the reaction membrane is on the left and the C line is on the right.

[0009] Furthermore, the sample pad is a glass fiber to which sodium borate buffer solution is added and dried.

[0010] Furthermore, the material of the binding pad and the absorbent paper is glass fiber, the material of the reaction membrane is nitrocellulose, and the material of the PVC bottom plate is polyvinyl chloride.

[0011] Furthermore, the bonding pad is per 1 cm 2 Contains 195ng of immune probe; each 1cm of the T line contains 300ng of anti-HbA1c monoclonal antibody; each 1cm of the C line contains 400ng of goat anti-mouse IgG.

[0012] Furthermore, the preparation method of the HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres is as follows:

[0013] 1) Synthesis of core-shell quantum dots:

[0014] ① Dissolve copper acetate and trioctylphosphine oxide in octadecene, degas at 100°C for 1 hour, blow nitrogen at 200°C for 5-15 minutes, add 1-dodecanethiol at 160°C, and after the solution turns brown, react at 200°C for 100 minutes. Cool, wash with butanol and methanol, centrifuge, and dissolve the crystals in toluene to obtain the stock solution.

[0015] ② Take the stock solution obtained in step ①, add toluene to dilute it 3 to 4 times, then add In(NO3)3·H2O methanol solution in the presence of sodium pyrrolidonecarboxylate, react at 17±2°C for 4 days, wash with butanol and methanol, take the purified CIS quantum dots, add toluene with 2 / 3 of the stock solution volume to dissolve, and obtain CIS quantum dot toluene solution;

[0016] ③ Take Zn(St)2, add 1-octadecene to dissolve, and then add TOP-S solution to mix to obtain a ZnS precursor solution; take the CIS quantum dot toluene solution obtained in step ② and mix it with 1-octadecene, remove toluene, heat to 200°C, add ZnS precursor solution, and then maintain at 200°C for 60 minutes. Cool, add acetone, centrifuge, take crystals and disperse them in toluene to obtain a dispersion;

[0017] ④ Take carboxyalkane sulfide, add water and tri(2-formylethyl)phosphine hydrochloride solution, mix, and then add tetramethylammonium hydroxide to pH 11.7 to obtain MUA solution; take the dispersion obtained in step ③, add chloroform and mix, then add MUA solution and stir for 8-12 hours, centrifuge, take the supernatant and centrifuge again, collect the solid and dissolve it in water at pH 7 to obtain CIS / ZnS core / shell quantum dot solution;

[0018] 2) Preparation of quantum dot fluorescent microspheres:

[0019] ⑤ Mix azobisisobutyronitrile and styrene to obtain a mixture; dissolve polyvinyl pyrrolidone in ethanol and water to obtain a mixed solution; add the mixture to the mixed solution, stir under a sealed nitrogen atmosphere for 12 to 20 hours, wash the solid with ethanol, and centrifuge to obtain linear polystyrene particles;

[0020] ⑥ Dispersing the linear polystyrene particles obtained in step ⑤ into an aqueous solution of sodium dodecyl sulfate, adding a cadmium solution, and swelling the mixture of styrene, divinylbenzene, methyl acetoacetate, and benzoyl peroxide, then adding an aqueous solution of polyvinyl alcohol, heating and reacting for 10 to 15 hours, washing the solid with ethanol, centrifuging, and finally extracting with dichloromethane to obtain a dichloromethane solution containing nanospheres;

[0021] ⑦ Take the CIS / ZnS core / shell quantum dot solution obtained in step ④, add methanol chloroform solution to mix, centrifuge, take the solid and disperse it in chloroform, then add the dichloromethane solution containing nanospheres obtained in step ⑥, stir and expand, then evaporate the chloroform and dichloromethane at room temperature, finally wash with xylene, centrifuge, and collect;

[0022] 3) Preparation of immunoprobes:

[0023] ⑧ Take the quantum dot fluorescent microspheres obtained in step ⑦, add 2-morpholineethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, activate for 20 to 60 minutes, then add anti-HbA1c antibody, react for 2 hours, centrifuge, take the precipitate, add glycine and mix for 1.5 hours, add tris(hydroxymethyl)aminomethane and mix to obtain.

[0024] Furthermore, in step ①, the ratio of copper acetate, trioctylphosphine oxide, octadecene, 1-dodecanethiol, and toluene is 0.6 mmol: 5.6 mmol: 50 mL: 3.5 ml: 5 mL; the volume ratio of butanol and methanol is 1:1; the centrifugation speed is 3000 rpm, and the time is 20 minutes;

[0025] In step ②, the amount of the In(NO3)3·H2O methanol solution added is 4 / 3 of the volume of the original solution; the concentration of In(NO3)3·H2O in the In(NO3)3·H2O methanol solution is 0.05mmolI / ml; the volume ratio of butanol to methanol is 1:1;

[0026] In step ③, the ratio of the Zn(St)2, 1-octadecene, and TOP-S solution is 0.2 mmol: 5 mL: 100 μL; the volume ratio of the CIS quantum dot toluene solution, 1-octadecene, ZnS precursor solution, acetone, and toluene is 1: 5: 5: 10: 10; the TOP-S solution is a solution containing 0.2 mmol sulfur per 100 μL TOP; the centrifugation speed is 3000 rpm, and the time is 15 min; the addition of the ZnS precursor solution is to add the ZnS precursor solution dropwise within 20 minutes;

[0027] In step ④, the ratio of the carboxyalkane sulfide, water, tris(2-formylethyl)phosphine hydrochloride solution, dispersion, chloroform and pH 7 water is 0.2 mmol: 3 mL: 500 μL: 5 ml: 5 ml: 1 ml;

[0028] The concentration of the tris(2-formylethyl)phosphine hydrochloride solution is 0.5 mol / L;

[0029] The centrifugation speed was 2500 rpm for 10 minutes, and then the centrifugation was continued at 10000 rpm for 20 minutes.

