Amylase activity fluorescence detection method, application and microfluidic detection device

Abstract

The invention relates to an amylase detection method, in particular to a fluorescence detection method and application of amylase activity and a microfluidic detection device.The fluorescence detection method comprises the following steps that a to-be-detected sample is diluted through a buffer solution, the diluted sample and a reaction reagent are fully mixed for a reaction, and a reaction solution is obtained; carrying out fluorescence detection on the reacted mixed solution; calculating the amylase activity based on the measured fluorescence intensity data; the sample to be detected is a sample containing amylase; the reaction reagent comprises starch and a fluorescent probe; the fluorescence intensity data and the starch concentration are in a linear relationship. Compared with the prior art, the defect that a detection result cannot be obtained in real time in the prior art is overcome, and the scheme realizes rapid detection of the activity value of amylase in detected solutions such as blood, urine and peritoneal drainage liquid through a fluorescence detection technology.

Description

Technical Field

[0001] The invention relates to an amylase detection method, and in particular to an amylase activity fluorescence detection method and application thereof and a microfluidic detection device. Background Art

[0002] Pancreatic cancer is one of the common malignant tumors of the digestive tract. It is known as the "king of cancer" in the field of tumors. Its annual incidence rate is almost equal to the annual mortality rate. The five-year survival rate after diagnosis is about 10%, which is one of the malignant tumors with the worst prognosis. The human pancreas is pear-shaped, about the size of a palm, and is located between the stomach and the spine in the abdominal cavity of the human body. The pancreas includes exocrine glands (composed of ducts and small sacs at the end of the ducts called acini. This part of the pancreas produces special proteins called enzymes, which are released into the small intestine to help the body digest and break down food, especially fat) and endocrine glands (composed of cells gathered in different parts of the pancreas, responsible for secreting insulin and various other hormones to help regulate human metabolism). In addition, the pancreas is composed of a head, a body, and a tail. Pancreatic cancer most often originates from the head of the pancreas, often called pancreatic head cancer, and surgical treatment is currently the only possible cure.

[0003] However, some complications such as pancreatic leakage, i.e., abdominal bleeding, are common after pancreatic head cancer surgery, which seriously affect the patient's postoperative survival rate. There are reports that the incidence of pancreatic leakage is 3% to 45%. At present, the means of judging whether a patient has pancreatic leakage after pancreatic head cancer surgery mainly rely on the measurement of amylase activity in the peritoneal drainage fluid. The dry chemical rate method and wet chemical analysis method mainly used by the hospital laboratory often require expensive equipment, complex sample preparation procedures, and time-consuming testing processes. Most importantly, the time from the clinical department submitting the test application, sampling, and testing to the patient receiving the test report (i.e., TAT time, turn-around time) takes at least 2-6 hours, or even longer. However, for diseases with a very short precious window period from the initial onset of symptoms to timely treatment, such as pancreatic leakage, i.e., abdominal bleeding, a common complication after pancreatic head cancer surgery, the longer α-amylase detection TAT time undoubtedly increases the difficulty of treatment and endangers the patient's life. In addition, due to the huge workload of medical staff, it is impossible to provide real-time care for a single patient.

[0004] Therefore, there is an urgent need for a technology or device that can be placed in a hospital bed and can perform rapid, point-of-care testing (POCT) of α-amylase. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the above problems and to provide a fluorescence detection method and application of amylase activity and a microfluidic detection device to solve the deficiency of the prior art that the detection results cannot be obtained in real time. This scheme uses fluorescence detection technology to achieve rapid testing of the activity value of amylase in test solutions such as blood, urine, and peritoneal drainage fluid.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention discloses a method for fluorescent detection of amylase activity, comprising the following steps:

[0008] Diluting the sample to be tested with a buffer solution, fully mixing the diluted sample with a reaction reagent to react, and performing fluorescence detection on the mixed solution after the reaction;

[0009] The amylase activity was calculated based on the measured fluorescence intensity data;

[0010] The sample to be tested is a sample containing amylase;

[0011] The reaction reagents include starch and fluorescent probe;

[0012] There is a linear relationship between the fluorescence intensity data and the starch concentration.

