A special paper chip for angiotensin converting enzyme inhibitors and a detection and analysis method

Abstract

The present invention relates to a paper chip and a detection method for detection and analysis, and the screening process of angiotensin converting enzyme inhibitors is realized on a paper chip. Angiotensin converting enzyme can decompose the substrate hippurylglycylglycine (HGG) into hippuric acid and glycine dipeptide, and glycine dipeptide can react with ninhydrin to produce a purple complex, and the color intensity is proportional to the enzyme activity, including 10 major steps. Compared with the prior art, the present invention has the advantages of fast detection speed, high accuracy, small sample dosage, low energy consumption, low pollution, low cost, etc., and the paper chip is easy to carry and simple to operate. The prepared 3DμPAD is used for the screening of angiotensin converting enzyme inhibitors without special sample pretreatment, and can be directly quantitatively analyzed. It can be used for the activity screening of enzyme inhibitor drugs, and has broad application prospects in the fields of clinical diagnosis, food quality control and environmental monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis, and relates to a paper chip and a detection method for detection and analysis, and in particular to a 3D microfluidic paper chip and a detection and analysis method for screening angiotensin converting enzyme inhibitors. Background Art

[0002] Angiotensin converting enzyme (ACE) is also known as kininase II or peptidyl-carboxypeptidase. Studies have shown that ACE can catalyze the hydrolysis of angiotensin I (decapeptide) into angiotensin II, an octapeptide, which further constricts blood vessels and increases blood pressure. Therefore, ACE inhibitors (ACEI) are widely used to treat hypertension, heart failure, myocardial infarction and other diseases. Since the various reference values ​​for ACE activity determination are different and related to age, gender and serum angiotensin converting enzyme (SACE), there is still controversy at home and abroad. At present, the commonly used methods for detecting ACE activity include spectrophotometry, fluorescence photometry, high performance liquid chromatography, enzyme coupling method, capillary electrophoresis, etc. The spectrophotometric method requires the use of ethyl acetate to extract the product hippuric acid, which is a cumbersome procedure; fluorescence spectrophotometry, HPLC and capillary electrophoresis have high requirements for instruments and equipment and are difficult to use in general laboratories; the enzyme coupling method is suitable for manual operation, but the substrate hippuric acid diglycine has the same ultraviolet absorption peak as the enzyme reaction product hippuric acid, and turbidity is easily produced when blood lipids are high.

[0003] Microfluidic paper chips, also known as microfluidic paper-based analytical devices (μPAD), were first proposed by the Whitesides group in 2007. They are another powerful separation and analysis method after HPLC and capillary electrophoresis (CE). They use paper as a substrate instead of glass, silicon, polymers and other materials. Through various processing technologies, hydrophilic / hydrophobic microchannel networks and related analytical devices with a certain structure are processed on paper to build a "paper micro-laboratory" (lab-on-paper). In addition to the characteristics of traditional microfluidic chips such as small sample usage and rapid analysis. The detection methods in μPAD analysis mainly include colorimetric detection, electrochemical detection, chemiluminescence detection and electrochemiluminescence detection. Among them, the colorimetric method does not require special instruments and equipment and is the most commonly used detection method in paper chip analysis. It mainly converts the component to be detected into a colored substance (or a colorless substance) through a color reaction (or the disappearance of color after the reaction), and uses the correlation between color intensity and the concentration of the component to be detected. It uses a digital camera or smart phone to take pictures and a scanner to scan them into images, and then uses image processing software such as Photoshop to process them, and conducts quantitative analysis based on the relationship between the image color intensity and the concentration of the substance to be detected.

[0004] Therefore, the present invention designs and manufactures a special 3D μPAD, and establishes a new rapid screening platform for natural antihypertensive active ingredients by selecting the corresponding color development system. Using ACE as a model and hippuryl-glycyl-glycine (HGG) as a substrate, the half-inhibitory concentration (IC50 value) of the drug is determined by the paper chip method, and its inhibitory activity is evaluated. So far, there are no related reports on the paper chip used in the present invention and the method for screening angiotensin converting enzyme inhibitors. Summary of the invention

[0005] In view of the above-mentioned prior art conditions, the purpose of the present invention is first to provide a paper chip specifically used for screening angiotensin converting enzyme inhibitors. Another purpose of the present invention is to provide a method for screening angiotensin converting enzyme inhibitors using the paper chip.