[0030] Furthermore, in step ⑤, the ratio of azobisisobutyronitrile, styrene, polyvinylpyrrolidone, ethanol and water is 0.3g:30g:2g:60g:8g;

[0031] In step ⑥, the ratio of the linear polystyrene particles, sodium lauryl sulfate aqueous solution, cadmium solution, styrene, divinylbenzene, methyl acetoacetate, benzoyl peroxide and dichloromethane is 10g:20ml:0.1ml:1.5ml:1ml:0.5ml:0.06ml:10ml; the final concentration of the polyvinyl alcohol aqueous solution after addition is 1wt%; the concentration of the cadmium solution is 100mg / L;

[0032] In step ⑦, the volume ratio of the CIS / ZnS core / shell quantum dot solution, the methanol chloroform solution, the chloroform, and the dichloromethane solution containing the nanospheres is 0.5:4:5:5;

[0033] The volume ratio of methanol to chloroform in the methanol-chloroform solution is 1:1;

[0034] In step ⑧, the ratio of the quantum dot fluorescent microspheres, 2-morpholineethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, anti-HbA1c antibody, and glycine solution is 13 μL:0.8 mL:0.8 mg:0.8 mg:100 μL:60 μL;

[0035] The pH of the 2-morpholinoethanesulfonic acid (MES) buffer was 7.0, the concentration of the anti-HbA1c antibody was 60 mg / mL, and the concentration of the glycine solution was 10 mg / mL.

[0036] The present invention also provides a method for preparing the aforementioned test strip, which comprises the following steps:

[0037] a. Preparation of sample pad: Take 20 mL of pH 8.0 sodium borate buffer and evenly add it dropwise onto the sample pad and let it dry;

[0038] b. Preparation of the conjugate pad: Spray the conjugate pad with HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres, and dry at 55-65°C for 2-8 hours to obtain a conjugate pad coated with HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres;

[0039] c. Preparation of reaction membrane: Take the reaction membrane, spray the monoclonal antibody on the T line and the goat anti-mouse IgG on the C line, and dry at 37℃ for 15 hours.

[0040] d. Preparation of test strips: stick the sample pad prepared in step a), the conjugate pad prepared in step b), the reaction membrane prepared in step c), and the absorbent paper pad on the PVC base plate from left to right in sequence.

[0041] Furthermore, the bonding pad is per 1 cm 2 Contains 195ng of immune probe; each 1cm of the T line contains 300ng of anti-HbA1c monoclonal antibody; each 1cm of the C line contains 400ng of goat anti-mouse IgG.

[0042] The immunochromatographic test strip for glycated hemoglobin (HbA1c) utilizes the rapidity and simplicity of immunochromatographic technology coupled with the easy excitation, strong luminescence, and photostability of quantum dots. Using the evaporation-swelling method and innovative reaction principles and synthesis steps, novel quantum dot fluorescent microspheres are prepared. Furthermore, the novel quantum dot fluorescent microspheres are used as markers to prepare immunoprobes using the EDC method. Based on this, an HbA1c chromatographic test strip is constructed, and HbA1c quantification is achieved by detecting the fluorescent signal from the test strip.

[0043] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0044] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Electron microscopy images of CIS / ZnS core / shell quantum dots

[0046] Figure 2 Electron microscopy of CIS / ZnS quantum dot fluorescent microspheres

[0047] Figure 3 Preparation of quantum dot fluorescent microspheres and synthesis of immune probes

[0048] Figure 4 Fluorescence intensity of fluorescent microspheres and antibody-fluorescent microsphere complexes at different emission wavelengths

[0049] Figure 5 Absorption values of fluorescent microspheres and antibody-fluorescent microsphere complexes at different wavelengths

[0050] Figure 6 Immunochromatographic test strip structure

[0051] Figure 7 Effect of monoclonal antibody labeling amount on the T / C ratio of the test strip

[0052] Figure 8 Effect of added amount of labeled microspheres on the change of T / C ratio of test strips over time

[0053] Figure 9 Optimal test strip T / C ratio determination time

[0054] Figure 10 Test strip signal detection principle

[0055] Figure 11 Test strip standard curve

[0056] Figure 12 Fluorescent microsphere test strips have a good correlation with HPLC DETAILED DESCRIPTION

[0057] Example 1. Preparation of the immunochromatographic test strip for glycated hemoglobin of the present invention

[0058] 1. Synthesis of core-shell quantum dots

[0059] 1)Cu 2-X Synthesis of S nanotemplate: 0.6 mmol of CuAc (copper acetate) and 5.6 mmol of TOPO (n-trioctylphosphine oxide) were added to 50 mL of ODE (octadecene) and degassed at 100 ° C for 1 hour, and then the reaction bottle was blown with N2 (nitrogen) at 200 ° C for 10 minutes. At 160 ° C, 3.5 mL of DDT (1-dodecanethiol) was quickly injected into the bottle to change the color of the solution from dark green to brown. Subsequently, it was grown at 200 ° C for 100 minutes, and the reaction mixture was naturally cooled to room temperature. The crude product was washed with 55 mL of a mixed solution of butanol and methanol (1: 1) and then centrifuged at 3000 rpm for 20 minutes. The washing step was repeated three times. Finally, the purified Cu 2-X The S nanocrystals were dissolved in 5 mL of anhydrous toluene to obtain a stock solution.

