Multichannel microfluidic chip and detection device for creatinine and uric acid detection

Abstract

This invention discloses a multi-channel microfluidic chip and device for detecting creatinine and uric acid. The invention is based on the principle that the H2O2 produced by the reaction of creatinine and creatinine hydrolase, and the H2O2 produced by the reaction of uric acid and uricase, rapidly quench the fluorescence of the aerogel. Sensitive detection of urinary creatinine and uric acid is achieved by detecting the fluorescence intensity. Because the sensor uses paper-based fiber material as a substrate and introduces quantum dot nanomaterials as sensitive units to construct a three-dimensional aerogel structure, it has characteristics such as a large specific surface area, providing more active sites for the adsorption of biological enzymes, thereby increasing the adsorption capacity. Simultaneously, the presence of a large amount of glutathione on the surface of the quantum dots constituting the aerogel provides good biocompatibility, which is beneficial for maintaining enzyme activity, thus improving the sensitivity and stability of the detection.

Description

Technical Field

[0001] This invention belongs to the field of creatinine / uric acid detection and relates to a glucose photoelectrochemical sensor and its preparation method. Background Technology

[0002] Currently, there is no cure for chronic kidney disease. Patients with chronic kidney disease can only maintain normal levels of various endocrine substances such as creatinine, uric acid, protein, and bilirubin through medication to ensure the health and normal functioning of their organs. Patients with chronic mild nephritis need to be especially careful, as nephritis can easily progress to kidney failure or even uremia. The most important characteristic indicators of kidney disease are creatinine, uric acid, and urinary protein levels; therefore, daily monitoring of creatinine and uric acid levels is essential and meaningful for patients with chronic kidney disease.

[0003] To date, numerous medical studies have shown that in patients with chronic kidney disease, the concentrations of creatinine and uric acid in the blood are directly proportional to the levels of creatinine and uric acid in the urine. Therefore, we can conclude that if elevated levels of creatinine and uric acid are found in the urine, it indicates that the kidneys are very likely experiencing some problems.

[0004] However, for patients with chronic kidney disease, going to the hospital for blood tests every few days is not very convenient. In addition, frequent needle pricks can cause great physical and mental trauma to patients.

[0005] Existing methods for detecting urinary creatinine and uric acid mostly employ titration, gas chromatography, high-performance liquid chromatography, and electrochemical analysis. However, while titration is simple to operate, it has a large margin of error; gas chromatography and high-performance liquid chromatography offer high sensitivity, but require long analysis times and expensive equipment; electrochemical analysis is convenient and portable, but is susceptible to interference from other substances. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a low-cost and convenient multi-channel microfluidic chip and device for the detection of creatinine and uric acid that can solve the above problems.

[0007] To solve the above-mentioned technical problems, the technical method adopted by the present invention is as follows: The present invention discloses a multi-channel microfluidic chip for creatinine and uric acid detection, including a paper-based chip and creatinine detection points and uric acid detection points disposed on the paper-based chip;

[0008] Glutathione-encapsulated cadmium telluride quantum dots were mixed with urinary creatinine hydrolase and attached to the creatinine detection points to construct a three-dimensional porous aerogel.

[0009] Glutathione-encapsulated cadmium telluride quantum dots were mixed with uric acid hydrolase and attached to the uric acid detection points to construct a three-dimensional porous aerogel.

[0010] Furthermore, the preparation method of the glutathione-encapsulated cadmium telluride quantum dots includes the following steps:

[0011] S1. After cleaning and drying the beaker, add 55mL of ultrapure water;

[0012] S2. Weigh 102.8 mg CdCl2·2.5H2O and 184.4 mg L-GSH and add them to a beaker;

[0013] S3. Use a pH meter to measure the pH value of the mixed solution in the beaker, while continuously stirring the solution with a magnetic stirrer; add 0.5M NaOH solution dropwise to the beaker until the pH of the solution becomes 10.5;

[0014] S4. Continue stirring the solution while adding 22.2 mg Na2TeO3 and 3.8 mg NaBH4 to the mixed solution in the beaker, and stir for 30 minutes.

[0015] S5. After stirring, pour the solution into the special container of the microwave synthesizer, set the reaction time to 40 min and the reaction temperature to 110℃; after the instrument has finished heating and stopped, take the quantum dot solution out of the special container.