[0013] Preferably, the samples to be tested include blood, urine and peritoneal drainage fluid, and processed blood, urine and peritoneal drainage fluid.

[0014] Preferably, the buffer solution includes physiological saline, PBS buffer solution and potassium chloride aqueous solution.

[0015] Preferably, the fluorescent probe is a fluorescent molecular rotor.

[0016] Preferably, the fluorescent molecular rotor comprises Thioflavin T and 9-(2-carboxy-2-cyanovinyl)julidine, and the wavelength of the excitation light for fluorescence detection is determined according to the fluorescent molecular rotor used.

[0017] Preferably, the amount of the fluorescent molecular rotor added is 40-60 μM; more preferably, the amount of the fluorescent molecular rotor added is 50 μM.

[0018] Preferably, the volume ratio of the diluted sample to the reaction reagent is 1:8-20; more preferably, the volume ratio of the diluted sample to the reaction reagent is 1:10.

[0019] Preferably, the relationship between the fluorescence intensity data and the starch concentration satisfies: y=ax+b, wherein a and b are both real numbers.

[0020] Preferably, the fluorescence intensity data and starch concentration, after taking their logarithms with base 10 respectively, satisfy the following relationship: y=kx+c, wherein k and c are both real numbers.

[0021] The second aspect of the present invention discloses an application of any of the above-mentioned methods for fluorescent detection of amylase activity in the field of medical detection.

[0022] The third aspect of the present invention discloses a microfluidic detection device, which uses any of the above-mentioned fluorescence detection methods for amylase activity for detection;

[0023] The microfluidic detection device comprises a microfluidic system and a fluorescence detector;

[0024] The microfluidic system comprises a first microfluidic mixer and a second microfluidic mixer connected to each other; the first microfluidic mixer is used to mix the sample to be tested with the buffer solution, and the second microfluidic mixer is used to mix the diluted sample flowing out of the first microfluidic mixer with the reaction reagent;

[0025] The fluorescence detector is arranged at the downstream of the microfluidic system and is used for performing fluorescence detection on the mixed solution after the reaction flowing out of the microfluidic system.

[0026] Preferably, the fluorescence detector is a fluorescence spectrophotometer.

[0027] Preferably, the microfluidic detection device further comprises a waste liquid collector, and the waste liquid collector is arranged downstream of the fluorescence detector and is used for collecting the mixed liquid detected by the fluorescence detector.

[0028] The working principle of the present invention is:

[0029] It is known that starch can form a transparent colloidal solution with a certain viscosity when heated in water (containing an appropriate amount of inorganic salts), and the viscosity increases with the increase of starch concentration. The higher the viscosity of the starch colloidal solution, the higher the viscosity. In the presence of certain viscosity-sensitive fluorescent probes (such as fluorescent molecular rotors), the fluorescence intensity of the system is correlated with the viscosity, and the fluorescence intensity can be quantitatively measured by a fluorescence spectrophotometer.

[0030] It is also known that after adding a certain amount of amylase activity to a starch colloid solution, the viscosity of the mixed solution will change over time due to the hydrolysis of starch by amylase. When different groups of starch colloid solutions with the same concentration are added with amylases of different activities, the viscosity of the solution is related to the activity of amylase within a certain reaction time.

[0031] In summary, the activity of amylase can be linked to the fluorescence intensity of the system, and the activity of amylase can be quantitatively detected by measuring the fluorescence intensity.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention aims to propose a method for realizing rapid testing of the activity value of amylase in the tested solutions such as blood, urine, and peritoneal drainage fluid by using fluorescence detection technology. The tested solutions can be continuously diluted, mixed, and sampled by traditional manual methods or by using microfluidic technology. According to the obtained results, a test report can be issued on-site in real time to evaluate the patient's physical condition and help doctors choose treatment or nursing plans. Therefore, the present invention has important applications in the field of medical testing (including but not limited to postoperative monitoring of patients with pancreatic head cancer).