[0060] 2) Synthesis of CIS quantum dots: Purified Cu 2-X 1.5 mL of the S nanocrystal stock solution was diluted into 5 mL of toluene. Then, 0.1 mmol of In(NO3)3·H2O was dissolved in 2 mL of methanol and added to the prepared Cu2O in the presence of a variable amount of TOP (sodium pyrrolidonecarboxylate). 2-X The reaction mixture was kept at room temperature (17 ± 2°C) for 4 days. The crude product was washed with 55 mL of a 1:1 mixture of butanol and methanol. Finally, the purified CIS quantum dots were dissolved in 1 mL of toluene and stored under nitrogen.

[0061] 3) Synthesis of CIS / ZnS core / shell quantum dots: 0.2 mmol of Zn(St)2 was dissolved in 5 mL of ODE (1-octadecene) at 150°C, and then 100 μL of TOP-S solution (0.2 mmol of elemental sulfur in 100 μL of TOP) was added to prepare a ZnS (zinc sulfide) precursor solution. Subsequently, 1 mL of purified CIS quantum dots was mixed with 1 mL of toluene solution and 5 mL of ODE (1-octadecene) and degassed at room temperature for 1 h to remove the toluene. The CIS QDs solution in ODE was then heated to 200°C under N2. When the temperature stabilized, the ZnS precursor solution (5 mL) was added dropwise over 20 minutes. The temperature was maintained at 200°C for 60 minutes, the flask was cooled to room temperature, and 10 mL of acetone was added to purify the nanocrystals. The nanocrystals were then centrifuged at 3000 rpm for 15 minutes and then dispersed in 10 mL of toluene. The entire process, from purification by acetone addition to resuspension by centrifugation, was repeated three times.

[0062] 4) Phase transfer of CIS / ZnS core / shell QDs (Quantum dots, QDs) into water: 0.2 mmol MUA (carboxyalkane sulfide) was dispersed in a mixture of 3 mL deionized water and 500 μL 0.5 M TCEP (tris(2-formylethyl)phosphine hydrochloride), resulting in white turbidity. 0.5 MTMAH (tetramethylammonium hydroxide) was added under vigorous stirring until pH 11.7 was reached, and the suspension became a clear solution. At the same time, 5 mL of the above CIS / ZnS QDs solution dispersed in toluene was taken in 5 mL chloroform. The solution was mixed with the MUA solution and then stirred (1000 rpm) at room temperature (20°C) overnight. Subsequently, the mixture was centrifuged at 2500 rpm for 10 minutes. The supernatant was centrifuged at 10000 rpm for 20 minutes. The CIS / ZnS core / shell quantum dots collected in the filter were redispersed in 1 mL of deionized water (pH 7) and stored in a refrigerator (4°C).

[0063] 2. Preparation of Quantum Dot Fluorescent Microspheres

[0064] 1) Preparation of linear polystyrene (PS) particles: A mixture of 0.3 g of an initiator (azobisisobutyronitrile (AIBN)) and 30 g of styrene was added dropwise to a mixture of 2 g of polyvinylpyrrolidone, 60 g of ethanol, and 8 g of pure water. The solution was sealed under a nitrogen atmosphere and stirred at 70°C and 120 rpm for 16 hours. The resulting linear PS particles were washed with ethanol and collected by centrifugation to produce linear polystyrene particles with a molecular weight of 87,900 g / mol.

[0065] 10 g of thread-like PS seed particles were re-dispersed in 20 ml of a 0.25 wt% sodium dodecyl sulfate aqueous solution using ultrasound. These seeds were swollen with 0.1 mL of cadmium (100 mg / L) in a 0.25 wt% sodium dodecyl sulfate aqueous solution at 30°C for 15 hours. After the cadmium droplets completely disappeared from the solution, the seeds were swollen for 15 hours with a mixture of 1.5 mL of St (styrene), 1 mL of DVB (divinylbenzene), 0.5 mL of MAA (methyl acetoacetate), and 0.06 mL of BPO (benzoyl peroxide), which had been pre-emulsified in a 0.25 wt% sodium dodecyl sulfate aqueous solution. Subsequently, an aqueous solution of polyvinyl alcohol (PVA) was added to a constant PVA concentration of 1 wt% of the total solution. The solution was then heated to 70°C under nitrogen for polymerization for 12 hours. The resulting microspheres were washed with ethanol and collected by centrifugation. Finally, the microspheres were extracted with 10 mL of dichloromethane for 48 hours to yield porous PSDM microspheres.

[0066] 2) Embedding quantum dots into nanospheres: Take 0.5 mL of the above quantum dots (stored in purified water), add 2 mL of methanol and 2 mL of chloroform (v / v = 1:1) to precipitate the quantum dots, centrifuge to obtain the quantum dots, and redisperse them in 5 mL of chloroform to form well-dispersed chloroform quantum dots (10 6 cells / mL) for further use.

[0067] 2 ml of the chloroform quantum dot solution was added to 2 ml of the dichloromethane microsphere solution. Chloroform and dichloromethane are completely miscible. Stirring was performed for 5 hours to allow the microspheres to expand and the quantum dots to embed into the nanospheres. The solution was then exposed to air at room temperature for an additional 5 hours to allow the chloroform (boiling point: 61.2°C) and dichloromethane (boiling point: 41°C) to completely evaporate. The microspheres were then washed with xylene and collected by centrifugation.