[0006] The present invention and technical solutions are described as follows:

[0007] The basic concept of the present invention is to design a paper chip specifically for the screening of angiotensin converting enzyme inhibitors by taking advantage of the low cost, simple preparation, and no need for complex peripheral equipment of paper chips, which can perform rapid, cheap, and convenient detection and analysis in a truly sense. The present invention implements the screening process of angiotensin converting enzyme inhibitors on a paper chip, based on the fact that angiotensin converting enzyme can decompose the substrate hippurylglycylglycine (HGG) into hippuric acid and glycine dipeptide, and glycine dipeptide can react with ninhydrin to produce a purple complex, and the color intensity is proportional to the enzyme activity. After adding the Chinese medicine extract, the enzyme activity is inhibited and the color intensity is reduced. The degree of color change is analyzed by Photoshop software to evaluate the drug inhibitory activity; 3D μPAD system detection process (attached Figure 1) is based on the strong capillary effect of cellulose paper itself. By folding, the color developer ninhydrin on the second layer of paper chip (2nd layer) is evenly loaded to the color development area loaded on the first layer of paper chip (1st layer). After the substrate and enzyme react for a certain period of time, they are loaded on the color development area of ​​the 1st layer; they are folded and pressed along the folding line, and the enzymatic hydrolysis products contact and react with ninhydrin. After the 3D μPAD is placed on a heating plate at 65℃ for 2 minutes, the experimental results are recorded by mobile phone (on the back of the 1st layer), and the RGB value is analyzed by Photoshop software. The stronger the drug inhibitory activity, the more significant the reduction of ACE activity, the less the content of hydrolysis products in the system, and the lower the RGB value (means value of superposition analysis of red, green and blue channels); conversely, the higher the RGB value; according to the inhibition rate of the gradient concentration drug on the enzymatic hydrolysis reaction, the IC50 value of each drug can be obtained. The μPAD platform can parallelly measure the inhibitory effects of multiple concentration gradient drugs at one time, and the IC50 value of the drug can be obtained in one operation. The present invention uses human blank serum as the source of ACE, and the content of cysteine ​​and the like is very small, so there is no interference with the detection of diGly peptide.

[0008] The invention discloses a paper chip specially used for screening angiotensin converting enzyme inhibitors, which is characterized in that the paper chip consists of two layers of chromatography paper; a parallel circular reaction area is designed on the first layer of chromatography paper; a liquid flow channel is designed at a position of the second layer of chromatography paper corresponding to the reaction area of ​​the first layer; the chromatography paper is black and printed with black wax, the circular sample loading area and the liquid flow channel are hydrophilic areas, and the rest are hydrophobic areas; the diameter of the circular reaction area is 0.60 cm to 1.0 cm; the liquid flow channel is 0.8 cm to 1.2 cm long and 0.25 cm to 0.45 cm wide.

[0009] The present invention also provides a method for screening angiotensin converting enzyme inhibitors using the paper chip, which is characterized by comprising the following steps:

[0010] Step 1: using a wax jet printer to print a paper chip specifically used for screening angiotensin converting enzyme inhibitors according to the design of the present invention, and heating it with a magnetic semi-automatic hot press to melt the solid paraffin and completely penetrate the paper to form a hydrophobic block or dam;

[0011] Step 2: Before testing, prepare HGG stock solution, ninhydrin solution, HCl solution, captopril stock solution, gastrodin stock solution, acetyl gastrodin stock solution and glycine dipeptide stock solution;

[0012] Step 3: Investigate the effects of sample loading, reaction time, color development time, substrate concentration, color developer concentration, and substrate terminator on the reaction results to obtain the optimal reaction conditions;

[0013] Step 4: Make a standard working curve;

[0014] Step 5: Determination of minimum detection limit;

[0015] Step 6: Repeat the experiment

[0016] Step 7: Determination of the inhibition of ACE activity by captopril;

[0017] Step 8: Determination of the inhibitory effect of gastrodin on ACE activity;

[0018] Step 9: Determination of the inhibition of ACE activity by acetyl gastrodin;

[0019] Step 10: Detect the inhibition of ACE by different drugs using UV spectrophotometry.