[0034] The present invention proposes a new method for quantitatively detecting the activity of amylase by fluorescence detection. The method innovatively and cleverly utilizes the physical and chemical correlation that "amylase activity" changes "starch concentration" and thus changes "starch viscosity", which ultimately causes the "fluorescence intensity" of the fluorescent molecular rotor in the sample to change. The method of testing the fluorescence of the sample (fluorescent molecular rotors include but are not limited to Thioflavin T, 9-(2-carboxy-2-cyanovinyl) julodidine, etc.) measures the change in the optical signal caused by the change in "fluorescence intensity", thereby quantitatively giving the activity value of the amylase. The method can ensure that the test results are given faster and at a lower cost on the basis of accuracy comparable to that of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a mechanism diagram of the detection method of the present invention;

[0036] Figure 2 Schematic diagram of the structure of the detection device of the present invention, wherein (a) is a manual operation case, and (b) is an automatic operation case;

[0037] Figure 3 The results of fluorescence detection of starch solutions of different concentrations after the reaction in Example 2, wherein (a) is the fluorescence intensity test result, (b) is the relationship between starch concentration and fluorescence intensity, and (c) is the relationship between starch concentration based on 10 and fluorescence intensity based on 10. DETAILED DESCRIPTION

[0038] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] In the following description, unless otherwise specified, the reagents used are conventional commercially available products, and the methods used are well known in the art.

[0040] A fluorescence detection method for amylase activity comprises the following steps:

[0041] Diluting the sample to be tested with a buffer solution, fully mixing the diluted sample with a reaction reagent to react, and performing fluorescence detection on the mixed solution after the reaction;

[0042] The amylase activity was calculated based on the measured fluorescence intensity data;

[0043] The sample to be tested is a sample containing amylase;

[0044] The reaction reagents include starch and fluorescent probe;

[0045] There is a linear relationship between the fluorescence intensity data and the starch concentration.

[0046] in:

[0047] The samples to be tested include blood, urine and peritoneal drainage fluid, as well as processed blood, urine and peritoneal drainage fluid.

[0048] The buffer solution includes physiological saline, PBS buffer solution and potassium chloride aqueous solution.

[0049] The fluorescent probe is a fluorescent molecular rotor, specifically comprising Thioflavin T and 9-(2-carboxyl-2-cyanovinyl) julodidine. The excitation light wavelength of the fluorescence detection is determined according to the fluorescent molecular rotor used.

[0050] The amount of the fluorescent molecular rotor added is 40-60 μM; more preferably, the amount of the fluorescent molecular rotor added is 50 μM.

[0051] The volume ratio of the diluted sample to the reaction reagent is 1:8-20; more preferably, the volume ratio of the diluted sample to the reaction reagent is 1:10.

[0052] The relationship between the fluorescence intensity data and the starch concentration satisfies: y=ax+b, wherein a and b are both real numbers.

[0053] The fluorescence intensity data and starch concentration, after taking the logarithm with base 10 respectively, satisfy the following relationship: y=kx+c, wherein k and c are both real numbers.

[0054] The above relationship is obtained by fitting based on actual measurement results.

[0055] Technical solutions such as Figure 1 As shown:

[0056] It is known that heating starch in water (containing an appropriate amount of inorganic salts) can form a transparent colloidal solution with a certain viscosity, and the higher the starch concentration, the greater the viscosity. The higher the viscosity of the starch colloidal solution, the higher the fluorescence intensity of the system is in the presence of certain viscosity-sensitive fluorescent probes (such as fluorescent molecular rotors), and the fluorescence intensity can be quantitatively measured by a fluorescence spectrophotometer. It is also known that after adding a certain amount of amylase activity to the starch colloidal solution, the viscosity of the mixed solution will change over time because the amylase has a hydrolytic effect on the starch. For different groups of starch colloidal solutions with the same concentration, amylases of different activities are added. Within a certain reaction time, the viscosity of the solution is related to the activity of the amylase.