[0068] 3. Preparation of immunoprobes:

[0069] Fluorescent microsphere conjugation with proteins: Quantum dot fluorescent microspheres (13 μL) were added to 2-morpholinoethanesulfonic acid (MES) buffer (pH 7.0) (0.8 mL), EDC (0.8 mg, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and NHS (0.8 mg, N-hydroxysuccinimide) and activated for 30 minutes. Anti-HbA1c antibody (60 mg / mL, 100 μL) was added to the activated microspheres. The conjugation reaction was allowed to proceed at room temperature for 2 hours. Unconjugated antibody was removed by centrifugation, and the remaining active groups on the microsphere surface were blocked with glycine (60 μL, 10 mg / mL) for 1.5 hours. The microspheres were then stored in Tris (tris(hydroxymethyl)aminomethane) buffer.

[0070] 4. Assembly of test strips

[0071] The composition of the immunochromatographic test strip mainly includes absorbent paper, NC (nitrocellulose) membrane with test line (T line) and quality control line (C line), conjugate pad (glass fiber is used as conjugate pad), sample pad (glass fiber is used as sample pad), and PVC (polyvinyl chloride) bottom plate. The purchased anti-HbA1c monoclonal antibody and goat anti-mouse IgG (HaiTide Biotechnology (Shanghai) Co., Ltd.) were used as test (T) and control (C) lines, respectively, and were sprayed on the NC membrane using a gold sprayer. The T line contained 300 ng of anti-HbA1c monoclonal antibody per 1 cm, and the C line contained 400 ng of goat anti-mouse IgG per 1 cm. The distance between the T line and the C line was approximately 5 mm. The membrane was then dried at 37°C for 15 hours. The sample pad was treated with sodium borate buffer (pH 8.0) (20 mL of sodium borate buffer was evenly dripped onto the entire sample pad, and the sample pad was ready for use after drying). The NC membrane was then attached to a PVC base plate. The affinity chromatography probe was streaked onto the conjugate pad at 5 μL / cm (i.e., 1 μg / cm) using a gold sprayer. 2After drying at 60°C for 5 hours, the sample pad, conjugate pad, NC membrane, and absorbent paper were attached to a plastic base. The strips were then cut into 4 mm widths and packaged in a plastic case.

[0072] The beneficial effects of the present invention are described below through test examples.

[0073] Experimental Example 1 Study on Immunochromatographic Test Strips for Quantitative Detection of Glycated Hemoglobin

[0074] 1. Synthesis of core-shell quantum dots

[0075] 1)Cu 2-X Synthesis of S nanotemplate: 0.6 mmol of CuAc (copper acetate) and 5.6 mmol of TOPO (n-trioctylphosphine oxide) were added to 50 mL of ODE (octadecene) and degassed at 100 ° C for 1 hour, and then the reaction bottle was blown with N2 (nitrogen) at 200 ° C for 10 minutes. At 160 ° C, 3.5 mL of DDT (1-dodecanethiol) was quickly injected into the bottle to change the color of the solution from dark green to brown. Subsequently, it was grown at 200 ° C for 100 minutes, and the reaction mixture was naturally cooled to room temperature. The crude product was washed with 55 mL of a mixed solution of butanol and methanol (1: 1) and then centrifuged at 3000 rpm for 20 minutes. The washing step was repeated three times. Finally, the purified Cu 2-X The S nanocrystals were dissolved in 5 mL of anhydrous toluene to obtain a stock solution.

[0076] 2) Synthesis of CIS quantum dots: On the template Cu 2-X In S nanocrystals, CIS quantum dots were synthesized by cation exchange of some Cu+ and In3+. 2-X 1.5 mL of the S nanocrystal stock solution was diluted into 5 mL of toluene. Then, 0.1 mmol of In(NO3)3·H2O was dissolved in 2 mL of methanol and added to the prepared Cu2O in the presence of a variable amount of TOP (sodium pyrrolidonecarboxylate). 2-X The reaction mixture was kept at room temperature (17 ± 2°C) for 4 days. The crude product was washed with 55 mL of a 1:1 mixture of butanol and methanol. Finally, the purified CIS quantum dots were dissolved in 1 mL of toluene and stored under nitrogen.

[0077] 3) Synthesis of CIS / ZnS core / shell quantum dots: Based on the above, the overgrowth of CIS quantum dots on the ZnS shell was achieved by the following steps. 0.2 mmol of Zn(St)2 was dissolved in 5 mL of ODE (1-octadecene) at 150°C, and then 100 μL of TOP-S solution (100 μL TOP containing 0.2 mmol of elemental sulfur) was added to prepare a ZnS (zinc sulfide) precursor solution. Subsequently, 1 mL of purified CIS quantum dots was mixed with 1 mL of toluene solution and 5 mL of ODE (1-octadecene) and degassed at room temperature for 1 h to remove toluene. The CIS QDs solution in ODE was then heated to 200°C under N2. When the temperature stabilized, ZnS precursor solution (5 mL) was added dropwise over 20 minutes. The mixture was kept at 200 °C for 60 min, the flask was cooled to room temperature, 10 mL of acetone was added to purify the nanocrystals, and then centrifuged at 3000 rpm for 15 min, and then dispersed in 10 mL of toluene. The entire process from adding acetone for purification to centrifugation and resuspension was repeated 3 times.

[0078] 4) Phase transfer of CIS / ZnS core / shell QDs into water: The purified CIS / ZnS core / shell quantum dots were transferred into water. 0.2 mmol MUA (carboxyl alkane sulfide) was dispersed in a mixture of 3 mL deionized water and 500 μL 0.5 M TCEP (tris(2-formylethyl)phosphine hydrochloride), resulting in white turbidity. 0.5 M TMAH (tetramethylammonium hydroxide) was added under vigorous stirring until pH 11.7 was reached, and the suspension became a clear solution. At the same time, 5 mL of the above-mentioned CIS / ZnS QDs solution dispersed in toluene was taken in 5 mL chloroform. The solution was mixed with the MUA solution and then stirred (1000 rpm) at room temperature (20°C) overnight. Subsequently, the mixture was centrifuged at 2500 rpm for 10 minutes. The supernatant was centrifuged at 10000 rpm for 20 minutes. The CIS / ZnS core / shell quantum dots collected in the filter were redispersed in 1 mL of deionized water (pH 7) and stored in a refrigerator (4°C). Figure 1 .

