Microfluidic board for combined quantitative detection of urinary albumin and urine sugar, detection reagent and use method thereof

Abstract

The invention discloses a urinary albumin and urine sugar combined quantitative detection microfluidic plate and a detection reagent and a use method thereof, and belongs to the technical field of detection.The microfluidic plate is characterized in that a 1 # sample adding hole is connected with a 1 # reaction tank, and a 2 # sample adding hole is connected with a 2 # reaction tank through a pretreatment channel; the 1 # reaction tank is connected with a substrate liquid A inlet hole, a substrate liquid B inlet hole and a washing liquid inlet hole, the 2 # reaction tank is connected with an HRP solution inlet hole, a reaction matrix liquid inlet hole and a substrate liquid B inlet hole, and the 1 # reaction tank and the 2 # reaction tank are connected to a waste liquid tank; an mAlb detection reagent is prefabricated in the 1 # reaction tank, and carboxyl is modified on the surface of the 1 # reaction tank; a urine glucose detection reagent is prepared in the second reaction tank, a filtering device is arranged on a pretreatment channel, the channel is coated with ascorbic acid oxidase, and the invention further discloses formulas of all the reagents and a detection method. By utilizing a polystyrene microfluidic plate microfluidic technology, high sensitivity of combined quantitative detection is realized, early screening and differential diagnosis of diabetic nephropathy are conveniently realized, and a very good effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and particularly to a microfluidic plate for combined quantitative detection of urinary albumin and urinary glucose, its detection reagent, and usage method. Background Art

[0002] Diabetic kidney disease (DKD) is a chronic kidney disease caused by diabetes. Studies have shown that DKD has surpassed other chronic kidney diseases such as glomerulonephritis to become the primary cause of hospitalization for kidney disease patients in China, and the main cause of dialysis treatment for end-stage renal disease (ESRD). Currently, for the clinical diagnosis of DKD, it is often based on albuminuria and whether the patient has diabetic retinopathy, etc. Clinically, the urinary albumin / creatinine ratio (UACR) ≥ 30 mg / g and / or the estimated glomerular filtration rate (eGFR) < 60 ml·min - 1·(1.73m 2 ) - 1, and if it persists for more than 3 months, it is diagnosed as DKD. Proteinuria is an important marker of kidney injury and an independent risk factor for the continuous progression of kidney disease. However, in the diagnosis of DKD, whether it is based on UACR or eGFR, there are inevitable limitations. Taking the most commonly used urinary UACR as an example, urinary albumin is a marker of early kidney damage in diabetic kidney disease, but the change in its level is often relatively slow. In diabetic patients, especially in the early stage of kidney injury, the excretion of urinary albumin may not have increased significantly, and even in some patients, the level of urinary albumin is normal or slightly elevated. Therefore, UACR may produce false-negative results in the early diagnosis of diabetic kidney disease and cannot identify the initial kidney injury in a timely manner. The excretion of urinary albumin is also easily affected by various factors, including blood glucose levels. In cases of large blood glucose fluctuations or poor long-term control, the level of urinary albumin may increase in the short term, reflecting the pressure of blood glucose fluctuations on the kidneys rather than kidney pathological changes.

[0003] Therefore, solely relying on UACR to judge the progression of diabetic kidney disease may be interfered by the status of blood glucose control, affecting its accuracy as a marker of long-term kidney damage. Moreover, the mechanisms of tubular and glomerular damage in diabetic kidney disease are complex, and the composition of urinary proteins also shows heterogeneity. In addition to urinary albumin, urine may also contain other types of proteins, such as urinary globulin, β2-microglobulin, etc. The excretion of these proteins may not be directly related to the severity of diabetic kidney disease, and the single indicator of UACR cannot comprehensively reflect the overall picture of kidney damage. Therefore, UACR may not be able to provide complete information about kidney lesions. More importantly, UACR mainly reflects glomerular function, while the early damage in diabetic kidney disease often first presents as tubular dysfunction, such as decreased tubular reabsorption function. UACR cannot effectively reflect the degree of tubular damage, which also plays an important role in the progression of diabetic kidney disease. Therefore, solely relying on UACR for kidney lesion assessment and ignoring the early tubular damage may underestimate the actual condition of diabetic kidney disease. Especially in the early stage of diabetic kidney disease, the kidney damage may be very slight, and the change in urinary albumin excretion is not obvious, making it difficult for UACR to detect these slight changes in a timely manner. Especially when the blood glucose of diabetic patients is well controlled or in the preclinical stage, the sensitivity of UACR is insufficient, and the best opportunity for early intervention may be missed.