[0057] In brief, the method of determining amylase activity by fluorescence detection includes that "amylase activity" changes "starch concentration" and thus changes "starch viscosity", which ultimately causes the "fluorescence intensity" of the fluorescent molecular rotor in the sample to change, and uses a method of testing sample fluorescence (fluorescent molecular rotors include but are not limited to Thioflavin T, 9-(2-carboxy-2-cyanovinyl) julidine, etc.) to measure the change in the optical signal caused by the change in "fluorescence intensity", thereby quantitatively giving the amylase activity value.

[0058] In summary, the activity of amylase can be linked to the fluorescence intensity of the system, and the activity of amylase can be quantitatively detected by measuring the fluorescence intensity.

[0059] Detection device such as Figure 2 As shown: It can be done by traditional manual ( Figure 2 (a)) can also be used with a microfluidic system ( Figure 2 (b)) A process for continuously diluting, mixing and sampling the solution to be tested.

[0060] A microfluidic detection device, using the above-mentioned fluorescence detection method of amylase activity for detection;

[0061] The microfluidic detection device comprises a microfluidic system and a fluorescence detector;

[0062] The microfluidic system comprises a first microfluidic mixer and a second microfluidic mixer connected to each other; the first microfluidic mixer is used to mix the sample to be tested with the buffer solution, and the second microfluidic mixer is used to mix the diluted sample flowing out of the first microfluidic mixer with the reaction reagent;

[0063] The fluorescence detector is arranged at the downstream of the microfluidic system and is used for performing fluorescence detection on the mixed solution after the reaction flowing out of the microfluidic system.

[0064] Preferably, the fluorescence detector is a fluorescence spectrophotometer.

[0065] Preferably, the microfluidic detection device further comprises a waste liquid collector, and the waste liquid collector is arranged downstream of the fluorescence detector and is used for collecting the mixed liquid detected by the fluorescence detector.

[0066] Specifically, the sample to be tested (including but not limited to blood, urine, peritoneal drainage fluid, etc. and samples thereof that have undergone various treatments) is diluted with a buffer solution (including but not limited to physiological saline, PBS buffer solution, potassium chloride aqueous solution with a certain concentration (determined according to test needs), etc.) (the dilution multiple includes but is not limited to dozens of times or even hundreds of times); the diluted sample to be tested is fully mixed with a reaction reagent (the reaction reagent contains starch and one or two viscosity-sensitive fluorescent molecular rotors, including but not limited to Thioflavin T, 9-(2-carboxy-2-cyanovinyl) julidine, etc.) for a certain time, and the reaction condition is room temperature and can also be adjusted to 37° C. by a heating device; finally, the sample to be tested after a certain time of reaction is transferred to a fluorescence detector through traditional manual or microfluidic system pipelines, and under the action of the corresponding wavelength excitation light of the fluorescent molecular rotor used, the fluorescence detector detects the fluorescence signal of the corresponding wavelength, and finally the test result is given after data processing.

[0067] Example 1

[0068] A microfluidic detection device, such as Figure 2 (b), the amylase activity was detected by the fluorescence detection method as described above;

[0069] The microfluidic detection device comprises a microfluidic system and a fluorescence detector;

[0070] The microfluidic system comprises a first microfluidic mixer and a second microfluidic mixer connected to each other; the first microfluidic mixer is used to mix the sample to be tested with the buffer solution, and the second microfluidic mixer is used to mix the diluted sample flowing out of the first microfluidic mixer with the reaction reagent;

[0071] The fluorescence detector is arranged at the downstream of the microfluidic system and is used for performing fluorescence detection on the mixed solution after the reaction flowing out of the microfluidic system.

