阅读说明：本技术 一种尿酸检测专用微流控纸芯片及检测分析方法 (Special micro-fluidic paper chip for uric acid detection and detection analysis method ) 是由 刘青叶 张剑 苗延青 靖会 于 2020-03-09 设计创作，主要内容包括：本发明涉及一种用于尿酸检测的微流控纸芯片及尿酸检测方法。纸质微流控芯片由纸基和沉积在纸基上的疏水材料构成；使用喷蜡打印机打印纸芯片文档,再于热压机上得测试用纸芯片。用纸质微流流控芯片对尿酸进行含量测定的方法采取微流控纸芯片技术对尿酸进行含量测定,利用尿酸的还原性,以三氯化铁为氧化剂组成氧化还原体系,以邻二氮菲对反应产物Fe<Sup>2+</Sup>进行显色,根据显色强度对尿酸含量进行检测,包括8大步骤：同现有技术相比,样品用量少、准确、简便、快速、重复性好,可用于血浆中尿酸的含量检测。表明此发明所用方法可靠、有效。此发明对尿酸的临床检测及相关疾病预防具有较为深远的意义。(The invention relates to a micro-fluidic paper chip for uric acid detection and a uric acid detection method. The paper microfluidic chip consists of a paper base and a hydrophobic material deposited on the paper base; and printing a paper chip document by using a wax-spraying printer, and then obtaining a paper chip for testing on a hot press. The method for measuring the content of uric acid by using a paper microfluidic chip adopts the microfluidic paper chip technology to measure the content of uric acid, utilizes the reducibility of uric acid, takes ferric chloride as an oxidant to form an oxidation-reduction system, and takes o-diazepine phenanthrene as a reaction product Fe 2+ Carrying out color development, and detecting the content of uric acid according to color development intensity, comprising 8 steps: compared with the prior art, the method has the advantages of less sample dosage, accuracy, simplicity, convenience, rapidness and good repeatability, and can be used for detecting the content of uric acid in blood plasma. The method is reliable and effective. The invention has far-reaching significance for clinical detection of uric acid and prevention of related diseases.)

1. A paper micro-fluidic chip special for uric acid determination is characterized in that: the paper microfluidic chip is composed of a paper base and a hydrophobic material deposited on the paper base; the paper base adopts chromatographic paper with low porosity, small aperture and uniform distribution, or adopts a nitrocellulose filter membrane to be suitable for detecting blood uric acid and uric acid; the hydrophobic material adopts wax; and printing a paper chip document on chromatographic paper according to the designed micro-channel network of the micro-fluidic paper chip by using a wax-spraying printer and drawing software installed in a computer, and then obtaining the paper chip for testing on a hot press.

2. The paper microfluidic chip special for uric acid determination according to claim 1, characterized in that: the porosity of the paper base is 65-85%; the aperture of the paper base is between 10 and 11 mu m; the chromatographic paper comprises: thickness 180 μm, basis weight 88g/m²Air flow rate of 10.5s/100m L/in²0.06 percent of ash content, 39.1N/15mm of tensile modulus and the lowest α -cellulose content of 98 percent, wherein the nitrocellulose filter membrane is cellulose paper with the pore diameter of 0.7-08 μm, a thickness of 105-140 μm, a burst strength of > 2psi, a weight of 3.6-5.5 mg/cm²The highest operation temperature is 80 ℃, the porosity is 66-84%, and the hydrophilic filter membrane is sterilized by steam autoclaving.

3. The paper microfluidic chip special for uric acid determination according to any one of claims 1 and 2, characterized in that: the diameters of the sample loading areas I and II are 4mm, the diameter of the detection area III is 6mm, the length of a channel between the areas I and III is 7mm, the width of the channel is 2mm, the uric acid solution and the color developing agent are loaded in the areas I and II respectively, move oppositely under the capillary action, and are mixed and reacted in the area III.