[0016] Furthermore, the construction of the three-dimensional porous aerogel includes the self-assembly of quantum dots with excess glutathione stabilizer removed to form a hydrogel, followed by freezing the hydrogel at -20°C, and then using a freeze dryer to directly sublimate the frozen water molecules into water vapor, thus forming the three-dimensional porous aerogel structure.

[0017] The present invention also discloses a detection device for creatinine and uric acid detection, comprising a camera for capturing images of the detection points of a multi-channel microfluidic chip for creatinine and uric acid detection, a light source, and the aforementioned multi-channel microfluidic chip for creatinine and uric acid detection.

[0018] Furthermore, the light source is positioned directly above the microfluidic chip, and the camera is positioned directly above the light source.

[0019] Furthermore, the light source is positioned 120 mm directly above the microfluidic chip.

[0020] Furthermore, the light source provides 350-380nm ultraviolet light irradiation.

[0021] Furthermore, the camera lens is equipped with a filter with a cutoff wavelength of 500nm.

[0022] Beneficial effects:

[0023] 1. This invention is based on the principle that the H2O2 produced by the reaction of creatinine and creatinine hydrolase, and the H2O2 produced by the reaction of uric acid and uricase, rapidly quench the fluorescence of the aerogel. Sensitive detection of urinary creatinine and uric acid is achieved by detecting the fluorescence intensity. Because the sensor uses paper-based fiber material as the substrate and introduces quantum dot nanomaterials as the sensitive unit to construct a three-dimensional aerogel structure, it has characteristics such as a large specific surface area, providing more active sites for the adsorption of biological enzymes, thereby increasing the adsorption capacity. Simultaneously, the presence of a large amount of glutathione on the surface of the quantum dots constituting the aerogel provides good biocompatibility, which is beneficial for maintaining enzyme activity, thus improving the sensitivity and stability of the detection.

[0024] 2. The invention relates to the design of an aerogel paper-based sensor chip that enables multi-channel detection and the construction of a portable detection instrument. Under optimal detection conditions, the relationship between urinary creatinine concentration and rate of change was obtained by detecting different concentrations of urinary creatinine / uric acid, and a corresponding detection model was established, achieving highly sensitive quantitative detection of urinary creatinine and uric acid.

[0025] 3. Microwave-assisted rapid synthesis is employed, which is simple to operate and low in cost. The synthesis time is shortened to tens of minutes or even a few minutes. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the overall structure of the detection device for detecting creatinine and uric acid in this invention;

[0027] Figure 2 This is a schematic diagram of the overall results of the multi-channel microfluidic chip used for creatinine and uric acid detection in this invention;

[0028] Figure 3 This is the detection standard curve for the creatinine detection point of the present invention;

[0029] Figure 4 This is the detection standard curve for the uric acid detection point of the present invention. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0031] A multichannel microfluidic chip for creatinine and uric acid detection directly measures urinary creatinine / uric acid concentrations using fluorescence changes. It includes a paper-based chip and creatinine and uric acid detection points mounted on the chip.

[0032] Glutathione-encapsulated cadmium telluride quantum dots were mixed with urinary creatinine hydrolase and attached to creatinine detection points to construct a three-dimensional porous aerogel.

[0033] Glutathione-encapsulated cadmium telluride quantum dots were mixed with uric acid hydrolase and attached to uric acid detection points to construct a three-dimensional porous aerogel.

[0034] like Figure 2 As shown, the multi-channel microfluidic chip for creatinine and uric acid detection in this invention has two sets of creatinine detection points and two sets of uric acid detection points.

[0035] The preparation method of glutathione-encapsulated cadmium telluride quantum dots includes the following steps:

[0036] S1. After cleaning and drying the beaker, add 55mL of ultrapure water;

[0037] S2. Weigh 102.8 mg CdCl2·2.5H2O and 184.4 mg L-GSH and add them to a beaker;

[0038] S3. Use a pH meter to measure the pH value of the mixed solution in the beaker, while continuously stirring the solution with a magnetic stirrer; add 0.5M NaOH solution dropwise to the beaker until the pH of the solution becomes 10.5;

[0039] S4. Continue stirring the solution while adding 22.2 mg Na2TeO3 and 3.8 mg NaBH4 to the mixed solution in the beaker, and stir for 30 minutes.

[0040] S5. After stirring, pour the solution into the special container of the microwave synthesizer, set the reaction time to 40 min and the reaction temperature to 110℃; after the instrument has finished heating and stopped, take the quantum dot solution out of the special container.