[0072] The microfluidic detection device consists of a microfluidic system and a fluorescence detector.

[0073] The microfluidic system is used to sequentially mix the sample to be tested, the buffer solution and the reaction reagent to achieve automated, controllable dilution, mixing and sample feeding. The microfluidic system may further include three microfluidic channels, whose input ends are sequentially used to input the sample to be tested, the buffer solution and the reaction reagent (a peristaltic pump or a metering pump or other conveying device may be connected to the microfluidic channel to provide a stable and controllable input), and the two microfluidic channels for inputting the sample to be tested and the buffer solution are first connected at the ends to mix the sample to be tested and the buffer solution to dilute the sample to be tested (the dilution multiple is controlled by controlling the input flow ratio), and then the microfluidic channel is connected to the microfluidic channel for inputting the reaction reagent to further mix and react. Since the microfluidic channel is used for input and mixing, the mixing effect is excellent, and rapid mixing and sufficient reaction can be achieved.

[0074] The fluorescence detector uses a fluorescence spectrophotometer to detect the fluorescence intensity of the liquid flow supplied by the microfluidic system.

[0075] A waste liquid collector is also provided downstream of the fluorescence detector to collect the detected waste liquid and subsequently process it in a centralized manner, thereby avoiding contamination caused by medical waste.

[0076] Example 2

[0077] A fluorescent detection method for amylase activity, such as Figure 1 As shown, the following steps are included:

[0078] Diluting the sample to be tested with a buffer solution, fully mixing the diluted sample with a reaction reagent to react, and performing fluorescence detection on the mixed solution after the reaction;

[0079] The amylase activity was calculated based on the measured fluorescence intensity data;

[0080] The sample to be tested is a sample containing amylase;

[0081] The reaction reagents include starch and fluorescent probe;

[0082] There is a linear relationship between the fluorescence intensity data and the starch concentration.

[0083] More specifically, in this embodiment:

[0084] The microfluidic detection device proposed in Example 1 was used for detection.

[0085] The sample to be tested (peritoneal drainage fluid) is diluted with a buffer solution (PBS buffer solution) (the dilution factor is 50 times); the diluted sample to be tested is fully mixed with a reaction reagent (the reaction reagent contains starch and a fluorescent molecular rotor, wherein the fluorescent molecular rotor is: Thioflavin T, 50 μM) (the volume ratio between the diluted sample to be tested and the reaction reagent is 1:10) and reacted for a certain time at room temperature; finally, the sample to be tested after a certain reaction time is transferred to a fluorescence detector for fluorescence intensity detection.

[0086] like Figure 3 As shown in (a)-3(c), the samples with different starch concentrations (0, 1, 2, 3, 4, 5 wt%) were excited with an excitation light of 423 nm, and the fluorescence was obtained at about 487 nm ( Figure 3 (a)). According to Figure 3 (b) and 3(c), the starch concentration value and fluorescence intensity and their logarithms with base 10 have a good linear fitting relationship, indicating that the fluorescence intensity measured by the fluorescence detector is linearly related to the starch concentration value, and the starch concentration directly affects the solution viscosity. The relationship between fluorescence intensity and starch concentration: y = 21666x + 30052, R 2 = 0.9484; take the logarithm of the fluorescence intensity with base 10 and the starch concentration: y = 0.5466x + 4.7413, R 2 =0.9838.

[0087] In addition, the rate of starch decomposition by amylase is constant within the same time (other conditions are the same, such as temperature, humidity, stirring, etc.), and the enzyme decomposition of starch affects the viscosity. Therefore, the fluorescence intensity measured by the fluorescence detector is linearly related to the activity of amylase.

[0088] In summary, the activity value of the inferred amylase can be quantified by fluorescence detection method.