4. A method for detecting the content of uric acid by using a paper microfluidic chip is characterized by comprising the following steps: the content of uric acid is measured by adopting a microfluidic paper chip technology, the reducibility of the uric acid is utilized, ferric trichloride is taken as an oxidant to form a redox system, and the o-diazaphenanthrene is used for reacting the product Fe²⁺Developing color, and detecting the content of uric acid according to the developing intensity, comprising the following steps:

step 1: designing a micro-channel network of the microfluidic paper chip by adopting drawing software, printing a designed paper chip document on chromatographic paper by using a wax-spraying printer, and heating on a hot press to obtain an experimental paper chip;

step 2: preparing uric acid stock solution, phosphate buffer solution and FeCl before detection₃Solution and phenanthroline solution;

and step 3: preparing a uric acid standard curve before detection;

and 4, step 4: drawing a curve of RGB (superposition of three color channels of red R, green G and blue B) value and concentration;

and 5: respectively carrying out detection under the conditions of reaction time of 1-20 min, pH 5-8 of a color developing agent and a ratio of 1: 1.2-1: 2 of uric acid and ferric trichloride to obtain optimal detection conditions;

step 6: detecting the content of uric acid in animal serum;

and 7: serum without adding uric acid solution is used as blank reference, and samples obtained after adding different uric acid standard substancesThe solution is respectively mixed with FeCl₃After mixing reaction, proportionally reacting with an o-phenanthroline color developing agent with the pH of 7.4 on a paper chip;

and 8: the recovery of uric acid in serum was calculated.

5. The method for detecting the content of uric acid by using the paper microfluidic chip according to claim 4, wherein the method comprises the following steps: and (3) completing the steps 3, 4 and 5, wherein the specific process of the minimum detection limit test required to be determined is as follows: respectively diluting the uric acid stock solution, labeling, reacting with ferric trichloride at a ratio of 1:1.6 for 10min, and reacting with an o-phenanthrene color developing agent with pH of 7.4 at an equal ratio on a paper chip to obtain the lowest detection limit of the paper chip for detecting uric acid.

6. The method for detecting the content of uric acid by using the paper microfluidic chip as claimed in claim 4, wherein the step 3, 4, and 5 is performed by diluting a uric acid stock solution into a uric acid solution with a certain concentration, taking 14 centrifuge tubes of 0.5m L with the labels 0-13 and 0 without adding coexisting ions, and respectively adding 1000 times of sodium chloride solution, potassium chloride solution, sodium sulfate solution, zinc sulfite solution, aluminum nitrate solution, 100 times of ammonium chloride solution, starch solution, glucose solution, 10 times of zinc sulfate solution, calcium chloride solution, barium chloride solution, anhydrous sodium carbonate solution, and equal times of vitamin C solution and dopamine solution to No. 1-13, wherein the interfering ions are respectively mixed with FeCl in a ratio of 1:1.6₃Reacting for 10min, and reacting with Ph7.4 phenanthroline color developing agent in equal proportion on a paper chip.

7. The method for detecting the content of uric acid by using the paper microfluidic chip as claimed in claim 4, wherein the reaction time required for completing the steps 3, 4 and 5 comprises precisely removing 200 μ L of uric acid stock solution and FeCl₃Mixing the solutions 400 mu L, reacting for 1min, 5min, 10min, 15min and 20min respectively, and determining the optimal reaction time for uric acid detection according to the color development intensity change on the paper chip along with the time changeIs 9min to 11 min.

8. The method for detecting the content of uric acid by using the paper microfluidic chip according to claim 4, wherein the method comprises the following steps: color developing agent FeCl determined by finishing steps 3, 4 and 5₃The specific process of the pH value of the o-phenanthroline solution comprises precisely transferring uric acid stock solution 200 mu L and FeCl₃And mixing the solution with 400 mu L for reaction for 10min, reacting with the phenanthroline on the paper chip in equal proportion, changing the color development intensity on the paper chip according to different pH values, and determining the pH value of the color development agent to be 7.3-7.5 according to the change of the color development intensity.

9. The method for detecting the content of uric acid by using the paper microfluidic chip as claimed in claim 4, wherein the specific process for detecting the content of uric acid in animal serum in step 6 comprises the steps of taking 4 parts of uric acid stock solution, adding 0.1m L serum into each of the 4 parts, adding 0.1m L uric acid and 0.2m L acetonitrile into each of the 4 parts, performing oscillation and centrifugation for 10min (1000r/min), taking supernatant, volatilizing a solvent with nitrogen, performing constant volume to 4m L, performing detection on a paper chip to obtain RGB values, substituting the RGB values into a working curve, and calculating the content of uric acid in serum.