[0004] Compared with UACR, eGFR has a worse ability to predict and diagnose early kidney damage. eGFR estimates the glomerular filtration rate through serum creatinine levels. In the early stage of diabetic kidney disease, the glomerular filtration rate may have already decreased, but the change in serum creatinine is relatively lagged. This is because creatinine, as a waste product, is mainly cleared through glomerular filtration. However, when tubular function is damaged, the increase in serum creatinine level does not appear immediately. Especially in the early stage of diabetic kidney disease, there may be a situation where tubular function is damaged while eGFR has not decreased significantly. Therefore, eGFR may show false negative results in the early diagnosis of diabetic kidney disease and cannot identify early kidney damage in a timely manner.

[0005] The urinary protein to urinary glucose ratio (UPGR) has unique advantages compared to the traditional urinary microalbumin to creatinine ratio (UACR) and eGFR, especially in the detection of early kidney injury in diabetic patients. Although the detection of urinary microalbumin can reflect partial impairment of kidney function, in diabetic patients, the increase in urinary microalbumin may be affected by various factors such as hyperglycemia and tubular dysfunction. The determination of urinary glucose, on the other hand, can directly reflect the status of blood glucose control. Hyperglycemia causes damage to the kidneys through multiple pathways, promoting dysfunction of renal tubular epithelial cells and exacerbating renal interstitial fibrosis through promoting renal glycotoxic effects. Therefore, as a core biomarker in diabetic patients, the detection of urinary glucose can not only reveal the direct impact of blood glucose out of control on the kidneys but also help identify early kidney injury caused by diabetes.

[0006] The urinary protein to urinary glucose ratio (UPGR) has great advantages in the early diagnosis of diabetic kidney disease, especially in the early stage when the urinary albumin level has not increased significantly. High urinary glucose levels are usually closely related to high blood glucose levels, which to a certain extent reflect the dual effects of kidney damage and blood glucose control imbalance. Compared with urinary microalbumin, the change in the concentration of urinary glucose can more directly reflect the continuous impact of blood glucose on the kidneys and reveal the potential risk of kidney injury before the significant increase in urinary albumin. Therefore, the urinary protein to urinary glucose ratio can provide a more sensitive indicator for the early screening of diabetic kidney disease. Especially in diabetic patients with poor blood glucose control, its change can be used as a key biomarker to evaluate the relationship between kidney function damage and poor diabetic metabolic control.

[0007] Combined quantitative detection of albumin and urinary glucose, which are commonly used markers for diagnosing DKD in clinical practice, by microfluidic technology for early screening and differential diagnosis of clinical DKD has better advantages of early diagnosis and early identification. Summary of the Invention

[0008] To solve the above technical problems, the present invention discloses a microfluidic plate for combined quantitative detection of urinary albumin and urinary glucose, its detection reagent, and usage method.

[0009] The technical solution of the present invention is as follows: A microfluidic plate for combined quantitative detection of urinary albumin and urinary glucose, the 1# sample adding hole is connected to the 1# reaction pool, and the 2# sample adding hole is connected to the 2# reaction pool through a pretreatment channel; the 1# reaction pool is connected to the substrate solution A inlet hole, the substrate solution B inlet hole, and the washing solution inlet hole, and the 2# reaction pool is connected to the HRP solution inlet hole, the reaction matrix solution inlet hole, and the substrate solution B inlet hole. The 1# reaction pool and the 2# reaction pool are connected to the waste liquid pool; the mAlb detection reagent is prefabricated in the 1# reaction pool, and the surface of the 1# reaction pool is modified with carboxyl; the urinary glucose detection reagent is prefabricated in the 2# reaction pool. A filtering device is provided on the pretreatment channel, and ascorbic acid oxidase is coated on the channel.

[0010] Further, the method for coating ascorbic acid oxidase on the pretreatment channel is as follows: Dilute the concentration of ascorbic acid oxidase to 1 - 3 μg / ml with 0.05 M phosphate buffer solution at pH 6.0. Aspirate a certain amount of the solution and spread it evenly on the pretreatment channel. After standing, pipette and then stand again. Then aspirate 10 - 30 μl of enzyme stabilizer and spread it evenly on the pretreatment channel, and dry it for standby.

[0011] The present invention also provides a detection reagent for a microfluidic plate for the combined quantitative detection of urinary albumin and urinary glucose, which is used for the aforementioned microfluidic plate. The detection reagent includes the mAlb detection reagent in the 1# reaction pool, and additionally added substrate solution A, substrate solution B, and washing solution; the urinary glucose detection reagent in the 2# reaction pool, and additionally added HRP solution, reaction matrix solution, and substrate solution B.

[0012] Further, the mAlb detection reagent includes coated streptavidin, biotin-labeled antibody, and HRP-labeled antibody, and the urinary glucose detection reagent includes glucose oxidase solution.

