A non-invasive test kit for glycosylated hemoglobin glycation rate in saliva and its application

Abstract

This invention relates to a non-invasive detection kit for glycated hemoglobin (HbA1c) glycation rate in saliva and its application. The kit includes an immunochromatographic test strip for simultaneous quantitative detection of hemoglobin and HbA1c, a saliva sampling device, and a saliva processing device. The kit can be used in conjunction with a fluorescence detection device. Compared with existing technologies, this invention's kit detects the HbA1c glycation rate in saliva with a simple saliva processing procedure. The test strip method simultaneously detects both hemoglobin and HbA1c in a single step, achieving quantitative determination of the glycation rate. The experimental steps are simple and the detection time is shortened. When used in conjunction with a fluorescence detection device, the kit allows for highly sensitive and accurate quantification of HbA1c glycation rate using a smartphone and the fluorescence detection device.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and in particular to a non-invasive test kit for the glycated hemoglobin glycation rate of saliva and its application. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. Clinically, the glycated hemoglobin (HbA1c) ratio, representing the percentage of HbA1c in total hemoglobin (Hb), is widely considered the gold standard biomarker for long-term glycemic control. HbA1c is a stable compound in human blood, formed by the covalent binding of glucose to the N-terminal valine residue of the hemoglobin β-chain (β-N-1-deoxyfructose hemoglobin). HbA1c levels reflect the average blood glucose concentration over the red blood cell lifespan (approximately 120 days).

[0003] Based on differences in detection principles, current detection methods mainly include ion-exchange high-performance liquid chromatography (HPLC), spectrophotometry, electrophoresis, borate affinity chromatography, immunoassay, colorimetry, and mass spectrometry. These methods typically require cumbersome procedures and expensive equipment. Due to the high concentration of hemoglobin (Hb) in blood samples, existing methods struggle to achieve high-sensitivity detection. Furthermore, current detection methods all rely on invasive blood collection procedures. Such invasive monitoring not only causes pain and infection risks for patients but also inconveniences specific populations requiring frequent monitoring, leading to decreased patient compliance. This underscores the urgent need to develop non-invasive detection technologies.

[0004] For example, patent CN113740543A discloses a rapid fluorescence immunochromatographic assay kit for detecting the proportion of glycated hemoglobin. The binding pad is coated with a time-resolved fluorescently labeled hemoglobin Hb1 monoclonal antibody, the T1 detection area is coated with a glycated hemoglobin HbA1c monoclonal antibody, and the T2 detection area is coated with a hemoglobin Hb2 monoclonal antibody. This method uses a double antibody sandwich method for detection and is only suitable for invasive samples such as serum and whole blood.

[0005] For example, patent CN115356488A discloses a fluorescent reagent strip for the combined detection of glycated hemoglobin and cortisol. The conjugate pad contains time-resolved fluorescent microspheres labeled with Cortisol antibody-biotin, time-resolved fluorescent microspheres labeled with a first HbA1c antibody, and time-resolved fluorescent microspheres labeled with chicken IgY antibody. A Cortisol detection line, an HbA1c detection line, and a control line are sequentially arranged on the nitrocellulose membrane. The Cortisol detection line is coated with Cortisol antigen-streptavidin, the HbA1c detection line is coated with a second HbA1c antibody, and the control line is coated with goat anti-chicken IgY antibody. Although this method uses a biotin-streptavidin signal amplification system for cortisol, it employs a double-antibody sandwich method for HbA1c detection, resulting in low sensitivity. Furthermore, this method still uses whole blood as the test sample, leading to cumbersome sampling, invasive procedures, and a high risk of cross-infection.

[0006] Saliva, as a core medium for oral health, serves multiple functions, including lubrication, buffering, antibacterial protection, and maintaining mucosal integrity. Notably, the proteomes of saliva and blood plasma share approximately 30% overlap, indicating its diagnostic potential in disease detection. Compared to invasive blood collection, saliva samples offer revolutionary advantages: the non-invasive procedure eliminates pain and the risk of needle infection, while also improving testing efficiency through convenient procedures and low processing costs. Furthermore, saliva collection reduces the occupational exposure risk to pathogens such as HIV and HPV for healthcare workers. However, as a "filtered product" of blood, the concentration of protein markers in saliva is typically 2-3 orders of magnitude lower than in serum. It is noteworthy that HbA1c accounts for only 5-15% of total Hb, and HbA1c levels in healthy, non-diabetic adults are typically below 6.5%. The absolute concentration of HbA1c in saliva is extremely low, thus requiring analytical methods with ultra-high sensitivity and accuracy to ensure the reliability and clinical significance of the results.