[0079] 2. Preparation of Quantum Dot Fluorescent Microspheres

[0080] 1) Synthesis of Porous PSDM Microspheres by Seed Copolymerization: Linear polystyrene (PS) particles were prepared by dispersion polymerization: A mixture of initiator (AIBN) and styrene was added dropwise to the continuous phase solution. The solution was sealed under a nitrogen atmosphere and stirred at 70°C and 120 rpm for 16 hours. The resulting linear PS particles were washed with ethanol and collected by centrifugation. The molecular weight of the linear polystyrene particles was controlled by adjusting the monomer concentration, continuous phase solution composition, and initiator in the polymerization system.

[0081] The thread-like PS seed particles were re-dispersed in a 0.25 wt% aqueous sodium dodecyl sulfate solution using ultrasound. These seeds were swollen with 0.1 ml of cadmium in a 0.25 wt% aqueous sodium dodecyl sulfate solution at 30°C for 15 hours. After the cadmium droplets completely disappeared from the solution, the seeds were swollen for 15 hours with a mixture of St, DVB, MAA, and BPO, pre-emulsified in a 0.25 wt% aqueous sodium dodecyl sulfate solution.

[0082] Subsequently, a PVA aqueous solution was added to maintain a fixed PVA concentration of 1 wt% of the total solution. The solution was then heated to 70°C under nitrogen for 12 hours to polymerize. The resulting microspheres were washed with ethanol and collected by centrifugation. Finally, the microspheres were extracted with dichloromethane for 48 hours to obtain porous PSDM microspheres.

[0083] 2) The quantum dots were embedded into the nanospheres using the swelling evaporation method. The specific steps are as follows: 2 ml of chloroform quantum dot solution was added to 2 ml of dichloromethane solution containing nanospheres. Stir for 5 hours to allow the microspheres to swell, then the solution was exposed to the air at room temperature for another 5 hours to allow the chloroform and dichloromethane to completely evaporate. The microspheres were then washed with xylene and collected by centrifugation. The results are shown in Figure 2. Figure 2 .

[0084] 3. Preparation of immunoprobes:

[0085] 3.1 The prepared fluorescent microspheres were coupled to proteins using the (EDC) one-step method: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) / N-hydroxysuccinimide (NHS) was used as a coupling agent to form an amide bond between the carboxyl group of the carboxylated microspheres and the amine group of the anti-HbA1c antibody, thereby covalently immobilizing anti-HAb1c on the microspheres. The microspheres were added to 2-morpholineethanesulfonic acid (MES) buffer (pH 7.0), EDC, and NHS and activated for 30 minutes. The anti-HbA1c antibody was added to the activated microspheres. The coupling reaction was carried out at room temperature for 2 hours, and the uncoupled antibody was removed by centrifugation. The remaining active groups on the surface of the microspheres were then blocked with glycine for 1.5 hours and stored in Tris (tris(hydroxymethyl)aminomethane) buffer. The specific principle is shown in [ 15 ]. Figure 3 .

[0086] 3.2Optimization of optimal absorption and emission wavelengths:

[0087] The excitation light characteristics of HbA1c labeled with fluorescent microspheres were detected using excitation light of 100-800 nm, and the absorbance of the sample was measured. HbA1c labeled with fluorescent microspheres showed a strong fluorescence signal and a narrow emission spectrum, just like the bare microspheres, with the emission peak at 425 nm ( Figure 4 In addition, HbA1c labeled with fluorescent microspheres also has an obvious small absorption peak near 350nm ( Figure 5In conclusion, the prepared fluorescent microspheres labeled HbA1c have the luminescence and excitation characteristics of monoclonal antibodies and quantum dots, and the fluorescent microspheres are suitable for the detection of HbA1c.

[0088] 4. Assembly of test strips

[0089] 4.1 Unlike traditional colloidal gold test strips, the prepared fluorescent microspheres can be directly sprayed onto the NC membrane without the need for additional fixing steps, which greatly simplifies the process flow. Therefore, it is only necessary to assemble the components of the immunochromatographic test strips in a certain order: the composition of the immunochromatographic test strips mainly includes absorbent paper, NC (nitrocellulose) membrane with test line (T line) and quality control line (C line), conjugate pad (glass fiber is used as conjugate pad), sample pad (glass fiber is used as sample pad), PVC (polyvinyl chloride) bottom plate, and the specific assembly form is as follows: Figure 2 As shown, the prepared monoclonal antibody and goat anti-mouse IgG were sprayed onto the NC membrane as test (T) and control (C) lines, respectively, using a gold sprayer. The distance between the T line and the C line was approximately 5 mm, and then dried at 37°C for 15 hours. The sample pad was treated with sodium borate buffer (pH 8.0) and dried at 60°C for 5 hours. The sample pad, conjugate pad, NC membrane, and absorbent paper were attached to a plastic base plate, and the strips were cut into 4 mm widths and packaged in plastic shells. They were stored at room temperature for future use. For details, see Figure 6 .