[0013] Further, the substrate solution A is 5.0 - 10.0 g / L of citric acid, 35.0 - 60.0 g / L of sodium acetate, 5 - 20 ml / L of Triton, and 0.1 - 0.5 ml / L of 3% hydrogen peroxide.

[0014] Further, the substrate solution B is 0.1 - 0.4 g / L of TMB, dissolved with dimethyl sulfoxide, 20 - 50 ml / L of glycerol, 50 - 100 g / L of polyoxyethylene lauryl ether, 1.5 - 3 g / L of citric acid, 5 - 10 g / L of sodium sulfite, and 0.05 - 0.2 g / L of EDTA-Na.

[0015] Further, the HRP solution is prepared by formulating HRP enzyme with HRP enzyme preservation solution to 0.1 μg - 0.3 μg / ml. The HRP enzyme preservation solution is in 0.1 M PBS buffer solution at pH 6.5, containing 0.05 - 0.2 g / L of sodium dodecyl sulfate, 10.0 - 30.0 g / L of trehalose, 10.0 - 30.0 g / L of BSA, 0.1 - 0.5% of mannitol, 5.0 - 10.0 g / L of polyethylene glycol, 0.5 - 1.0 ml / L of Tween-20, 0.1 - 0.2 g / L of MgCl2, and 0.01 - 0.05 g / L of ZnCl2.

[0016] Further, the reaction matrix solution is 5.0 - 10.0 g / L of citric acid, 35.0 - 60.0 g / L of sodium acetate, and 5 - 20 ml / L of Triton.

[0017] Further, the glucose oxidase solution is prepared with a glucose oxidase preservation solution to a concentration of 5.0 - 20.0 mg / ml. The formula of the glucose oxidase preservation solution is as follows: in a 0.1M PBS buffer solution with a pH of 6.0, 0.1 - 0.5 mg / L α-cyclodextrin, 1.0 - 5.0 g / L mannitol, 0.01 - 0.03 g / L sodium bicarbonate, 0.05 - 0.2 g / L sodium dodecyl sulfate, 10.0 - 30.0 g / L trehalose, 0.5 - 1.0 ml / L Tween-20, 4.5 - 9.0 g / L NaCl.

[0018] The present invention also provides a method for using a microfluidic plate for the combined quantitative detection of urinary albumin and urinary glucose. Using the aforementioned microfluidic plate for the combined quantitative detection of urinary albumin and urinary glucose, and the detection reagent for the aforementioned microfluidic plate for the combined quantitative detection of urinary albumin and urinary glucose, the method for use includes:

[0019] First step: Add urine into the 1# sample injection hole and the 2# sample injection hole respectively.

[0020] Second step: The sample in the 1# sample injection hole flows into the 1# reaction pool through the liquid channel, reacts with the coated antibody and the HRP-labeled antibody in the reaction pool to generate an antigen-antibody complex. Add a washing solution through the liquid inlet hole to wash the 1# reaction pool, and drain the waste liquid into the waste liquid pool; the sample in the 2# sample injection hole flows into the 2# reaction pool through the pre-treated channel, reacts with the glucose oxidase in the reaction pool, and then adds an HRP solution through the liquid inlet hole to react.

[0021] Third step: Add substrate solution A and substrate solution B to the 1# reaction pool successively through the liquid inlet hole, add reaction matrix solution and substrate solution B to the 2# reaction pool successively through the liquid inlet hole, and detect the fluorescence value after the reaction.

[0022] Fourth step: Using the calibration curve with known concentrations, substitute the obtained fluorescence value into the standard curve equation to calculate the concentrations of albumin and urinary glucose in the measured sample.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention has developed a detection reagent for the combined quantitative detection of urinary albumin and urinary glucose, with high sensitivity, facilitating the early screening and differential diagnosis of diabetic nephropathy. (2) The present invention utilizes the microfluidic technology of a polystyrene microfluidic plate to achieve combined quantitative detection; (3) The present invention proposes a scheme for modifying carboxyl groups in specific regions of the microfluidic plate, coating streptavidin, performing pretreatment on the liquid inlet channel, and optimizing the stability of glucose oxidase, improving sensitivity, accuracy, enhancing specificity, and achieving good results. Description of the Drawings

[0024] Figure 1 is a schematic diagram of the microfluidic plate;

[0025] Figure 2 It is a correlation analysis diagram of mALB;

[0026] Figure 3 It is a correlation analysis diagram of urine sugar.

[0027] Among them, 1 is the sample loading hole 1#; 2 is the reaction pool 1#; 3 is the inlet hole of substrate solution A; 4 is the inlet hole of washing solution; 5 is the inlet hole of substrate solution B; 6 is the waste liquid pool; 7 is the sample loading hole 2#; 8 is the reaction pool 2#; 9 is the inlet hole of HRP solution; 10 is the inlet hole of reaction matrix solution. Specific implementation mode

[0028] The technical solution of the present invention will be further explained below in conjunction with the accompanying drawings.