10. The method for detecting the content of uric acid by using the paper microfluidic chip as claimed in claim 4, wherein the step 8 of determining the recovery rate of uric acid in serum comprises the steps of collecting 3 parts of uric acid stock solution, adding 0.1m L and 0.2m L acetonitrile into each part of serum, performing oscillation centrifugation for 10min (1000r/min), collecting supernatant, volatilizing solvent with nitrogen, and fixing the volume to 4m L, wherein serum without uric acid solution is used as blank control No. 0, and A-C is a sample solution obtained after adding different uric acid standards and FeCl respectively₃After reacting for 10min with the mixture of 1:1.6, the mixture reacts with the pH7.4 phenanthroline color developing agent in equal proportion on a paper chip, and the recovery rate of uric acid in serum is determined.

Technical Field

The invention belongs to the technical field of drug analysis, relates to a special microfluidic paper chip for detection and analysis and a detection method, and particularly relates to a microfluidic paper chip for uric acid detection and a uric acid detection method.

Background

In the pharmaceutical analysis technology, there are many detection methods according to various analytes. Only with respect to the uric acid detection method, in the prior art before the present invention, there are enzyme methods, uric acid biosensor detection methods, voltammetry, high performance liquid chromatography, phosphotungstic acid reduction methods, and the like, which have been reported in the literature. The enzyme method is simple and convenient to operate, the sample is simple to process, but the operation process is complex. The development of uric acid biosensor detection method is closely related to the rapid development of enzyme immobilization technology. The enzyme immobilization technology is the key for manufacturing enzyme electrodes, plays a role in determining various performances of the electrodes, and various immobilization methods such as an adsorption method, a carrier coupling method, a cross-linking method, an embedding method and the like are developed at present. The voltammetry is a commonly used method for detecting uric acid at present, and in recent years, the voltammetry for detecting uric acid has been greatly developed in theoretical research and practical application to clinical diagnosis. High performance liquid chromatography detection methods, while simple, often require cumbersome sample pre-treatment or use of complex column switching equipment. The phosphotungstic acid reduction method is simple to operate, has good accuracy, is a classic clinical examination method, and is the most common detection method. In recent years, the technology of microfluidic chips for various analysis and detection purposes, such as electrophoresis chips, silicon-based microchannel chips, droplet generation chips, cell chips, paper chips, etc., has been developed rapidly. Compared with the microfluidic chip in the common sense, the paper microfluidic chip has the advantages of low cost, simple preparation, no need of complex peripheral equipment, capability of performing disposable, low-cost and portable analysis in the true sense, and has attracted more and more attention, and is generally regarded as one of the development trends of field real-time diagnosis in the future. Compared with other traditional methods, the method has the advantages of high analysis speed, low consumption, low material consumption, low pollution, low price, safety and the like. Due to its great potential in the fields of biology, chemistry, medicine and the like, the method has been developed into a new research field crossing the disciplines of biology, chemistry, medicine, fluid, electronics, materials, machinery and the like. At present, the paper chip and the method for detecting uric acid used by the invention have not been reported yet.

Disclosure of Invention

In view of the above prior art, the present invention provides a paper microfluidic chip for uric acid detection, and another object of the present invention is to provide a method for uric acid detection and analysis using the paper microfluidic chip.

The concept and technical solution of the present invention will now be described as follows:

the invention has the basic concept that the paper microfluidic chip special for uric acid detection and analysis of animal urine is designed by utilizing the characteristics that the paper microfluidic chip has low cost, is simple and convenient to prepare, does not need complex peripheral equipment, and can carry out disposable, low-price and portable detection and analysis in the true sense; meanwhile, the reducibility of uric acid is utilized, ferric trichloride is taken as an oxidant to form a redox system, and the o-diazaphenanthrene is used as a reaction product Fe²⁺Developing color, and detecting and analyzing the uric acid content according to the color development intensity.

The invention relates to a paper microfluidic chip special for uric acid determination, which is characterized in that: consists of a paper base and a hydrophobic material deposited on the paper base; the paper base adopts chromatographic paper with low porosity, small aperture and uniform distribution, or adopts a nitrocellulose filter membrane to be suitable for the detection of blood uric acid and uric acid; the hydrophobic material adopts wax; and (3) printing a paper chip document on chromatographic paper according to the designed micro-channel network of the micro-fluidic paper chip (shown in figure 1) by using a wax-spraying printer and drawing software installed in a computer, and then obtaining a paper chip for testing on a hot press.