[0007] Therefore, developing a low-cost, easy-to-operate, highly sensitive, and non-invasive sampling technology for detecting glycated hemoglobin glycation rate is of significant practical need and application value. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems of low sensitivity, cumbersome operation steps, high cost and invasiveness in the existing technology, and to provide a non-invasive detection kit for glycated hemoglobin glycation rate in saliva and its application.

[0009] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a non-invasive detection kit for the glycated hemoglobin glycation rate of saliva, characterized in that it includes an immunochromatographic test strip for simultaneously assisting in the quantitative detection of hemoglobin and glycated hemoglobin, a saliva collection device, and a saliva processing device. The immunochromatographic test strip for simultaneous quantitative detection of hemoglobin and glycated hemoglobin includes a base plate and a sample pad, a conjugation pad, an NC membrane and an absorbent pad placed sequentially on the base plate. The bonding pad is loaded with the following components: (a) Biotinylated anti-glycated hemoglobin capture antibody, (b) Antibody for detecting glycated hemoglobin labeled with quantum dot microspheres. (c) Antibody for detecting hemoglobin labeled with quantum dot microspheres; The NC membrane is arranged in parallel with the first detection line T1, the second detection line T2, and the control line C. All three lines are perpendicular to the chromatographic flow direction. The T1 line is coated with streptavidin; the T2 line is coated with Hb antibody; and the C line is coated with goat anti-mouse IgG. The T1 line is close to the conjugation pad, the T2 line is between the T1 line and the C line, and the C line is close to the absorbent pad. When the sample to be tested contains glycated hemoglobin, the T1 line will produce a fluorescent signal; When the sample contains hemoglobin, the T2 line will produce a fluorescent signal. When the immunochromatographic test strip is used in conjunction with a fluorescence detection device, the fluorescence detection device achieves quantitative detection of glycated hemoglobin and hemoglobin based on the intensity of the fluorescence signal on the immunochromatographic test strip.

[0010] In a preferred embodiment of the present invention, the sample to be tested is saliva.

[0011] In a preferred embodiment of the present invention, the biotinylated anti-glycated hemoglobin capture antibody is a substance obtained by biotinylating a glycated hemoglobin detection antibody with biotin-NHS, wherein biotin-NHS is selected from one of D-biotin-N-hydroxysuccinimide ester, 6-biotinaminohexanoate-N-hydroxysuccinimide ester, or biotin-polyethylene glycol-N-hydroxysuccinimide ester.

[0012] In a preferred embodiment of the present invention, the quantum dot microsphere-labeled anti-glycated hemoglobin detection antibody is a substance obtained by activating quantum dot microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then adding glycated hemoglobin detection antibody (HbA1c detection antibody); The quantum dot microsphere-labeled anti-hemoglobin detection antibody was obtained by activating quantum dot microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, followed by the addition of hemoglobin detection antibody (Hb detection antibody).

[0013] In a preferred embodiment of the present invention, the anti-glycated hemoglobin capture antibody was purchased from Wuhan Aokebotai Biotechnology Co., Ltd., product number A4101; The anti-hemoglobin capture antibody was purchased from Wuhan Aokebotai Biotechnology Co., Ltd., product number A1501; The antibody for detecting glycated hemoglobin was purchased from Wuhan Aokebotai Biotechnology Co., Ltd., product number A1509; The antihemoglobin detection antibody was purchased from Wuhan Aokebotai Biotechnology Co., Ltd., product number A1507.

[0014] In a preferred embodiment of the present invention, the particle size of the quantum dot microspheres is 100-300 nanometers; the mass-to-volume ratio of HbA1c antibody or Hb antibody to quantum dot microspheres is 0.5-2 μg:1 μL.

[0015] In a preferred embodiment of the present invention, the concentration of the coating solution of streptavidin coated on the T1 line is 0.5-2 mg / mL and the amount used is 1-2 μL / cm; The concentration of the coating solution for Hb capture antibodies coated on the T2 line is 0.5-2 mg / mL, and the volume is 1-2 μL / cm. The concentration of the coating solution for sheep anti-mouse IgG coated on the C-line was 0.5-2 mg / mL, and the volume was 1-2 μL / cm.