[0029] As Figure 1 shown, a microfluidic plate for combined quantitative detection of urinary albumin and urine sugar, the sample loading hole 1# 1 is connected to the reaction pool 1# 2, and the sample loading hole 2# 7 is connected to the reaction pool 2# 8 through a pretreatment channel; the reaction pool 1# 2 is connected to the inlet hole of substrate solution A 3, the inlet hole of substrate solution B 5 and the inlet hole of washing solution 4, the reaction pool 2# 8 is connected to the inlet hole of HRP solution 9, the inlet hole of reaction matrix solution 10, and the inlet hole of substrate solution B 5, and the reaction pool 1# 2 and the reaction pool 2# 8 are connected to the waste liquid pool 6; the mAlb detection reagent is prefabricated in the reaction pool 1# 2.

[0030] The surface of the reaction pool 1# 2 is modified with carboxyl groups, and the surface modification method is used to modify the carboxyl groups on the polystyrene surface:

[0031] 1. Surface pretreatment

[0032] The microfluidic plate is ultrasonically cleaned with isopropanol for 5 - 10 minutes to remove surface organic impurities, then rinsed with deionized water 3 - 5 times and dried, and then ultrasonically cleaned with ethanol for 5 minutes, rinsed with deionized water 3 - 5 times and dried.

[0033] 2. Introduction of carboxyl groups by chemical coupling method

[0034] (1) Silanization reaction:

[0035] Add 30 ul of ethanol solution containing 0.5% - 5% 3 - carboxypropyltrimethoxysilane to the reaction pool 1# 2 of the pretreated microfluidic plate, and react at room temperature for 4 - 6 hours.

[0036] (2) Cleaning and curing:

[0037] The reaction pool 1# 2 of the microfluidic plate after the reaction is successively cleaned 3 - 5 times with ethanol and deionized water to remove the unreacted silane reagent.

[0038] The microfluidic plate is dried and solidified at 50-70°C to enhance bonding stability. The prepared microfluidic plate is sealed in an aluminum foil bag and stored for later use.

[0039] The surface-modified carboxyl groups can be activated by reagents to form stable amide bonds with amino (-NH2) biomolecules (such as antibodies), achieving directional fixation and improving the sensitivity of immunoassays.

[0040] The urine sugar detection reagent is prefabricated in the 2# reaction pool 2, a filtering device is arranged on the pretreatment channel, and the channel is coated with ascorbic acid oxidase.

[0041] A detection reagent for a microfluidic plate for quantitatively detecting urine albumin and urine sugar, used for the aforementioned microfluidic plate, the detection reagent comprising a mAlb detection reagent in a 1# reaction pool, as well as an added substrate solution A, a substrate solution B, and a washing solution, a urine sugar detection reagent in a 2# reaction pool, as well as an added HRP solution, a reaction matrix solution, and a substrate solution B.

[0042] Example 1 mAlb detection reagent

[0043] 1. Coating of Streptavidin in the Detection Pool

[0044] Pretreatment of reaction pool #1: Prepare 10% 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 10% N-Hydroxysuccinimide (NHS) as activators. Add 50ul EDC and 30ul NHS to reaction pool #1 and place on a shaker for 30min to fully expose the carboxyl groups on the surface of reaction pool #1. Discard the reaction solution and set aside.

[0045] Streptavidin pretreatment: Prepare 0.1M MES buffer with pH 4.0-7.0, and replace the original storage solution of streptavidin with the prepared MES buffer using an ultrafiltration centrifuge tube. Centrifuge at 8000-12000r / min for 5-6min, and repeat 3-5 times.

[0046] Prepare the pretreated streptavidin with 0.1M MES buffer at pH 4.0-7.0 to a concentration of 4.0-6.0ug / mL, pipette 50-100ul into the 1# reaction pool, and place on a shaker to react for 1-2 hours. After discarding the reaction solution, add 100ul Tris buffer containing 2% BSA, react for 1 hour, and then wash 2-5 times with 2% BSA Tris buffer.

[0047] 2. Biotinylated Antibodies

[0048] Weigh 2 - 4 mg of NHS - Biotin and dissolve it in 600 μL of DMSO to prepare a stock solution. Take 100 μL of the stock solution and add it to 900 μL of 10 mM PBS buffer to make the reaction concentration of NHS - Biotin. Take 100 μL of the NHS - Biotin with the reaction concentration and place it in a 1.5 mL centrifuge tube. Add 0.1 mg of mALB antibody purified with 10 mM PBS buffer (pH 7.0 - 8.0), and mix well by pipetting. Incubate at room temperature in the dark with rotation for 1 hour. Transfer the reacted biotinylated antibody to an ultrafiltration centrifuge tube and ultrafilter it 3 - 5 times with 10 mM PBS buffer (pH 7.0 - 8.0), and collect it into a new centrifuge tube.