The invention further provides a paper microfluidic chip special for uric acid determination, which is characterized in that: the porosity of the paper base is 65-85%; the aperture of the paper base is between 10 and 11 mu m; the chromatographic paper comprises: thickness 180 μm, basis weight 88g/m²Air flow rate of 10.5s/100m L/in²0.06 percent of ash content, 39.1N/15mm of tensile modulus and 98 percent of minimum α -cellulose content, wherein the nitrocellulose filter membrane is cellulose paper with the pore diameter of 0.7-0.8 mu m, the thickness of 105-140 mu m, the burst strength of more than 2psi and the weight of 3.6-5.5 mg/cm²The highest operation temperature is 80 ℃, the porosity is 66-84%, and the hydrophilic filter membrane is sterilized by steam autoclaving.

The invention further provides a paper microfluidic chip special for uric acid determination, which is characterized in that: the diameters of the sample loading areas I and II are 4mm, the diameter of the detection area III is 6mm, the length of a channel between the areas I and III is 7mm, the width of the channel is 2mm, the uric acid solution and the color developing agent are loaded in the areas I and II respectively, move oppositely under the capillary action, and are mixed and reacted in the area III.

The invention also provides a method for measuring the content of uric acid by using the paper microfluidic chip, which comprises the following steps:

step 1: designing a micro-channel network of the microfluidic paper chip by adopting drawing software, printing a designed paper chip document on chromatographic paper by using a wax-spraying printer, and heating on a hot press to obtain an experimental paper chip;

step 2: preparing uric acid stock solution, phosphate buffer solution and FeCl before detection₃Solution and phenanthroline solution;

and step 3: respectively carrying out detection under the conditions of reaction time of 1-20 min, pH 5-8 of a color developing agent and a ratio of 1: 1.2-1: 2 of uric acid and ferric trichloride to obtain optimal detection conditions;

and 4, step 4: preparation of uric acid standard curve before detection

Taking uric acid stock solution to be respectively diluted into 1.19 mmol/L, 2.38 mmol/L, 3.57 mmol/L, 4.76 mmol/L and 5.95 mmol/L, and then mixing with FeCl according to the ratio of 1:1.6₃Reacting for 10min, and reacting with the Ph7.4 phenanthroline color developing agent in equal proportion on the paper chip;

and 5: drawing a working curve between RGB (red R, green G, blue B) values and concentration;

step 6: detecting the content of uric acid in animal serum;

and 7: serum without adding uric acid solution is used as blank reference, and sample solutions obtained after adding different uric acid standard substances are respectively mixed with FeCl₃After mixing reaction, proportionally reacting with an o-phenanthroline color developing agent with the pH of 7.4 on a paper chip;

and 8: the recovery of uric acid in serum was calculated.

Compared with the prior art, the paper chip prepared by the invention is used for measuring the uric acid, does not need to carry out special sample pretreatment, can directly carry out quantitative analysis, is compared with the result of detecting the uric acid content based on an ultraviolet spectrophotometry, has the advantages of less sample dosage, accuracy, simplicity, convenience, rapidness and good repeatability, and can be used for detecting the content of the uric acid in serum. The method is reliable and effective. The invention has far-reaching significance for clinical detection of uric acid and prevention of related diseases.

Drawings

FIG. 1: micro-channel paper chip design (A) Single-channel paper chip design (B) blank control paper chip (C) sample detection paper chip (D) six-channel paper chip (E) eight-channel paper chip (F)

FIG. 2: effect of reaction time on color development reaction

FIG. 3: influence of pH value on color reaction

FIG. 4: FeCl₃Effect of the ratio to uric acid on color development reaction

FIG. 5: determination of minimum detection limit

FIG. 6: establishing a standard curve based on a paper chip

FIG. 7: influence of interfering ions

FIG. 8: repetitive experiment for uric acid detection

FIG. 9: sample addition recovery

FIG. 10: determination of uric acid content in animal serum

Detailed Description

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.