[0090] 4.2 Optimization of test strip reaction conditions:

[0091] 4.2.1 Monoclonal Antibody Labeling Amount

[0092] Monoclonal antibodies with different final concentrations were conjugated with fluorescent microspheres. The sample to be tested and the conjugated label mixture were added dropwise to the sample well of the test strip. The timer was started when the sample solution completely infiltrated the NC membrane. The fluorescence intensity values of the T and C lines of the test strip and the T / C ratio were read using an immunofluorescence analyzer. The results of the effect of the antibody labeling amount on the T / C ratio of the test strip are shown in Figure 7 .

[0093] from Figure 7 It can be seen that when the monoclonal antibody labeling amount is 60ug / mL, the T / C ratio reaches the maximum and its fluorescence intensity is the maximum, so the optimal monoclonal antibody labeling amount is 60ug / mL.

[0094] 4.2.2 Optimal amount of labeled microspheres added

[0095] 1-20 μL of fluorescent microsphere-labeled antibody was mixed with 15% HbA1c and incubated. The mixture was then added to the sample well of the test strip. The timer was started when the sample solution completely soaked the NC membrane. The immunofluorescence analyzer was used to read the result after 10 minutes. The change of the T / C ratio of the test strip over time is shown in Figure 8 .

[0096] from Figure 8 It can be seen that when the amount of microspheres added is 13 μL, the T / C ratio reaches the maximum and the fluorescence intensity is the maximum, so the optimal amount of labeled microspheres added is 13 μL.

[0097] 4.2.3 Determination of the optimal result determination time

[0098] Add the mixture dropwise to the sample well of the test strip. Start timing when the sample solution completely soaks the NC membrane. Use an immunofluorescence analyzer to read the fluorescence intensity values of the T and C lines of the test strip and the T / C ratio. Record the T / C ratio of the test strip every 1 minute. The trend of the change of the T / C ratio of the test strip over time is shown in Figure 9 . 9 minutes after the test strip was added with the sample, the T / C ratio gradually increased and stabilized around 16 minutes, so the optimal detection time of the test strip was 16 minutes.

[0099] 5. Test strip signal detection

[0100] Use a fluorescence quantitative detector to collect the fluorescence intensity of the T and C lines and calculate the T / C ratio: add the sample solution containing the required concentration of HbA1c to the sample pad. Fetal calf serum (FCS) is used as a control. Both the sample and control solutions migrate to the absorbent paper through capillary action. The higher the concentration of the analyte in the sample, the stronger the fluorescence signal generated by the T line. Then record the fluorescence intensity of the test line and the control line, and calculate the ratio between the two. First, use a standard to test and draw a standard curve of concentration-fluorescence intensity. Then test the fluorescence intensity of the analyte and substitute it into the standard curve to convert it into concentration to quantify the analyte. For specific principles, see Figure 10 .

[0101] 6.Test strip performance evaluation

[0102] 6.1 Establishment of standard curve:

[0103] Under optimal conditions, a series of HbA1c standard solutions (with a 1% concentration gradient) were tested using the constructed immunochromatographic test strips. A standard curve was plotted with HbA1c concentration as the horizontal axis and the T / C line fluorescence intensity ratio as the vertical axis. The concentration corresponding to the fluorescence intensity generated by the blank control was the lowest detection limit, and a linear range of 4%-15% was observed. The linear regression equation was y = 0.1838X + 0.0752, R 2 =0.9956, which proves that the test strip has good linearity and can meet the needs of clinical HbA1c quantitative detection. See Table 1 for details. Figure 11 .

[0104] Table 1, Standard curve measurement data

[0105] HbA1c concentration (%) T-line fluorescence signal intensity C-line fluorescence signal intensity T / C ratio 4.0 397 475 0.836 5.0 483 524 0.922 6.0 624 559 1.116 7.0 786 562 1.399 8.0 845 553 1.592 9.0 983 547 1.795 10.0 1036 546 1.897 11.0 1129 539 2.093 12.0 1265 551 2.297 13.0 1354 542 2.496 14.0 1434 550 2.609 15.0 1501 536 2.802

[0106] 6.2 Precision inspection:

[0107] Six sets of HbA1c standards of varying concentrations were selected, with 10 test strips in each set subjected to replicate measurements under optimal reaction conditions. Fluorescence signals from the T line were read, and concentrations were calculated using the standard curve. The mean, standard deviation, and coefficient of variation were also calculated. The CV% values for the six sets of HbA1c standards, from low to high, were 4.29%, 3.54%, 1.74%, 1.79%, 2.91%, and 3.16%, respectively. These values were all less than 5%, indicating good precision and high reliability of the single-test strip measurement results. See Table 2 for details.

[0108] Table 2, Test strip precision measurement data

[0109]

[0110] 6.3 Clinical Sample Testing

[0111] To determine the effectiveness of the test strips on actual sample detection, we tested clinical specimens using the test strips and HPLC methods, and obtained data for correlation analysis. The horizontal axis represents the test results of the fluorescent immunochromatographic test strips, and the vertical axis represents the test results of the HPLC method. The obtained linear regression equation is y = 0.863X + 0.0765, R 2 =0.9881. The results show that the fluorescent microsphere test strip of the present invention has a good correlation with HPLC and has a high clinical application value. Figure 12 .

[0112] 7. Discussion on the effects

[0113] 1) Quantification of the analyte: Traditional immunochromatographic techniques, such as colloidal gold, can only provide qualitative or semi-quantitative results, making interpretation subjective and sensitive. This invention incorporates quantum dot fluorescent microspheres into chromatographic test strips. Instrumental detection of the signal generated by the markers avoids the low repeatability caused by subjective factors while improving the detection limit and sensitivity of the test strips, enabling rapid, quantitative detection of analytes.