[0049] 3. React the biotin - labeled antibody with the streptavidin coated in the 1# reaction pool

[0050] Dilute the biotin - labeled antibody with the reaction buffer to a concentration of 0.5 mg / ml. Pipette 10 - 30 μl of the diluted antibody and add it to the 1# reaction pool coated with streptavidin. React at room temperature with shaking for 1 - 2 hours. Discard the reaction solution. Set aside for later use.

[0051] 4. HRP - labeled antibody

[0052] Prepare a 0.02 - 0.08 M NaIO4 solution. Weigh 1 mg of HRP and dissolve it in 0.1 - 0.5 mL of distilled water. Use a dropper to gradually add 0.1 - 0.5 mL of the NaIO4 solution drop - by - drop to the HRP solution, and react at 2 - 8℃ in the dark for 30 min. Prepare a 0.1 - 0.2 M ethylene glycol solution. Take 0.1 - 0.5 mL of the prepared ethylene glycol and add it to the HRP - NaIO4 mixed solution, and stir at room temperature in the dark for 30 min. Pretreat the mALB antibody in an ultrafiltration centrifuge tube with 0.2 M pH 9.0 CBS by ultrafiltration. Take 0.3 - 1.0 mg of the pretreated mALB antibody and add it to the reacted HRP - NaIO4 mixed solution, and stir at room temperature in the dark for 2 hours. Transfer the reacted labeled antibody into a dialysis bag and dialyze it in 0.05 M carbonate buffer, and stir at 2 - 8℃ in the dark overnight. Transfer the dialyzed labeled antibody to a centrifuge tube, add 0.1 - 0.2 mL of freshly prepared 5 mg / mL NaBH4 solution, mix well and keep it in the dark at 2 - 8℃ for 2 hours. After purification through a purification column, dilute it with the HRP - labeled antibody preservation solution to a working concentration of 0.005 mg - 0.02 mg / ml. Pipette 5 μl of the diluted antibody and add it to the 1# reaction pool treated in step 3.

[0053] 5. Solution A and Solution B

[0054] Substrate solution A: 5.0 - 10.0 g / L of citric acid, 35.0 - 60.0 g / L of sodium acetate, 5 - 20 ml / L of Triton, 0.1 - 0.5 ml / L of 3% hydrogen peroxide.

[0055] Substrate solution B: TMB 0.1 - 0.4 g / L (dissolved in dimethyl sulfoxide), glycerol 20 - 50 ml / L, polyoxyethylene lauryl ether 50 - 100 g / L, citric acid 1.5 - 3 g / L, sodium sulfite 5 - 10 g / L, EDTA-Na 0.05 - 0.2 g / L.

[0056] Example 2 Urine Glucose Detection Reagent

[0057] 1. Preparation of Sample Pretreatment Channel

[0058] A filtering device is provided on the channel between the 2# sampling hole and the 2# reaction pool of the polystyrene microfluidic plate to filter out interfering substances such as urate crystals and flocculants in urine. Ascorbic acid oxidase is coated on the channel to eliminate ascorbic acid interference. Dilute the concentration of ascorbic acid oxidase to 1 - 3 μg / ml with 0.05M PH6.0 phosphate buffer solution, and use a pipette to suck 100 - 200 μl and spread it evenly on the channel between the 2# sampling hole and the 2# reaction pool. After standing for 1 hour, blow it 5 - 10 times with a pipette and then stand for another 1 hour. Discard the reaction solution, suck 10 - 30 μl of enzyme stabilizer (formula: α-cyclodextrin) and spread it evenly on the pretreatment channel between the 2# sampling hole and the 2# reaction pool, and dry it at 37°C for standby.

[0059] 2. Preparation of Glucose Oxidase

[0060] Formulation of glucose oxidase preservation solution:

[0061] Glucose oxidase preservation solution 1: In 0.1M PH 6.0 PBS buffer solution, 0.1 - 0.5 mg / L α-cyclodextrin, 1.0 - 5.0 g / L mannitol, 0.01 - 0.03 g / L sodium bicarbonate, 0.05 - 0.2 g / L sodium dodecyl sulfate, 10.0 - 30.0 g / L trehalose, 0.5 - 1.0 ml / L Tween-20, 4.5 - 9.0 g / L NaCl.