[0016] In a preferred embodiment of the present invention, the spraying amount of the biotinylated anti-glycated hemoglobin capture antibody, the quantum dot microsphere-labeled anti-glycated hemoglobin detection antibody, and the quantum dot microsphere-labeled anti-hemoglobin detection antibody loaded on the pad is 1-2 μL / cm, the spraying solution concentration is 0.5-2 mg / mL, and the spraying speed is 40-80 mm / s. Preferably, the spraying amount is 1 μL / cm, the spraying solution concentration is 1 mg / mL, and the spraying speed is 60 mm / s.

[0017] Preferably, the T1 line, T2 line and C line are parallel to each other and are spaced 3 to 7 mm apart.

[0018] In a preferred embodiment of the present invention, the base plate is made of polyvinyl chloride, the sample pad and the bonding pad are made of glass cellulose membrane, the absorbent pad is made of cotton cellulose membrane, and the NC membrane is made of nitrocellulose membrane.

[0019] The quantum dot microspheres of this invention are coated with a large number of quantum dots, enabling amplification of fluorescence signals. For hemoglobin in saliva at low concentrations, fluorescence signal amplification detection is achieved using anti-hemoglobin detection antibodies labeled with quantum dot microspheres. For glycated hemoglobin in saliva at extremely low concentrations, the biotinylated anti-glycated hemoglobin capture antibody-streptavidin system is combined with anti-glycated hemoglobin detection antibodies labeled with quantum dot microspheres for detection, significantly improving detection sensitivity and specificity. This invention pre-places different immune probes in the conjugate pad, and three parallel lines are arranged on the NC membrane, each line modified with a different antibody. By using a combination of Hb antibodies and HbA1c antibodies with different recognition sites, the abnormalities caused by mutual interference during Hb and HbA1c detection can be minimized. A novel immunochromatographic test strip was designed to simultaneously run streptavidin and hemoglobin antibody onto a single strip. Two T lines quantify glycated hemoglobin concentration and hemoglobin concentration, respectively, thereby achieving quantitative analysis of glycated hemoglobin glycation rate in one step. The test is fast and highly accurate.

[0020] In a preferred embodiment of the present invention, the saliva collection device is a medical cotton swab; the saliva processing device includes a syringe and a needle filter membrane, wherein the lower end of the syringe is connected to the needle filter membrane.

[0021] After collecting saliva with a medical cotton swab, the swab is placed into a syringe. A needle-type filter membrane is connected to the lower end of the syringe. Pushing the piston squeezes and filters out mucin, obtaining saliva with lower viscosity. This saliva is then dropped onto the test strip and allowed to stand until chromatography is complete. This method allows for rapid and simple pretreatment of saliva, reducing its viscosity and making it a homogeneous and stable liquid that can overcome chromatographic resistance on the test strip, allowing it to flow smoothly.

[0022] In a preferred embodiment of the present invention, the mouth is rinsed with water 5 minutes before saliva collection; each collection session lasts no more than 5 minutes.

[0023] In a preferred embodiment of the present invention, the length of the medical cotton swab is 0.5-4 cm and the diameter is 0.2-1.5 cm, and the saliva collection is 200-1000 microliters; the pore size of the needle filter membrane is 0.1-0.8 μm; the amount of saliva added to the test strip is 1-5 drops, and the time for standing for chromatography to be completed is 1-30 minutes.

[0024] The present invention further provides a method for using a reagent kit in conjunction with a fluorescence detection device, the method being for non-disease diagnostic purposes, comprising the following steps: After collecting saliva with a medical cotton swab, the swab is placed into a syringe. The lower end of the syringe is connected to a needle-type filter membrane. The piston is pushed to squeeze and filter the mucoprotein, resulting in saliva with lower viscosity. Add a drop of low-viscosity saliva onto the test strip and let it stand until the chromatography is complete; then place the test strip into the fluorescence detection device and use an image acquisition device to take a picture and read the fluorescence signal.