[0020] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: combining steps 1, 2, and 3: detecting under the conditions of reaction substrate loading amount 2-6 μL, color developer loading amount 30-50 μL, reaction time 10-50 min, color development time 30-150 s, substrate concentration 6-30 mmol / L, color developer concentration 0.2%-1.0% and reaction terminator 1-9 μL, respectively, to obtain the optimal detection conditions.

[0021] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "preparation of a standard working curve" in step 4 is specifically as follows:

[0022] Take five centrifuge tubes and add different concentrations of glycine dipeptide solution (5, 9, 13, 17, 21×10 -3 mol / L) 10μL, serum 10μL, 1× PBS buffer solution 25μL, HCl (1mmol / L) 5μL, after mixing, take 4μL of the mixture and add it to the 1st layer color development area of ​​the paper chip, add 50μL of ninhydrin (8mg / mL) solution to the 2nd layer loading area, fold and press, heat at 65℃ for 2min to develop color, take pictures and analyze the RGB values ​​of glycine dipeptide of different concentrations, and plot the RGB value against the glycine dipeptide concentration to obtain the standard working curve.

[0023] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "minimum detection limit test" described in step 5 is specifically as follows:

[0024] The glycine dipeptide solution was continuously diluted for color development until the difference between RGB and the blank value reached 10, and the glycine dipeptide concentration corresponding to the glycine dipeptide concentration detection limit was obtained.

[0025] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "repetitive experiment" described in step 6 is specifically as follows:

[0026] 10 μL serum and 5 μL captopril solution (100.46 nmol / L) were mixed in 20 μL PBS buffer solution, 10 μL substrate solution (24 mmol / L) was added to react for 20 min, and then 5 μL HCl (1 mmol / L) was added to terminate the reaction. 4 μL of the reaction solution was added to the 1st layer color development area of ​​the paper chip, and 50 μL ninhydrin (8 mg / mL) solution was added to the 2nd layer loading area. The paper chip was folded and pressed tightly, and heated at 65°C for 2 min to develop color and examine the repeatability. Repeat five times to examine the repeatability of the activity inhibition of gastrodin (2.48 mmol / L) and acetyl gastrodin (2.56 mmol / L).

[0027] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "captopril inhibition of ACE activity determination" in step 7 is specifically as follows:

[0028] 5 μL of different concentrations (1.0046×10 -1 μmol / L, 1.0046×10 -2 μmol / L, 1.0046×10 -3 μmol / L, 1.0046×10 -4 μmol / L, 1.0046×10 -5 Captopril solution (24 μmol / L) was mixed with 10 μL serum in 20 μL PBS buffer solution, 10 μL substrate solution (24 mmol / L) was added and reacted for 20 min, then 5 μL HCl (1 mmol / L) was added to terminate the reaction, 4 μL of reaction solution was added to the 1st layer color development area of ​​the paper chip, 50 μL ninhydrin (8 mg / mL) solution was added to the 2nd layer loading area, the paper chip was folded and pressed, heated at 65℃ for 2 min for color development, the RGB values ​​of captopril at different concentrations were photographed and analyzed, and the captopril concentration was nonlinearly fitted with the inhibition rate to obtain the IC50 value of captopril inhibition on ACE.

[0029] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "determination of the inhibition of ACE activity by gastrodin" in step 8 is specifically as follows:

[0030] Prepare 2.48×10 -2 mol / L,2.48×10 -3mol / L,2.48×10 -4 mol / L,2.48×10 -5 mol / L,2.48×10 -6 mol / L gastrodin solution was mixed with 10μL serum in 20μL PBS buffer solution, 10μL substrate solution (24 mmol / L) was added and reacted for 20min, then 5μL HCl (1mmol / L) was added to terminate the reaction, 4μL of reaction solution was added to the 1st layer color development area of ​​the paper chip, 50μL ninhydrin (8mg / mL) solution was added to the 2nd layer loading area, the paper chip was folded and pressed, heated at 65℃ for 2min for color development, the RGB values ​​of gastrodin solutions with different concentrations were photographed and analyzed, the inhibition rate was used to perform nonlinear fitting on the gastrodin concentration, and the IC50 value of gastrodin for ACE inhibition was obtained.