[0041] The construction of a three-dimensional porous aerogel involves the self-assembly of quantum dots with excess glutathione stabilizer removed to form a hydrogel, followed by freezing the hydrogel at -20°C, and then using a freeze dryer to directly sublimate the frozen water molecules into water vapor, thus forming a three-dimensional porous aerogel structure.

[0042] This invention provides a detection device for detecting creatinine and uric acid, such as... Figure 1As shown, the system includes a camera 1 for capturing images of the detection points of a multi-channel microfluidic chip used for creatinine and uric acid detection, a light source 2, a multi-channel microfluidic chip 3 for creatinine and uric acid detection, an industrial computer 4, a light source controller 5, and a power module 6. The light source 2 is a ring-shaped ultraviolet excitation light source, specifically the VL-365 model from Weilang Optics, which offers advantages such as low price, small size, light weight, low power consumption, long lifespan, and adjustable emission intensity via an external controller. The UV excitation light source is positioned 120mm directly above the detection area of ​​the biochip, ensuring that the center of the light source coincides with the center of multiple detection areas on the chip. The UV intensity of the ring-shaped light source can be adjusted via software. The ring-shaped UV lamp is positioned directly above the detection area of ​​the paper-based sensor, ensuring good uniformity of UV light in the detection area.

[0043] To avoid interference from ultraviolet light, the camera incorporates a 500nm cutoff wavelength filter in the lens to block ultraviolet light and other interfering light sources below 500nm. Specifically, a Hikvision industrial camera, model MV-CE120-10GC, can be used. The industrial camera is positioned 120m above the paper-based sensor, and focusing at this location ensures clear images.

[0044] A creatinine biosensor aerogel includes the following steps:

[0045] (1) Dilute the reserve quantum dot solution by 10 times as needed, and then add 40U of each of creatinine / uricase, creatine enzyme, and sarcosine oxidase to a 100μL CdTe solution.

[0046] (2) Use a pipette to inject the mixed CdTe-CRx solution into the circular paper-based channels, injecting 8 μl into each channel;

[0047] (3) Place the paper-based sensor in a refrigerator at -20°C for 24 hours to freeze and solidify the mixed solution;

[0048] (4) After freezing, take it out and immediately put it into a vacuum dryer with a freezer for freeze drying. The drying process lasts for 6 hours, and a paper-based sensor modified with CdTe-CRx aerogel can be obtained.

[0049] The rate of change of the R-value of a circular paper-based sensor under 365nm ultraviolet light was calculated, and a linear detection model was established using the rate of change and creatinine concentration. The linear curve results are shown below. Figure 3 As shown, within the creatinine concentration range of 0.22-27.2 mmol, the relative change value of fluorescence of the circular paper-based sensor (R0-R) / R0 has a good linear relationship with the logarithmic concentration of creatinine log10(C), and the linear range fully meets the requirements for creatinine detection and screening.

[0050] Among these, the optimal volume for adding a sample to the creatinine detection paper-based chip to detect creatinine concentration is 8 μL; the optimal concentration for quantum dot dilution in the creatinine experiment is 10 times the original solution; and 6 minutes is selected as the optimal reaction time for creatinine detection to improve sensor sensitivity.

[0051] A uric acid biosensor aerogel includes the following steps:

[0052] (1) Dilute the stored quantum dot solution by 10 times as needed, and then add 60U of uricase to each 100μL CdTe solution.

[0053] (2) Use a pipette to inject the mixed CdTe-UAx solution into the circular paper-based channels, injecting 10 μL into each channel;

[0054] (3) Place the paper-based sensor in a refrigerator at -20°C for 24 hours to freeze and solidify the mixed solution;

[0055] (4) After freezing, take it out and immediately put it into a vacuum dryer with a freezer for freeze drying. The drying process lasts for 6 hours, and the paper-based sensor modified with CdTe-UAx aerogel can be obtained.

[0056] The rate of change of the R-value of a circular paper-based sensor under 365nm ultraviolet light was calculated, and a linear detection model was established using the rate of change and uric acid concentration. The linear curve results are shown below. Figure 4 As shown, within the uric acid concentration range of 0.08-0.8 mM, the relative change value of fluorescence of the circular paper-based sensor (R0-R) / R0 has a good linear relationship with the uric acid concentration (C), and the linear range fully meets the requirements for uric acid detection and screening.