[0025] In a preferred embodiment of the present invention, the fluorescence detection device includes a housing, an ultraviolet light source, an excitation light filter, an emission light filter, a detection drawer, and a power supply; The detection drawer is located on one side of the base inside the housing; the excitation light source is fixed inside the upper part of the housing, facing the detection drawer at an angle, and the light-emitting end of the excitation light source is equipped with an excitation light filter; the emission light filter is fixed inside the top of the housing, vertically facing the detection drawer below.

[0026] The image acquisition device is located at the top of the outer casing, directly above the emission filter, with the light path vertically aligned with the test paper detection area of ​​the detection drawer below.

[0027] After chromatography, the test strip is placed on the detection drawer in the fluorescence detection device, the ultraviolet light source is turned on, and an image acquisition device is used to take a picture.

[0028] Preferably, the outer shell material is black and can be prepared by 3D printing.

[0029] Preferably, the excitation light filter is a 365 nm narrowband filter with a bandwidth of 40-50 nm and a cutoff depth of OD3-OD4; Preferably, the emitted light filter is a 615 nm narrowband filter with a bandwidth of 40-50 nm and a cutoff depth of OD3-OD4.

[0030] The image acquisition device is preferably a smartphone, which is easy to use for taking pictures.

[0031] After taking the photo, the image of the sample to be tested is imported into ImageJ software for data processing; the blank, unreacted area of ​​the test strip in the image is selected as the blank background; the average signal intensity of each channel (R, G, B) in the blank background area is calculated as the blank background fluorescence signal; Select the effective region (ROI) of the fluorescent spot in the figure, extract the average signal intensity of the R, G, and B channels in the region as the fluorescence signal, and obtain the fluorescence signal of glycated hemoglobin after subtracting the blank background or the fluorescence signal of hemoglobin after subtracting the blank background; The glycation rate of glycated hemoglobin is quantified based on a pre-established standard curve and linear fitting slope of fluorescence signal versus glycated hemoglobin concentration or hemoglobin concentration. The calculation method for the glycation rate of glycated hemoglobin is as follows: Where R is the glycation rate, k1 is the slope of the glycated hemoglobin standard curve, k2 is the slope of the hemoglobin standard curve, ΔT1 is the fluorescence signal of glycated hemoglobin after subtracting the blank background, ΔT2 is the fluorescence signal of hemoglobin after subtracting the blank background, [HbA1c] is the HbA1c concentration, and [NHb] is the concentration of non-glycated Hb.

[0032] In a preferred embodiment of the present invention, the standard curve is established as follows: Standard samples of glycated hemoglobin at varying concentrations were prepared sequentially. The test strips were then photographed and analyzed. The images of the standard glycated hemoglobin samples were imported into ImageJ software for data processing. The blank, unreacted areas of the test strips in the images were selected as the blank background. The average signal intensity of each channel (R, G, B) in the blank background area was calculated as the blank background fluorescence signal. The effective region (ROI) of the fluorescent spot in the images was selected, and the average signal intensity of each channel (R, G, B) within the region was extracted as the fluorescence signal. A linear regression was performed with the concentration of the standard glycated hemoglobin samples as the x-axis and the fluorescence signal intensity of glycated hemoglobin after subtracting the blank background as the y-axis to obtain the linear regression equation.

[0033] Standard test samples of hemoglobin with varying concentrations were prepared sequentially. The test strips described above were used to photograph and test the samples. The images of the standard test samples of hemoglobin were imported into ImageJ software for data processing. The blank, unreacted area of ​​the test strip in the image was selected as the blank background. The average signal intensity of each channel (R, G, B) in the blank background area was calculated as the blank background fluorescence signal. The effective region (ROI) of the fluorescent spot in the image was selected, and the average signal intensity of each channel (R, G, B) within the region was extracted as the fluorescence signal. A linear regression was performed with the concentration of the standard test sample of hemoglobin as the x-axis and the fluorescence signal of the blank hemoglobin sample after subtracting the blank background as the y-axis to obtain the linear regression equation.

[0034] In a preferred embodiment of the present invention, the absolute concentration range of the standard test sample for glycated hemoglobin is 0-20 ng / mL; the absolute concentration range of the standard test sample for hemoglobin is 0-300 ng / mL.

[0035] In a preferred embodiment of the present invention, the object of the photograph is the test strip.

[0036] In a preferred embodiment of the present invention, a smartphone is placed above a fluorescence detection device to form a smartphone imaging system.