[0031] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "determination of ACE activity inhibition by acetyl gastrodin" in step 9 is specifically as follows:

[0032] Prepare 2.56×10 -2 mol / L,2.56×10 -3 mol / L,2.56×10 -4 mol / L,2.56×10 -5 mol / L,2.56×10 -6 mol / L acetyl gastrodin solution was mixed with 10 μL serum in 20 μL PBS buffer solution, 10 μL substrate solution (24 mmol / L) was added and reacted for 20 min, then 5 μL HCl (1 mmol / L) was added to terminate the reaction, 4 μL of the reaction solution was added to the 1st layer color development area of ​​the paper chip, 50 μL ninhydrin (8 mg / mL) solution was added to the 2nd layer loading area, the chips were folded and pressed, heated at 65°C for 2 min for color development, the RGB values ​​of acetyl gastrodin solutions of different concentrations were photographed and analyzed, the acetyl gastrodin concentration was nonlinearly fitted with the inhibition rate, and the IC50 value of acetyl gastrodin inhibition on ACE was obtained.

[0033] The present invention further provides a method for screening angiotensin converting enzyme inhibitors using 3D μPAD, characterized in that: the "ultraviolet spectrophotometry to detect the inhibition of ACE by different drugs" in step 10 is as follows:

[0034] 25 μL of different concentrations (2.48×10 --2 mol / L,2.48×10 -3 mol / L,2.48×10 -4mol / L,2.48×10 -5 mol / L,2.48×10 -6 mol / L) of gastrodin solution and 50μL of serum were mixed in 100μL PBS buffer solution, 50μL of substrate solution (24 mmol / L) was added, reacted for 20min, and then 25μL of HCl (1mmol / L) was added to terminate the reaction, 200μL of ninhydrin solution (8 mg / mL) was added for color development and then diluted, and the absorbance value was measured at 568nm. The inhibition rate was fitted to the concentration of gastrodin to obtain the IC value of gastrodin for ACE inhibition by UV method. 50 Value; Preparation 2.56×10 -2 mol / L,2.56×10 -3 mol / L,2.56×10 -4 mol / L,2.56×10 -5 mol / L,2.56×10 -6 mol / L acetyl gastrodin solution, the rest of the operation is the same as gastrodin; prepare 1.0046×10 -1 μmol / L, 1.0046×10 -2 μmol / L, 1.0046×10 -3 μmol / L, 1.0046×10 -4 μmol / L, 1.0046×10 -5 μmol / L captopril solution, and the rest of the operation is the same as that of gastrodin.

[0035] Compared with the prior art, the present invention has the advantages of fast detection speed, high accuracy, small sample dosage, low energy consumption, low pollution, low cost, etc., and the paper chip is easy to carry and simple to operate. The prepared 3D μPAD does not require special sample pretreatment for the screening of angiotensin-converting enzyme inhibitors, and can be directly used for quantitative analysis. It can be used for the activity screening of enzyme inhibitor drugs, and has broad application prospects in the fields of clinical diagnosis, food quality control, and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :Color reaction principle detection flow chart

[0037] Figure 2 :Paper chip appearance DETAILED DESCRIPTION

[0038] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0039] Embodiment 1:

[0040] See also Figure 1, combined with step 1: use Inkscape 0.91 software to design the shape and channels of the paper chip, print it on A4-sized Whatman grade 1 chromatography paper using a Xerox Phaser 8570 wax jet printer, heat it at 176.7℃ for 105 s on a magnetic semi-automatic hot press to melt the solid paraffin and completely penetrate the paper to form a hydrophobic block (or dam), and then place it on the first layer of chromatography paper (1 st Parallel circular reaction areas (diameter 0.80 cm) were designed on the first layer (2 layer) and loaded with substrate hippurylglycylglycine (HGG) and angiotensin converting enzyme (ACE) solution. nd layer), and the first layer (1 st The liquid flow channel (length 1.00 cm, width 0.35 cm) is designed at the position corresponding to the reaction area of ​​1 layer. st layer chromatography paper is printed with black wax;