[0062] Glucose oxidase preservation solution 2: In 0.1M PH 6.0 PBS buffer solution, 1.0 - 5.0 g / L mannitol, 0.005 - 0.02 g / L sodium bicarbonate, 0.05 - 0.2 g / L sodium dodecyl sulfate, 10.0 - 30.0 g / L trehalose, 0.5 - 1.0 ml / L Tween-20, 4.5 - 9.0 g / L NaCl.

[0063] Glucose oxidase preservation solution 3: In 0.1M PBS buffer with pH 6.0, 0.1 - 0.5 mg / L α-cyclodextrin, 0.05 - 0.2 g / L sodium dodecyl sulfate, 10.0 - 30.0 g / L trehalose, 0.5 - 1.0 ml / L Tween-20, 4.5 - 9.0 g / L NaCl.

[0064] Preparation of glucose oxidase: Use the prepared glucose oxidase preservation solution to prepare the glucose oxidase at a working concentration of 5.0 - 20.0 mg / ml, and pipette 5 μl and place it in the reaction cell #2.

[0065] 3. Preparation of HRP enzyme

[0066] Preparation of HRP enzyme preservation solution: In 0.1M PBS buffer with pH 6.5, 0.05 - 0.2 g / L sodium dodecyl sulfate, 10.0 - 30.0 g / L trehalose, 10.0 - 30.0 g / L BSA, 0.1 - 0.5% mannitol, 5.0 - 10.0 g / L polyethylene glycol, 0.5 - 1.0 ml / L Tween-20, 0.1 - 0.2 g / L MgCl2, 0.01 - 0.05 g / L ZnCl2.

[0067] Preparation of HRP enzyme: Use the prepared HRP enzyme preservation solution to prepare the HRP enzyme at a working concentration of 0.1 μg - 0.3 μg / ml, and pipette 5 μl and place it in the HRP solution cell.

[0068] 4. Preparation of reaction substrate solution

[0069] Citric acid 5.0 - 10.0 g / L, sodium acetate 35.0 - 60.0 g / L, Triton 5 - 20 ml / L.

[0070] Example 3

[0071] The first step during use: Add urine to the sample wells #1 and #2 respectively.

[0072] The second step: The sample in the sample well #1 flows into the reaction cell #1 through the liquid channel, reacts with the coated antibody and HRP-labeled antibody in the reaction cell to form an antigen-antibody complex, add washing solution to the reaction cell #1 through the liquid inlet hole to wash, and discharge the waste liquid to the waste liquid cell; the sample in the sample well #2 flows into the reaction cell #2 through the pre-treated channel, reacts with the glucose oxidase in the reaction cell, and then adds the HRP solution to react through the liquid inlet hole.

[0073] The third step: Add substrate solution A and substrate solution B to the reaction cell #1 successively through the liquid inlet hole, add reaction substrate solution and substrate solution B to the reaction cell #2 successively through the liquid inlet hole, and detect the fluorescence value after the reaction.

[0074] Step 4: Using the calibration curve with known concentrations, substitute the obtained fluorescence values into the standard curve equation to calculate the concentrations of albumin and urine glucose in the measured samples.

[0075] Example 4 Performance Evaluation

[0076] 1. Glucose Oxidase Stability Assessment

[0077] Prepare 3 kinds of glucose oxidase preservation solutions to formulate glucose oxidase into a working concentration of 5.0 - 20.0 mg / ml, and divide each into two parts and place them at 2 - 8°C and 37°C respectively. Taking the results at day 0 (detected on the day of preparation) and the results at 2 - 8°C as references, on days 1, 3, 5, 7, 9, 11, 13, and 15, use a quality control product with a concentration of 2 mg / ml to detect the glucose oxidase of the three formulations respectively. The results are shown in Tables 1, 2, and 3. After being placed at 2 - 8°C for 15 days, compared with the results at day 0, the maximum differences of the glucose oxidase of the three formulations are 3.4%, 2.5%, and 3.7% respectively, and the stabilities are all good. After being placed at 37°C for 15 days, compared with the results at 2 - 8°C, the maximum differences are 4.1%, 10.9%, and 5.4% respectively. For formulation 2, there is a degradation trend after being placed at 37°C for 13 days, and for formulation 3, there is a degradation trend after being placed at 37°C for 11 days. The glucose oxidase prepared with formulation 1 shows good stability after being placed at 2 - 8°C and 37°C for 15 days.