[0037] Thirdly, the application of the above-mentioned kit in the preparation of diabetes screening products is provided.

[0038] This invention presents a non-invasive assay kit for glycosylated hemoglobin glycation rate in saliva. Biotin-streptavidin is incorporated into the test strip, improving the detection limit sensitivity. Simultaneously, a smartphone fluorescence imaging system enables the quantification of HbA1c glycation rate in saliva. The entire method requires no specific equipment, is simple to operate, and is conducive to its application and widespread use in immunoassay.

[0039] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is for saliva samples. The samples do not need to undergo complicated pretreatment and purification processes. The simple pretreatment steps reduce the viscosity of saliva and provide a stable matrix for the flow of saliva on the test strip.

[0040] (2) A novel immunochromatographic test strip is designed to simultaneously run streptavidin and hemoglobin antibody on one test strip. The two T lines quantify the glycated hemoglobin concentration and hemoglobin concentration, respectively, thereby realizing the quantification of glycated hemoglobin glycation rate in one step. The detection time is fast and the accuracy is high.

[0041] (3) The present invention has extremely high sensitivity in detecting glycated hemoglobin and hemoglobin in saliva.

[0042] (4) By combining a low-cost fluorescence detection device with a smartphone, an economical, portable and efficient POCT solution was constructed. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of saliva processing and a schematic diagram of the test strip.

[0044] Figure 2 This is a schematic diagram of a smartphone imaging system.

[0045] Figure 3 This is the standard curve for glycated hemoglobin.

[0046] Figure 4 This is the standard curve for hemoglobin.

[0047] Figure 5 This is the standard curve for glycated hemoglobin in the kit for detecting glycated hemoglobin using the direct double antibody sandwich method, as shown in Comparative Example 1.

[0048] Figure 6 This is a physical image of a sample for detecting glycated hemoglobin in saliva.

[0049] 1 is a medical cotton swab; 2 is a syringe; 3 is a needle filter membrane; 4 is saliva; 5 is hemoglobin; 6 is glycated hemoglobin; 7 is quantum dot microspheres; 8 is an anti-glycated hemoglobin capture antibody; 9 is an anti-glycated hemoglobin detection antibody; 10 is an anti-hemoglobin capture antibody; 11 is an anti-hemoglobin detection antibody; 12 is streptavidin; 13 is biotin; 14 is goat anti-mouse IgG; 15 is a smartphone; 16 is an emission light filter; 17 is an excitation light filter; 18 is an excitation light source; 19 is a detection drawer; and 20 is the outer casing. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0051] Example 1 A non-invasive detection kit for glycosylated hemoglobin in saliva and a matching fluorescence detection device: preparation and detection method. A non-invasive test kit for glycated hemoglobin in saliva includes a saliva sampling device, a saliva processing device, and an immunochromatographic test strip that simultaneously assists in the quantitative detection of hemoglobin and glycated hemoglobin.

[0052] 1. Preparation of the above-mentioned non-invasive test kit for glycated hemoglobin glycation rate in saliva 1.1 Preparation of quantum dot microsphere immunoprobes a. Add 10 μL of quantum dot microspheres (Suzhou Xingshuo Nanotechnology Co., Ltd., CdSe-NS-R-300) to 500 μL of MES buffer and wash 3 times.

[0053] b. Prepare 10 mg / mL NHS solution and EDC solution respectively.

[0054] c. Slowly add NHS and EDC to the quantum dot microspheres at twice the amount of carboxyl groups on the surface. Mix thoroughly and react at room temperature for 20 min, then centrifuge at 15000 g at 4℃ for 15 min.

[0055] d. After reconstituted with borate buffer, quantum dot microspheres were mixed with 15 μg of HbA1c detection antibody (Wuhan Aokebotai Biotechnology Co., Ltd., A1509) or Hb detection antibody (Wuhan Aokebotai Biotechnology Co., Ltd., A1507). After reacting at room temperature for 3 h, an equal volume of 2% BSA was added to the system for blocking for 1 h.

[0056] e. After the reaction is complete, centrifuge at 15000 g at 4℃ for 10 min, and reconstitute with probe preservation solution (PBS + 1% BSA + 1% trehalose + 0.1% Tween 20) to obtain quantum dot microsphere-labeled anti-glycated hemoglobin detection antibody or quantum dot microsphere-labeled anti-hemoglobin detection antibody, i.e., quantum dot microsphere immune probe.