[0041] Embodiment 2:

[0042] Combined with step 2: Before the test, prepare HGG stock solution (30 mmol / L), ninhydrin solution (10 mg / mL), HCl solution (1 mmol / L), captopril stock solution (1.0046×10 -3 mol / L), gastrodin mother solution (2.48×10 -2 mol / L), acetyl gastrodin mother solution (2.56×10 -2 mol / L) and glycine dipeptide stock solution (6.0×10 -2 mol / L);

[0043] Embodiment 3:

[0044] Combined with step 3:

[0045] Sample loading: 10 μL substrate solution (30 mmol / L) and 10 μL serum were mixed in 20 μL PBS buffer solution. After 20 min of reaction, 10 μL HCl (1 mmol / L) was added to terminate the reaction. 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL of the reaction solution were respectively added to the paper chip 1. st layer color area, 2 ndAdd 50μL of ninhydrin (10 mg / mL) solution to the layer loading area, fold and press tightly, and fully develop the color. When the reaction solution is 2-6μL, the color development area can be covered, and the color intensity increases with the increase of the amount of reaction solution loaded. However, when more than 4μL is loaded and the solution is easy to be lost during the folding process, it affects the stability of the experiment. Add 30μL, 35μL, 40μL, 45μL, and 50μL of ninhydrin (10 mg / mL) solution to the loading area, respectively. As the amount of ninhydrin loaded increases, the loading area is gradually filled, and 50μL just fills the entire color development area. Therefore, this study determined that the optimal loading amounts of the reaction solution and the color developer are 4μL and 50μL, respectively.

[0046] Investigation of reaction time: Take five centrifuge tubes, number them 1-5, add 20μL PBS buffer solution, 10μL substrate solution (30 mmol / L) and 10μL serum into the centrifuge tubes, react for 10min, 20min, 30min, 40min, 50min respectively. After the reaction time is reached, add 10μL HCl (1mmol / L) to terminate the reaction. Take 4μL of the reaction solution from each of the five centrifuge tubes and add it to the paper chip 1. st layer color area, 2 nd 50 μL of ninhydrin (10 mg / mL) solution was added to the layer loading area, folded and pressed, fully colored, and photographed and analyzed the mean values ​​of the RGB channels at different reaction times (hereinafter referred to as RGB values). The results showed that when the reaction time was 20 min, the activity of ACE enzyme in human serum was good, and the color intensity increased slightly after 20 min. However, based on the principles of economy and high efficiency, the present invention selected the optimal reaction time of 20 min;

[0047] Study on color development time: 10 μL substrate solution (30 mmol / L) and 10 μL serum were mixed in 20 μL PBS buffer solution. After 20 min of reaction, 10 μL HCl (1 mmol / L) was added to terminate the reaction. 4 μL of the reaction solution was added to the paper chip 1. st layer color area, 2 nd 50 μL of ninhydrin (10 mg / mL) solution was added to the layer loading area, folded and pressed, heated on a 65°C heating plate, photographed every 30 seconds, and analyzed the RGB values ​​at different color development times. The results showed that under the 65°C heating condition, the color development intensity increased with time, reached the maximum at 2 minutes, and then tended to be stable. Therefore, the present invention adopts 65°C heating for 2 minutes for color development;

[0048] Investigation of substrate concentration: Take the substrate mother solution and dilute it with substrate buffer solution to prepare 6, 12, 18, 24, and 30 mmol / L substrate solutions; Take five centrifuge tubes, numbered 1-5, add 10 μL of substrate solutions of different concentrations and 10 μL of serum to 20 μL PBS buffer solution, react for 20 minutes, add 10 μL HCl (1 mmol / L) to terminate the reaction, and take 4 μL of the reaction solution from each of the five centrifuge tubes and add it to paper chip 1 st layer color area, 2 nd 50 μL of ninhydrin (10 mg / mL) solution was added to the layer loading area, folded and pressed, heated at 65°C for 2 min to develop color, and the RGB values ​​of different substrate concentrations were photographed and analyzed. The results showed that with the increase of substrate concentration, the color intensity gradually increased, and when the substrate was 24 mmol / L, the color intensity reached the maximum; the substrate concentration continued to increase, and the color intensity remained basically unchanged, so the substrate concentration used in the present invention was 24 mmol / L;