[0057] Among them, the optimal volume for adding a sample to the uric acid detection paper-based chip is 10 μL; the optimal concentration for quantum dot dilution in the uric acid experiment is 6 times the original solution; and 8 minutes is selected as the optimal reaction time for uric acid detection to improve sensor sensitivity.

[0058] A cadmium telluride quantum dot is rapidly synthesized using a simple and low-cost microwave-assisted method. The method includes the following steps:

[0059] (1) Take a 100mL beaker, clean and dry it, and add 55mL of ultrapure water;

[0060] (2) Weigh 102.8 mg CdCl2·2.5H2O and 184.4 mg L-GSH and add them to a beaker;

[0061] (3) Use a Mettler pH meter to measure the pH of the mixed solution in the beaker, while continuously stirring the solution with a magnetic stirrer. Add 0.5M NaOH solution dropwise to the beaker until the pH of the solution becomes 10.5. The entire solution will become turbid during pH adjustment, and then gradually become transparent when the pH rises to 7.5;

[0062] (4) Continue stirring the solution while adding 22.2 mg Na2TeO3 and 3.8 mg NaBH4 to the mixed solution in the beaker and stirring for 30 minutes.

[0063] (5) After stirring, pour the solution into the special container of the microwave synthesizer, set the reaction time to 40 min and the reaction temperature to 110 °C. After the instrument has finished heating and stopped, take the quantum dot solution out of the special container.

[0064] Among these methods, microwave-assisted heating provides uniform heating and rapid temperature rise from the inside out, significantly reducing the original reaction time to just a few minutes or even tens of minutes. The optical properties of the quantum dots are characterized using a fluorescence spectrophotometer and ultraviolet absorption spectroscopy.

[0065] The foregoing description illustrates the design principles and advantages of this invention. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A multichannel microfluidic chip for detecting creatinine and uric acid, characterized in that: It includes a paper-based chip and creatinine and uric acid detection points disposed on the paper-based chip; Glutathione-encapsulated cadmium telluride quantum dots were mixed with urinary creatinine hydrolase and attached to the creatinine detection points to construct a three-dimensional porous aerogel. Glutathione-encapsulated cadmium telluride quantum dots were mixed with uric acid hydrolase and attached to the uric acid detection points to construct a three-dimensional porous aerogel. The method for preparing the glutathione-encapsulated cadmium telluride quantum dots includes the following steps: S1. After cleaning and drying the beaker, add 55 mL of ultrapure water; S2. Weigh 102.8 mg CdCl2·2.5H2O and 184.4 mg L-GSH and add them to a beaker; S3. Use a pH meter to measure the pH value of the mixed solution in the beaker, while continuously stirring the solution with a magnetic stirrer; add 0.5M NaOH solution dropwise to the beaker until the pH of the solution becomes 10.5; S4. Continue stirring the solution while adding 22.2 mg Na2TeO3 and 3.8 mg NaBH4 to the mixed solution in the beaker, and stir for 30 minutes. S5. After stirring, pour the solution into the special container of the microwave synthesizer, set the reaction time to 40 min and the reaction temperature to 110℃; after the instrument has finished heating and stopped, take the quantum dot solution out of the special container. The construction of the three-dimensional porous aerogel includes the self-assembly of quantum dots with excess glutathione stabilizer removed to form a hydrogel, followed by freezing the hydrogel at -20°C, and then using a freeze dryer to directly sublimate the frozen water molecules into water vapor, thus forming the three-dimensional porous aerogel structure.

2. A detection device for detecting creatinine and uric acid, characterized in that: The invention includes a camera (1) for capturing images of the detection points of a multichannel microfluidic chip for creatinine and uric acid detection, a light source (2), and the multichannel microfluidic chip for creatinine and uric acid detection as described in claim 1.

3. The detection device for detecting creatinine and uric acid according to claim 2, characterized in that: The light source (2) is positioned directly above the microfluidic chip, and the camera (1) is positioned directly above the light source (2).

4. The detection device for detecting creatinine and uric acid according to claim 3, characterized in that: The light source (2) is positioned 120 mm directly above the microfluidic chip.

5. The detection device for detecting creatinine and uric acid according to claim 2, characterized in that: The light source (2) provides 350-380nm ultraviolet light irradiation.

6. The detection device for detecting creatinine and uric acid according to claim 2, characterized in that: The camera (1) lens is equipped with a filter with a cutoff wavelength of 500nm.

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