[0057] 1.2 Preparation of biotinylated HbA1c capture antibody a. Replace 500 μg of HbA1c capture antibody (Wuhan Aokebotai Biotechnology Co., Ltd., A4101) with PBS buffer, and then dilute with PBS to a final concentration of 1 mg / mL.

[0058] b. Dissolve D-biotin-N-hydroxysuccinimide ester in DMSO.

[0059] c. Slowly add 20 times the amount of D-biotin-N-hydroxysuccinimide to 500 μg of 1 mg / mL HbA1c capture antibody. React at room temperature for 2 h.

[0060] d. After the reaction was completed, the biotinylated HbA1c capture antibody was purified by ultrafiltration using a 10 kDa MWCO ultrafiltration device, and the antibody was replaced with PBS buffer five times consecutively. The biotinylated HbA1c capture antibody was then concentrated to a concentration of 1 mg / mL and stored at -20°C.

[0061] 1.3 Preparation of immunochromatographic test strips for simultaneous quantitative detection of hemoglobin 5 and glycated hemoglobin 6 ( Figure 1 ) a. Streptavidin 12, anti-hemoglobin capture antibody 10 (Wuhan Aokebotai Biotechnology Co., Ltd., A1501), and goat anti-mouse IgG14 were sequentially streaked onto an NC membrane at concentrations of 1 mg / mL, 0.2 mg / mL, and 1 mg / mL, respectively, at a streaking speed of 0.8 μL / cm. The membranes were then dried overnight at 37°C.

[0062] b. The prepared quantum dot microsphere 7-labeled anti-glycated hemoglobin detection antibody 9, quantum dot microsphere 7-labeled anti-hemoglobin detection antibody 11, and biotinylated 13-labeled anti-glycated hemoglobin capture antibody 8 were sprayed onto the conjugate pad using a gold spraying apparatus. The spraying volume was 1 μL / cm, the spraying solution concentration was 1 mg / mL, and the spraying speed was 60 mm / s. After spraying, the mixture was dried overnight at 37°C.

[0063] c. Assemble the sample pad, conjugate pad, NC membrane and absorbent pad in sequence on the base plate to obtain the test strip plate. Cut the assembled card into strips with a width of 4.0 mm using a high-precision paper cutter and store them in a desiccator at room temperature.

[0064] 2. Preparation of the fluorescence detection device The device integrates a 365 nm LED light source (excitation source 18), a 365 nm narrowband excitation filter 17 (bandwidth 40-45 nm, cutoff depth OD3-OD4), a 615 nm narrowband emission filter 16 (bandwidth 50 nm, cutoff depth OD3), a detection drawer 19, and a housing 20 to form a fluorescence detection device. Figure 2 During testing, place the test strip on the test drawer 19, then place the test drawer 19 inside the fluorescence detection device, turn on the LED light source, and take a picture with a smartphone.

[0065] A smartphone 15 is placed above a fluorescence detection device to form a smartphone imaging system.

[0066] Fluorescence detection device 3. The glycation rate of glycated hemoglobin was detected using the kit prepared above in conjunction with a fluorescence detection device. 3.1 Test strip testing Add saliva to the sample pad of the test strip, let it stand until the chromatography is complete, and take a picture with the smartphone imaging system within 10-30 minutes.

[0067] 3.2 Data Processing Creating a standard curve: Dilute the standard glycated hemoglobin sample to an absolute concentration range of 0-20 ng / mL with diluent (PBS + 1% BSA) or to an absolute concentration range of 0-300 ng / mL with diluent (PBS + 1% BSA). Add the sample to the sample pad of the test strip and allow it to stand until chromatography is complete. Within 10-30 minutes, place it in the fluorescence detection device and take a picture using the smartphone imaging system. Import the image into ImageJ software for data processing. Select the blank, unreacted area of ​​the test strip in the image as the blank background. Calculate the average signal intensity of each channel (R, G, B) in the blank background area as the blank background fluorescence signal. Select the effective region (ROI) of the fluorescent spot in the image and extract the average signal intensity of each channel (R, G, B) within the region as the fluorescence signal. Obtain the fluorescence signal of glycated hemoglobin after subtracting the blank background or the fluorescence signal of hemoglobin after subtracting the blank background, and obtain the HbA1c standard curve (…). Figure 3 ) and Hb standard curve ( Figure 4 ).