[0049] Study on the concentration of the colorimetric reagent: 20 μL of substrate solution (24 mmol / L) and 20 μL of serum were mixed in 40 μL of PBS buffer solution. After reacting for 20 min, 20 μL of HCl (1 mmol / L) was added to terminate the reaction. 50 μL of the reaction solution was drawn and added to the paper chip 2. nd layer loading area, 1 st 4 μL of ninhydrin solution of different concentrations was added to the color development area of ​​the layer, folded and pressed, heated at 65°C for 2 min, and the RGB values ​​of different color developer concentrations were photographed and analyzed. The results showed that as the concentration of the color developer increased, the color intensity increased accordingly, reaching the maximum at a concentration of 0.8%, and then tended to be stable. Therefore, the optimal concentration of the ninhydrin solution in the present invention is 0.8%;

[0050] Investigation of the amount of reaction terminator added: Take 5 centrifuge tubes, number 1-5, mix 10μL substrate solution (30mmol / L) with 10μL serum, react for 20 min, add 1μL, 3μL, 5μL, 7μL, 9μL HCl (1mmol / L) to terminate the reaction, and take 4μL of the reaction solution from each tube and add it to paper chip 1 st layer color area, 2 nd 50 μL of ninhydrin (8 mg / mL) solution was added to the layer loading area, folded and pressed, heated on a 65°C heating plate, and photographed and analyzed the RGB values ​​of different amounts of reaction terminators. The results showed that as the volume of terminator HCl added increased, the ACE activity gradually decreased, the amount of enzymatic hydrolysis anti-products gradually decreased, and the color intensity gradually weakened; when the amount of HCl added was 5 μL, the color intensity was the smallest, and then remained basically constant, so the present invention adopted the addition of 5 μL HCl (1 mmol / L) to terminate the enzymatic hydrolysis reaction;

[0051] Embodiment 4:

[0052] Combined with step 4:

[0053] Before the test, a glycine dipeptide standard curve was prepared; different concentrations of glycine dipeptide solutions (5, 9, 13, 17, 21×10 -3 mol / L) 10μL, reacted with the color developer, and plotted the RGB value against the glycine dipeptide standard solution. The results showed that the color intensity increased with the increase of the glycine dipeptide solution concentration. The linear fitting of the RGB value against the glycine dipeptide solution concentration was obtained, and the linear regression equation y =1.5223 x + 107.32, its linear correlation coefficient R 2 It is 0.9981, indicating that the linear relationship is good in the concentration range of 4 to 21 mmom / L;

[0054] Embodiment 5:

[0055] Step 5: Determination of minimum detection limit: Continuously dilute the glycine dipeptide solution for color development until the RGB value differs from the blank (74.25) by 10, and the detection limit is 0.001 mmol / L;

[0056] Embodiment 6:

[0057] Combined with step 6: Repeatable experiment

[0058] Mix 10 μL of serum and 5 μL of captopril solution (100.46 nmol / L) in 20 μL of PBS buffer solution, add 10 μL of substrate solution (24 mmol / L) and react for 20 min, then add 5 μL of HCl (1 mmol / L) to terminate the reaction. Pipette 4 μL of the reaction solution and add it to paper chip 1. st layer color area, 2 nd 50 μL of ninhydrin (8 mg / mL) solution was added to the layer loading area, folded and pressed, and heated at 65°C for 2 min to develop color. The method for gastrodin solution (2.48 mmol / L) and acetyl gastrodin solution (2.56 mmol / L) was the same as that for captopril. The results showed that the relative standard deviations (RSDs) of captopril, gastrodin and acetyl gastrodin on ACE inhibition were 1.35%, 3.02% and 1.78%, respectively (n = 5).