[0078] Table 1 Stability Experiment of Glucose Oxidase Preservation Solution 1

[0079]

[0080] Table 2 Stability Experiment of Glucose Oxidase Preservation Solution 2

[0081]

[0082] Table 3 Stability Experiment of Glucose Oxidase Preservation Solution 3

[0083]

[0084]

[0085] 2. Sensitivity

[0086] Repeatedly measure the zero - concentration standard product 20 times, calculate the average value (Mean) and standard deviation (SD) of the 20 detected concentrations according to the standard curve, and calculate the concentration value of Mean + 2SD, which is the blank limit of the kit. The blank limit of the mALB reagent is 0.19 mg / L, and the blank limit of urine glucose is 0.009 mg / ml, as shown in Table 4.

[0087] Detect mALB with known concentrations of 0.5 mg / L, 1.0 mg / L, 2 mg / L, 4 mg / L and urine glucose with concentrations of 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, and repeat the detection 5 times for each concentration. The results are shown in Table 5. The sensitivity of mALB is 1.0 mg / L, and the sensitivity of urine glucose is 0.05 mg / ml.

[0088] Table 4 Reagent Blank Limit

[0089]

[0090] Table 5 Reagent Sensitivity

[0091]

[0092] 3. Accuracy

[0093] Add 5.0 mg / L, 8.0 mg / L of mALB and 1.5 mg / ml, 9.0 mg / ml of glucose to two portions of urine with known concentrations (Urine 1: mALB is 10.3 mg / L, urine glucose is 0.82 mg / ml; Urine 2: mALB is 56.7 mg / L, urine glucose is 2.17 mg / ml), and repeat the detection 3 times for each concentration, then calculate the mean value. The recovery rates are as follows: for mALB in Urine 1: 92% - 104%, for urine glucose in Urine 1: 95% - 104%. For mALB in Urine 2: 95% - 96%, for urine glucose in Urine 2: 93% - 96%.

[0094] 4. Repeatability

[0095] Repeat the determination 10 times for each of the two portions of urine with known concentrations (Urine 1: mALB is 10.3 mg / L, urine glucose is 0.82 mg / ml; Urine 2: mALB is 56.7 mg / L, urine glucose is 2.17 mg / ml), and calculate the repeatability CV. For mALB, it is 2.30% and 1.35%, and for urine glucose, it is 4.93% and 2.57%. The results are shown in Table 6.

[0096] Table 6 Results of Reagent Repeatability Detection

[0097]

[0098] 5. Specificity

[0099] Add 3.0 mg / ml creatinine (Cr), 400 ng / ml neutrophil gelatinase-associated lipocalin (NGAL), and 0.5 mg / ml vitamin C to two urine samples with known concentrations (urine 1: mALB is 10.3 mg / L, urine glucose is 0.82 mg / ml; urine 2: mALB is 56.7 mg / L, urine glucose is 2.17 mg / ml) to obtain the test samples. The mean of the measurement results should be within the range of the target concentration ±10%.

[0100] Test results (Table 7): For urine 1, mALB is between 10.16 mg / L and 10.52 mg / L, and urine glucose is between 0.79 mg / ml and 0.84 mg / ml. For urine 2, mALB is between 52.21 mg / L and 56.81 mg / L, and urine glucose is between 2.04 mg / ml and 2.14 mg / ml, all within the target concentration range.

[0101] Table 7 Specificity test results

[0102]

[0103] 6. Detection of clinical samples

[0104] Collect urine samples from 52 normal people and 47 diabetic patients. Use the reagent developed by this patent for combined detection and perform a correlation analysis with the results of the control reagent. The results are shown in Figure 3 . The correlation coefficient R of mALB 2 = 0.9896, and the correlation coefficient R of urine glucose 2 = 0.9141.

Claims

1. A microfluidic plate for quantitative detection of urine albumin and urine glucose, characterized in that: The 1# sample loading hole is connected to the 1# reaction pool, and the 2# sample loading hole is connected to the 2# reaction pool through a pretreatment channel; the 1# reaction pool is connected to the substrate solution A inlet hole, the substrate solution B inlet hole and the washing solution inlet hole, the 2# reaction pool is connected to the HRP solution inlet hole, the reaction matrix solution inlet hole, and the substrate solution B inlet hole, and the 1# reaction pool and the 2# reaction pool are connected to the waste liquid pool; the mAlb detection reagent is prefabricated in the 1# reaction pool, and the surface of the 1# reaction pool is modified with a carboxyl group; the urine sugar detection reagent is prefabricated in the 2# reaction pool, and a filtering device is arranged on the pretreatment channel, and ascorbic acid oxidase is coated on the channel.

2. The microfluidic plate for quantitative detection of urine albumin and urine glucose according to claim 1, characterized in that: The method for coating the pretreatment channel with ascorbic acid oxidase is to dilute the ascorbic acid oxidase concentration to 1-3ug / ml with 0.05M pH6.0 phosphate buffer, absorb some of the solution and spread it on the pretreatment channel, let it stand, blow it, let it stand again, then absorb 10-30ul of enzyme stabilizer and spread it on the pretreatment channel, and dry it for later use.