[0068] Glycated hemoglobin standard curve: y = 10.53x + 48.53, R 2 =0.9990.

[0069] Hemoglobin standard curve: y = 0.6676x + 32.29, R 2 =0.9974 Sample data processing: Add saliva droplets to the sample pad of the test strip and allow it to stand until chromatography is complete. Within 10-30 minutes, place it in the fluorescence detection device and take a picture using the smartphone imaging system. Import the image into ImageJ software for data processing. Select the blank, unreacted area of ​​the test strip in the image as the blank background. Calculate the average signal intensity of each channel (R, G, B) in the blank background area as the blank background fluorescence signal. Select the effective region (ROI) of the fluorescent spot in the image and extract the average signal intensity of each channel (R, G, B) within the region as the fluorescence signal. Obtain the fluorescence signal of glycated hemoglobin after subtracting the blank background and the fluorescence signal of hemoglobin after subtracting the blank background. The method for calculating the glycation rate of glycated hemoglobin is as follows: Where R is the glycation rate, k1 is the slope of the glycated hemoglobin standard curve, k2 is the slope of the hemoglobin standard curve, ΔT1 is the fluorescence signal of glycated hemoglobin after subtracting the blank background, ΔT2 is the fluorescence signal of hemoglobin after subtracting the blank background, [HbA1c] is the HbA1c concentration, and [NHb] is the concentration of non-glycated Hb.

[0070] Comparative Example 1 Similar to Example 1, except that this comparative example does not use the streptavidin-biotin system, but instead uses an HbA1c capture antibody instead of streptavidin, and employs a direct double-antibody sandwich method to detect glycated hemoglobin concentration.

[0071] Preparation of a kit for detecting glycated hemoglobin using a direct double-antibody sandwich method Similar to Example 1, except that the T1 line is coated with glycated hemoglobin capture antibody (the coating method and coating concentration of glycated hemoglobin capture antibody are the same as the streptavidin coating method in 1.3).

[0072] The standard glycated hemoglobin samples were diluted with diluent (PBS + 1% BSA) to an absolute concentration range of 0-120 ng / mL, and then detected to obtain the HbA1c standard curve. Figure 5Compared to the direct double antibody sandwich method for detecting glycated hemoglobin, the streptavidin-biotin system has significantly improved detection sensitivity and a lower detection limit.

[0073] HbA1c comparison standard curve: y = 1.536x + 32.98, R 2 =0.9972 Example 2 The glycation rate of glycated hemoglobin in saliva was detected using the non-invasive detection kit for saliva prepared above, in conjunction with a fluorescence detection device. The procedure is as follows: Figure 1 As shown.

[0074] 1.1 Saliva Pretreatment Use a medical cotton swab 1 to draw 200-1000 microliters of saliva, then put the cotton swab into a syringe 2, push the piston to squeeze, and connect a needle filter membrane 3 at the lower end of the syringe to filter mucin, obtaining saliva 4 with lower viscosity.

[0075] 1.2 Test strip detection Add 4 drops of saliva to 3 drops of the sample pad on the test strip, let it stand until the chromatography is complete, and place it in the fluorescence detection device within 10-30 minutes and take a picture with the smartphone imaging system.

[0076] 1.3 Data Processing After taking a photo with a smartphone, obtain Figure 6 The image was imported into ImageJ software for data processing. The blank, unreacted area of ​​the test strip in the image was selected as the blank background. The average signal intensity of each channel (R, G, B) in the blank background area was calculated as the blank background fluorescence signal. The effective region (ROI) of the fluorescent spot in the image was selected, and the average signal intensity of each channel (R, G, B) within the region was extracted as the fluorescence signal. The fluorescence signals of glycated hemoglobin after subtracting the blank background and hemoglobin after subtracting the blank background were obtained. The method for calculating the glycation rate of glycated hemoglobin is as follows: Where R is the glycation rate, k1 is the slope of the glycated hemoglobin standard curve, k2 is the slope of the hemoglobin standard curve, ΔT1 is the fluorescence signal of glycated hemoglobin after subtracting the blank background, ΔT2 is the fluorescence signal of hemoglobin after subtracting the blank background, [HbA1c] is the HbA1c concentration, and [NHb] is the concentration of non-glycated Hb.