[0089] Example 3

[0090] This embodiment is basically the same as embodiment 2, except that: the sample to be tested is blood, the buffer solution is physiological saline, the dilution factor is 100 times, and the fluorescent molecular rotor is 9-(2-carboxy-2-cyanovinyl) julodidine.

[0091] Example 4

[0092] This embodiment is basically the same as embodiment 2, except that the sample to be tested is urine, the buffer solution is a potassium chloride aqueous solution, and the dilution multiple is 200 times.

[0093] Example 5

[0094] This embodiment is basically the same as embodiment 2, except that the fluorescent molecular rotor is a combination of 9-(2-carboxyl-2-cyanovinyl)julidine and thioflavin T, and the reaction is carried out at a controlled temperature of 37°C.

[0095] In summary, the present invention aims to propose a method for rapidly testing the activity value of amylase in tested solutions such as blood, urine, and peritoneal drainage fluid using fluorescence detection technology. The tested solution can be continuously diluted, mixed, and sampled by traditional manual methods or by using microfluidic technology. Based on the obtained results, a test report can be issued on-site in real time to evaluate the patient's physical condition and help doctors choose treatment or nursing plans. Therefore, the present invention has important applications in the field of medical testing (including but not limited to postoperative monitoring of patients with pancreatic head cancer).

[0096] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. A fluorescence detection method for amylase activity, characterized in that: The steps include: Diluting the sample to be tested with a buffer solution, fully mixing the diluted sample with a reaction reagent to react, and performing fluorescence detection on the mixed solution after the reaction; The amylase activity was calculated based on the measured fluorescence intensity data; The sample to be tested is a sample containing amylase; The reaction reagents include starch and fluorescent probe; There is a linear relationship between the fluorescence intensity data and the starch concentration.

2. The method for fluorescent detection of amylase activity according to claim 1, characterized in that: The samples to be tested include blood, urine and peritoneal drainage fluid, as well as processed blood, urine and peritoneal drainage fluid.

3. The method for fluorescent detection of amylase activity according to claim 1, characterized in that: The buffer solution includes physiological saline, PBS buffer solution and potassium chloride aqueous solution.

4. The method for fluorescent detection of amylase activity according to claim 1, characterized in that: The fluorescent probe is a fluorescent molecular rotor.

5. The method for fluorescent detection of amylase activity according to claim 4, characterized in that: The fluorescent molecular rotor comprises thioflavin T and 9-(2-carboxyl-2-cyanovinyl) julodidine.

6. The method for fluorescent detection of amylase activity according to claim 1, characterized in that: The relationship between the fluorescence intensity data and the starch concentration satisfies: y=ax+b, wherein a and b are both real numbers.

7. The method for fluorescent detection of amylase activity according to claim 1, characterized in that: The fluorescence intensity data and starch concentration, after taking the logarithm with base 10 respectively, satisfy the following relationship: y=kx+c, wherein k and c are both real numbers.

8. Use of the fluorescent detection method for amylase activity according to any one of claims 1 to 7 in the field of medical detection.

9. A microfluidic detection device, characterized in that: Detection is performed using the fluorescence detection method for amylase activity as described in any one of claims 1 to 7; The microfluidic detection device comprises a microfluidic system and a fluorescence detector; The microfluidic system comprises a first microfluidic mixer and a second microfluidic mixer connected to each other; the first microfluidic mixer is used to mix the sample to be tested with the buffer solution, and the second microfluidic mixer is used to mix the diluted sample flowing out of the first microfluidic mixer with the reaction reagent; The fluorescence detector is arranged at the downstream of the microfluidic system and is used for performing fluorescence detection on the mixed solution after the reaction flowing out of the microfluidic system.

10. A microfluidic detection device according to claim 9, characterized in that: The microfluidic detection device further comprises a waste liquid collector, which is arranged downstream of the fluorescence detector and is used to collect the mixed liquid detected by the fluorescence detector.

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