3. A detection reagent for quantitative detection of urine albumin and urine sugar on a microfluidic plate, characterized in that: A microfluidic plate for the combined quantitative detection of urine albumin and urine sugar according to any one of claims 1 to 2, wherein the detection reagent comprises the mAlb detection reagent in the 1# reaction pool, as well as the added substrate solution A, substrate solution B, and washing solution, and the urine sugar detection reagent in the 2# reaction pool, as well as the added HRP solution, reaction matrix solution, and substrate solution B.

4. The detection reagent for the microfluidic plate for quantitative detection of urine albumin and urine sugar according to claim 2, characterized in that: The mAlb detection reagent includes coated streptavidin and biotin labeled antibody, HRP labeled antibody, and the urine sugar detection reagent includes glucose oxidase solution.

5. The detection reagent for the microfluidic plate for quantitative detection of urine albumin and urine sugar according to claim 2, characterized in that: The substrate solution A comprises 5.0-10.0 g / L of citric acid, 35.0-60.0 g / L of sodium acetate, 5-20 ml / L of Triton, and 0.1-0.5 ml / L of 3% hydrogen peroxide.

6. The detection reagent for the microfluidic plate for quantitative detection of urine albumin and urine glucose according to claim 2, characterized in that: The substrate solution B is TMB 0.1-0.4 g / L, dissolved in dimethyl sulfoxide, glycerol 20-50 ml / L, polyoxyethylene lauryl ether 50-100 g / L, citric acid 1.5-3 g / L, sodium sulfite 5-10 g / L, and EDTA-Na 0.05-0.2 g / L.

7. The detection reagent for the microfluidic plate for quantitative detection of urine albumin and urine glucose according to claim 2, characterized in that: The HRP solution is prepared by using HRP enzyme preservation solution to prepare HRP enzyme into 0.1ug-0.3ug / ml, and the HRP enzyme preservation solution is in 0.1M PBS buffer with pH 6.5, containing 0.05-0.2g / L sodium dodecyl sulfate, 10.0-30.0g / L trehalose, 10.0-30.0g / L BSA, 0.1-0.5% mannitol, 5.0-10.0g / L polyethylene glycol, 0.5-1.0ml / L Tween-20, 0.1-0.2g / L MgCl2, and 0.01-0.05g / L ZnCl2.

8. The detection reagent for the microfluidic plate for quantitative detection of urine albumin and urine sugar according to claim 2, characterized in that: The reaction matrix liquid comprises 5.0-10.0 g / L citric acid, 35.0-60.0 g / L sodium acetate, and 5-20 ml / L Triton.

9. The detection reagent for the microfluidic plate for quantitative detection of urine albumin and urine glucose according to claim 2, characterized in that: The glucose oxidase solution is prepared with a glucose oxidase preserving solution to prepare glucose oxidase at 5.0-20.0 mg / ml. The formula of the glucose oxidase preserving solution is as follows: 0.1M PBS buffer with a pH of 6.0, 0.1-0.5 mg / L α-cyclodextrin, 1.0-5.0 g / L mannitol, 0.01-0.03 g / L sodium bicarbonate, 0.05-0.2 g / L sodium dodecyl sulfate, 10.0-30.0 g / L trehalose, 0.5-1.0 ml / L Tween-20, and 4.5-9.0 g / L NaCl.

10. A method for using a microfluidic plate for quantitative detection of urine albumin and urine glucose, characterized in that: Using the microfluidic plate for combined quantitative detection of urine albumin and urine sugar according to claims 1 to 2, and the detection reagent for the microfluidic plate for combined quantitative detection of urine albumin and urine sugar according to any one of claims 3 to 9, the method of use comprises: Step 1: Add urine to sample well 1# and sample well 2# respectively; Step 2: The sample in the 1# sample well flows into the 1# reaction pool through the liquid channel, reacts with the coated antibody and HRP labeled antibody in the reaction pool to generate an antigen-antibody complex, and then adds a wash solution to the 1# reaction pool through the liquid inlet hole for cleaning, and discharges the waste liquid into the waste liquid pool; the sample in the 2# sample well flows into the 2# reaction pool through the pre-treated channel, reacts with the glucose oxidase in the reaction pool, and then adds the HRP solution through the liquid inlet hole for reaction; Step 3: Add substrate solution A and substrate solution B to reaction pool #1 through the inlet hole, add reaction matrix solution and substrate solution B to reaction pool #2 through the inlet hole, and detect the fluorescence value after the reaction; Step 4: Using the calibration curve of known concentration, substitute the obtained fluorescence value into the standard curve equation to calculate the concentration of albumin and urine sugar in the measured sample.

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