[0077] Using the standard curve and linear fitting slope established in Example 1, the glycosylation rate of this saliva sample was found to be 9.1%, achieving accurate quantification.

[0078] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A non-invasive test kit for the glycated hemoglobin glycation rate in saliva, characterized in that, It includes immunochromatographic test strips that simultaneously assist in the quantitative detection of hemoglobin and glycated hemoglobin, saliva collection devices, and saliva processing devices; The immunochromatographic test strip for simultaneous quantitative detection of hemoglobin and glycated hemoglobin includes a base plate and a sample pad, a conjugation pad, an NC membrane and an absorbent pad placed sequentially on the base plate. The bonding pad is loaded with the following components: (a) Biotinylated anti-glycated hemoglobin capture antibody, (b) Antibody for detecting glycated hemoglobin labeled with quantum dot microspheres. (c) Antibody for detecting hemoglobin labeled with quantum dot microspheres; The NC membrane is arranged in parallel with the first detection line T1, the second detection line T2, and the control line C. All three lines are perpendicular to the chromatographic flow direction. The T1 line is coated with streptavidin; the T2 line is coated with Hb antibody; and the C line is coated with goat anti-mouse IgG. The T1 line is close to the conjugation pad, the T2 line is between the T1 line and the C line, and the C line is close to the absorbent pad. When the sample to be tested contains glycated hemoglobin, the T1 line will produce a fluorescent signal; When the sample contains hemoglobin, the T2 line will produce a fluorescent signal. When the immunochromatographic test strip is used in conjunction with a fluorescence detection device, the fluorescence detection device achieves quantitative detection of glycated hemoglobin and hemoglobin based on the intensity of the fluorescence signal on the immunochromatographic test strip.

2. The non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, The sample to be tested is saliva.

3. The non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, Biotinylated anti-glycated hemoglobin capture antibody is a substance obtained by biotinylation of glycated hemoglobin detection antibody with biotin-NHS, wherein biotin-NHS is selected from one of D-biotin-N-hydroxysuccinimide ester, 6-biotinaminohexanoate-N-hydroxysuccinimide ester or biotin-polyethylene glycol-N-hydroxysuccinimide ester.

4. The non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, The quantum dot microsphere-labeled anti-glycated hemoglobin detection antibody is a substance obtained by activating quantum dot microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then adding the glycated hemoglobin detection antibody. The quantum dot microsphere-labeled anti-hemoglobin detection antibody is a substance obtained by activating quantum dot microspheres with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then adding the hemoglobin detection antibody.

5. A non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, The quantum dot microspheres have a particle size of 100-300 nanometers; the mass-to-volume ratio of HbA1c antibody or Hb antibody to quantum dot microspheres is 0.5-2 μg:1 μL.

6. The non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, The concentration of the coating solution for streptavidin on the T1 line was 0.5-2 mg / mL, and the volume was 1-2 μL / cm. The concentration of the coating solution for Hb capture antibodies coated on the T2 line is 0.5-2 mg / mL, and the volume is 1-2 μL / cm. The concentration of the coating solution for sheep anti-mouse IgG coated on the C-line was 0.5-2 mg / mL, and the volume was 1-2 μL / cm.

7. The non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, The combined pad-loaded biotinylated anti-glycated hemoglobin capture antibody, quantum dot microsphere-labeled anti-glycated hemoglobin detection antibody, and quantum dot microsphere-labeled anti-hemoglobin detection antibody were all sprayed at a rate of 1-2 μL / cm, with a spray solution concentration of 0.5-2 mg / mL and a spraying speed of 40-80 mm / s.

8. The non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, The saliva collection device is a medical cotton swab; the saliva processing device includes a syringe and a needle-type filter membrane, with the lower end of the syringe connected to the needle-type filter membrane.

9. A non-invasive detection kit for glycated hemoglobin glycation rate in saliva according to claim 1, characterized in that, Medical cotton swabs are 0.5-4 cm long and 0.2-1.5 cm in diameter; needle-type filter membranes have a pore size of 0.1-0.8 μm.

10. The use of the kit according to any one of claims 1-9 in the preparation of a diabetes screening product.

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