[0059] Embodiment 7:

[0060] Combined with step 7: Determination of the inhibition of ACE activity by captopril

[0061] 5 μL of different concentrations (1.0046×10 -1μmol / L, 1.0046×10 -2 μmol / L, 1.0046×10 -3 μmol / L, 1.0046×10 -4 μmol / L, 1.0046×10 -5 The captopril solution of 1 μmol / L (24 mmol / L) was mixed with 10 μL serum in 20 μL PBS buffer solution, and 10 μL substrate solution (24 mmol / L) was added to react for 20 min, and then 5 μL HCl (1 mmol / L) was added to terminate the reaction. 4 μL of the reaction solution was added to the 1st layer color development area of ​​the paper chip, and 50 μL ninhydrin (8 mg / mL) solution was added to the 2nd layer loading area. The paper chip was folded and pressed, and heated at 65°C for 2 min to develop color. The RGB values ​​of different concentrations of captopril were analyzed by taking pictures, and the captopril concentration was nonlinearly fitted with the inhibition rate. With the increase of the concentration of the captopril solution, the color intensity decreased, indicating the enhancement of ACE activity. The inhibition rate was plotted against the logarithm of the captopril concentration, and it was found that when the HGG concentration was 24 mmol / L, the IC value of captopril for ACE was 0. 50 The value is 1.98 nmol / L, which is within the range of IC 50 The values ​​ranged from 0.75 to 22 nmol / L, indicating that the method was feasible.

[0062] Embodiment 8:

[0063] Combined with step 8: Determination of the inhibitory activity of gastrodin on ACE

[0064] Prepare 2.48×10 -2 mol / L,2.48×10 -3 mol / L,2.48×10 -4 mol / L,2.48×10 -5 mol / L,2.48×10 -6mol / L of gastrodin solution, respectively, and 10μL of serum were mixed in 20μL PBS buffer solution, 10μL of substrate solution (24 mmol / L) was added to react for 20min, and then 5μL HCl (1mmol / L) was added to terminate the reaction, 4μL of the reaction solution was taken and added to the 1st layer color development area of ​​the paper chip, and 50μL of ninhydrin (8mg / mL) solution was added to the 2nd layer loading area, folded and pressed, heated at 65℃ for 2min to develop color, and the RGB values ​​of gastrodin solutions of different concentrations were photographed and analyzed. The gastrodin concentration was nonlinearly fitted with the inhibition rate. The inhibition degree of gastrodin solutions of different concentrations on ACE also changed with the color intensity on the paper chip. As the concentration of gastrodin increased, the inhibitory effect on ACE also increased, and the color gradually became lighter. It was concluded that when the HGG concentration was 24 mmol / L, the IC of gastrodin on ACE was 2. 50 The value is 506.12 μmol / L.

[0065] Embodiment 9:

[0066] Combined with step 9: Determination of ACE activity inhibition by acetyl gastrodin

[0067] Prepare 2.56×10 -2 mol / L,2.56×10 -3 mol / L,2.56×10 -4 mol / L,2.56×10 -5 mol / L,2.56×10 -6 mol / L acetyl gastrodin solution was mixed with 10μL serum in 20μL PBS buffer solution, 10μL substrate solution (24 mmol / L) was added and reacted for 20min, then 5μL HCl (1mmol / L) was added to terminate the reaction, 4μL of the reaction solution was added to the 1st layer color development area of ​​the paper chip, 50μL ninhydrin (8mg / mL) solution was added to the 2nd layer loading area, the paper chip was folded and pressed, heated at 65℃ for 2min for color development, and the RGB values ​​of acetyl gastrodin solutions of different concentrations were photographed and analyzed. The acetyl gastrodin concentration was nonlinearly fitted with the inhibition rate. With the increase of acetyl gastrodin concentration, the inhibitory effect on ACE also increased, and the color gradually became lighter. When the HGG concentration was 24mmol / L, the IC of acetyl gastrodin on ACE was 2. 50 The value is 304.88μmol / L.