[0114] 2) Reduce the biotoxicity of quantum dots and improve the photoluminescence quantum yield (PLQY): Most of the currently used near-infrared emitting quantum dots contain highly toxic elements such as Cd, Pb and As (e.g. CdTe, CdSe). The present invention uses CIS / ZnS core / shell quantum dots as a substitute. The quantum dots have low toxicity, a large absorption coefficient in a wide spectral range, and fluorescence tunability. Specifically, Cu2 -X S nanocrystals are used as templates to partially exchange In through topologically oriented Cu+ ions. 3+ , Cu 2-XS nanocrystals are converted into CIS quantum dots, which are then used as the core of an overgrown zinc sulfide (ZnS) shell to synthesize CIS / ZnS core / shell quantum dots. These quantum dots exhibit excellent colloidal stability and high, tunable fluorescence properties. Their PLQY surpasses that of commonly used water-soluble near-infrared fluorophores (dyes and quantum dots), enabling immunochromatographic test strips using them as markers to exhibit increased sensitivity.

[0115] 3) Improve the embedding rate of microspheres: This is reflected in two aspects: 1. The present invention adopts a seed copolymerization method to synthesize narrowly dispersed microspheres (PSDM microspheres) with a specific size, surface carboxyl groups and a porous structure. The porous structure helps the quantum dots penetrate into the interior of the microspheres. 2. In view of the fact that the incorrect use of solvents will damage the fluorescence properties of quantum dots, the present invention adopts a method that combines swelling and evaporation, that is, the swelling process is combined with the gradual evaporation of the solvent, so that the quantum dots are gradually concentrated in the dispersion. Compared with the swelling method alone, this method is an effective method to increase the fluorescence intensity of microspheres. Through these two methods, microspheres encoded with single or multiple wavelength emission quantum dots can be effectively prepared.

[0116] 4) Assisting blood glucose monitoring: CIS / ZnS quantum dot fluorescent microspheres are used as fluorescent markers to label anti-HbA1c antibodies. Combined with immunochromatographic technology and a corresponding fluorescence detector, rapid and quantitative HbA1c detection is achieved. This eliminates the instrument and personnel dependence of traditional methods such as high-performance liquid chromatography (HPLC), microcolumn chromatography, electrophoresis, and chemiluminescence, facilitating HbA1c self-testing at home and self-monitoring for diabetic patients. This provides technical support for determining the efficacy of diabetes treatment and adjusting treatment plans, and also offers a new methodological foundation and approach for the detection of large molecular proteins, with significant medical significance and economic benefits.

Claims

1. An immunochromatographic test strip for glycated hemoglobin, characterized in that: It is composed of a base plate, a sample pad, a conjugate pad, a reaction membrane and an absorbent paper pad; The reaction membrane is marked with a T line and a C line; the T line is fixed with an anti-HbA1c monoclonal antibody; the C line is fixed with a goat anti-mouse IgG; The conjugate pad contains an immune probe; the immune probe is an HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres; The preparation method of the HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres is as follows: 1) Synthesis of core-shell quantum dots: ① Dissolve copper acetate and trioctylphosphine oxide in octadecene, degas at 100°C for 1 hour, blow nitrogen at 200°C for 5-15 minutes, add 1-dodecanethiol at 160°C, and after the solution turns brown, react at 200°C for 100 minutes. Cool, wash with butanol and methanol, centrifuge, and dissolve the crystals in toluene to obtain the stock solution. ② Take the stock solution obtained in step ①, add toluene to dilute it 3 to 4 times, then add In(NO3)3·H2O methanol solution in the presence of sodium pyrrolidonecarboxylate, react at 17±2°C for 4 days, wash with butanol and methanol, take the purified CIS quantum dots, add toluene with 2 / 3 of the stock solution volume to dissolve, and obtain CIS quantum dot toluene solution; ③ Take Zn(St)2, add 1-octadecene to dissolve, and then add TOP-S solution to mix to obtain a ZnS precursor solution; take the CIS quantum dot toluene solution obtained in step ② and mix it with 1-octadecene, remove toluene, heat to 200°C, add ZnS precursor solution, and then maintain at 200°C for 60 minutes. Cool, add acetone, centrifuge, take crystals and disperse them in toluene to obtain a dispersion; ④ Take carboxyalkane sulfide, add water and tri(2-formylethyl)phosphine hydrochloride solution, mix, and then add tetramethylammonium hydroxide to pH 11.7 to obtain MUA solution; take the dispersion obtained in step ③, add chloroform and mix, then add MUA solution and stir for 8-12 hours, centrifuge, take the supernatant and centrifuge again, collect the solid and dissolve it in water at pH 7 to obtain CIS / ZnS core / shell quantum dot solution; 2) Preparation of quantum dot fluorescent microspheres: ⑤ Mix azobisisobutyronitrile and styrene to obtain a mixture; dissolve polyvinyl pyrrolidone in ethanol and water to obtain a mixed solution; add the mixture to the mixed solution, stir under a sealed nitrogen atmosphere for 12 to 20 hours, wash the solid with ethanol, and centrifuge to obtain linear polystyrene particles; ⑥ Dispersing the linear polystyrene particles obtained in step ⑤ into an aqueous solution of sodium dodecyl sulfate, adding a cadmium solution, and swelling the mixture of styrene, divinylbenzene, methyl acetoacetate, and benzoyl peroxide, then adding an aqueous solution of polyvinyl alcohol, heating and reacting for 10 to 15 hours, washing the solid with ethanol, centrifuging, and finally extracting with dichloromethane to obtain a dichloromethane solution containing nanospheres; ⑦ Take the CIS / ZnS core / shell quantum dot solution obtained in step ④, add methanol chloroform solution to mix, centrifuge, take the solid and disperse it in chloroform, then add the dichloromethane solution containing nanospheres obtained in step ⑥, stir and expand, then evaporate the chloroform and dichloromethane at room temperature, finally wash with xylene, centrifuge, and collect; 3) Preparation of immunoprobes: ⑧ Take the quantum dot fluorescent microspheres obtained in step ⑦, add 2-morpholineethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, activate for 20 to 60 minutes, then add anti-HbA1c antibody, react for 2 hours, centrifuge, take the precipitate, add glycine and mix for 1.5 hours, add tris(hydroxymethyl)aminomethane and mix to obtain.

2. The test strip according to claim 1, wherein: The bottom plate is a PVC bottom plate; the sample pad, conjugate pad, reaction membrane and absorbent paper pad are sequentially adhered to the PVC bottom plate from left to right; the T line in the reaction membrane is on the left and the C line is on the right.

3. The test strip according to claim 1, wherein: The sample pad is a glass fiber to which sodium borate buffer solution is added and dried.

4. The test strip according to claim 1, wherein: The material of the binding pad and the absorbent paper is glass fiber, the material of the reaction membrane is nitrocellulose, and the material of the PVC bottom plate is polyvinyl chloride.

5. The test strip according to claim 1, wherein: The conjugate pad is 1 cm 2 Contains 195ng of immune probe; each 1cm of the T line contains 300ng of anti-HbA1c monoclonal antibody; each 1cm of the C line contains 400ng of goat anti-mouse IgG.

6. The test strip according to claim 1, wherein: In step ①, the ratio of copper acetate, trioctylphosphine oxide, octadecene, 1-dodecanethiol, and toluene is 0.6 mmol: 5.6 mmol: 50 mL: 3.5 ml: 5 mL; the volume ratio of butanol and methanol is 1:1; the centrifugation speed is 3000 rpm, and the time is 20 minutes; In step ②, the amount of the In(NO3)3·H2O methanol solution added is 4 / 3 of the volume of the original solution; the concentration of In(NO3)3·H2O in the In(NO3)3·H2O methanol solution is 0.05mmolI / ml; the volume ratio of butanol to methanol is 1:1; In step ③, the ratio of the Zn(St)2, 1-octadecene, and TOP-S solution is 0.2 mmol: 5 mL: 100 μL; the volume ratio of the CIS quantum dot toluene solution, 1-octadecene, ZnS precursor solution, acetone, and toluene is 1: 5: 5: 10: 10; the TOP-S solution is a solution containing 0.2 mmol sulfur per 100 μL TOP; the centrifugation speed is 3000 rpm, and the time is 15 min; the addition of the ZnS precursor solution is to add the ZnS precursor solution dropwise within 20 minutes; In step ④, the ratio of the carboxyalkane sulfide, water, tris(2-formylethyl)phosphine hydrochloride solution, dispersion, chloroform and pH 7 water is 0.2 mmol: 3 mL: 500 μL: 5 ml: 5 ml: 1 ml; The concentration of the tris(2-formylethyl)phosphine hydrochloride solution is 0.5 mol / L; The centrifugation speed was 2500 rpm for 10 minutes, and then the centrifugation was continued at 10000 rpm for 20 minutes.

7. The test strip according to claim 1, wherein: In step ⑤, the ratio of azobisisobutyronitrile, styrene, polyvinylpyrrolidone, ethanol and water is 0.3g:30g:2g:60g:8g; In step ⑥, the ratio of the linear polystyrene particles, sodium lauryl sulfate aqueous solution, cadmium solution, styrene, divinylbenzene, methyl acetoacetate, benzoyl peroxide and dichloromethane is 10g:20ml:0.1ml:1.5ml:1ml:0.5ml:0.06ml:10ml; the final concentration of the polyvinyl alcohol aqueous solution after addition is 1wt%; the concentration of the cadmium solution is 100mg / L; In step ⑦, the volume ratio of the CIS / ZnS core / shell quantum dot solution, the methanol-chloroform solution, chloroform, and the dichloromethane solution containing the nanospheres is 0.5:4:5:5; the volume ratio of methanol to chloroform in the methanol-chloroform solution is 1:1; In step ⑧, the ratio of the quantum dot fluorescent microspheres, 2-morpholineethanesulfonic acid (MES) buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, anti-HbA1c antibody, and glycine solution is 13 μL:0.8 mL:0.8 mg:0.8 mg:100 μL:60 μL; The pH of the 2-morpholinoethanesulfonic acid (MES) buffer 7.0, anti-HbA1c antibody concentration was 60 mg / mL, and glycine solution concentration was 10 mg / mL.

8. A method for preparing the test strip according to any one of claims 1 to 7, characterized in that: It includes the following steps: a. Preparation of sample pad: Take 20 mL of pH 8.0 sodium borate buffer and evenly add it dropwise onto the sample pad and let it dry; b. Preparation of the conjugate pad: Spray the conjugate pad with HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres, and dry at 55-65°C for 2-8 hours to obtain a conjugate pad coated with HbA1c antibody labeled with CIS / ZnS core / shell quantum dot fluorescent microspheres; c. Preparation of reaction membrane: Take the reaction membrane, spray the monoclonal antibody on the T line and the goat anti-mouse IgG on the C line, and dry at 37°C for 15 hours. d. Preparation of test strips: stick the sample pad prepared in step a), the conjugate pad prepared in step b), the reaction membrane prepared in step c), and the absorbent paper pad on the PVC base plate from left to right in sequence.

9. The preparation method according to claim 8, characterized in that: The conjugate pad is 1 cm 2 Contains 195ng of immune probe; each 1cm of the T line contains 300ng of anti-HbA1c monoclonal antibody; each 1cm of the C line contains 400ng of goat anti-mouse IgG.

Images