[0068] Embodiment 10:

[0069] Combined with step 10: UV spectrophotometry to detect the inhibition of ACE by different drugs

[0070] 25 μL of different concentrations (2.48×10 --2mol / L,2.48×10 -3 mol / L,2.48×10 -4 mol / L,2.48×10 -5 mol / L,2.48×10 -6 mol / L) of gastrodin solution and 50μL of serum were mixed in 100μL PBS buffer solution, 50μL of substrate solution (24 mmol / L) was added, reacted for 20min, and then 25μL of HCl (1mmol / L) was added to terminate the reaction, 200μL of ninhydrin solution (8 mg / mL) was added for color development and then diluted, and the absorbance value was measured at 568nm. The inhibition rate was fitted to the concentration of gastrodin to obtain the IC value of gastrodin for ACE inhibition by UV method. 50 Value; Preparation 2.56×10 -2 mol / L,2.56×10 -3 mol / L,2.56×10 -4 mol / L,2.56×10 -5 mol / L,2.56×10 -6 mol / L acetyl gastrodin solution, the rest of the operation is the same as gastrodin; prepare 1.0046×10 -1 μmol / L, 1.0046×10 -2 μmol / L, 1.0046×10 -3 μmol / L, 1.0046×10 -4 μmol / L, 1.0046×10 -5 μmol / L captopril solution, and the rest of the operation was the same as that of gastrodin. The absorbance of the blank group at 568 nm was 0.2967, and that of the control group was 0.4939. The inhibition of ACE by ACEI solutions of different concentrations was measured at 568 nm with different absorbance values. When the HGG concentration was 24mmol / L, the IC value of captopril on ACE was 50 The value is 2.08 nmol / L, and the IC of gastrodin to ACE 50 The value is 514μmol / L, and the IC of acetyl gastrodin to ACE 50 The value was 309 μmol / L. Compared with the 3D μPAD method, the relative errors were 4.8%, 1.5%, and 1.3%, respectively. The errors were within 5%, which met the requirements, indicating that the 3D μPAD method can be used for the activity screening of enzyme inhibitor drugs.

Claims

1. A method for detecting and analyzing angiotensin converting enzyme inhibitors using a microfluidic paper chip, characterized in that: A paper chip for screening angiotensin converting enzyme inhibitors is printed out by a wax jet printer, and heated by a magnetic semi-automatic hot stamping machine to melt the solid paraffin and completely penetrate the paper to form a hydrophobic block or dam; the paper chip is composed of two layers of chromatography paper; a parallel circular reaction area is designed on the first layer of chromatography paper; a liquid flow channel is designed at a position corresponding to the first layer of reaction area on the second layer of chromatography paper; the first layer of chromatography paper is black and printed with black wax, the circular sample loading area and the liquid flow channel are hydrophilic areas, and the rest are hydrophobic areas; the diameter of the circular reaction area is 0.60cm to 1.0cm; the liquid flow channel is 0.8cm to 1.2cm long and 0.25cm to 0.45cm wide; The detection and analysis method is to implement the angiotensin converting enzyme inhibitor screening process on a paper chip, based on the angiotensin converting enzyme decomposing the substrate hippurylglycylglycine into hippuric acid and glycine dipeptide, and the glycine dipeptide can react with ninhydrin to produce a purple complex, and the color intensity is proportional to the enzyme activity; after adding the traditional Chinese medicine extract, the enzyme activity is inhibited and the color intensity is reduced, and the degree of color change is analyzed by Photoshop software to evaluate the drug inhibitory activity; Among them, after the substrate and enzyme react for a certain period of time, they are loaded on the color development area of ​​the first layer of paper chip; they are folded in half along the folding line and pressed tightly, and the enzymatic hydrolysis product contacts and reacts with ninhydrin; the microfluidic paper chip is placed on a heating plate at 65°C for heating and color development for 2 minutes, and then a mobile phone photo is taken to record the experimental results on the back of the first layer of paper chip, and Photoshop software is used to analyze the RGB value; the stronger the drug inhibitory activity, the more significant the reduction in angiotensin converting enzyme activity, the less the content of hydrolysis product in the system, the lower the RGB value, and vice versa, the higher the RGB value; the IC50 value of each drug is obtained according to the inhibition rate of the gradient concentration drug on the enzymatic hydrolysis reaction.

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