A whole blood separation paper chip and its application

Abstract

The present invention discloses a whole blood separation paper chip and its application, which belongs to the detection field. The SZ0883 whole blood separation membrane intercepts red blood cells to filter and adsorb cell components in the whole blood sample, thereby quickly separating serum; the reducing property of uric acid is used to react with FeCl3 to generate FeCl3. 2+ The color intensity RGB value is positively correlated with the uric acid concentration by complexing with o-phenanthroline, thereby detecting the uric acid content. The present invention uses a whole blood separation paper chip to establish a channel bridge type whole blood separation color development system, realizes the separation of serum from whole blood and connects it to the color development system, establishes a sample pretreatment method that can be used for whole blood uric acid detection, and lays an experimental foundation for the use of microfluidic paper chips for real-time detection of uric acid.

Description

Technical Field

[0001] The invention relates to the field of detection, and in particular to a whole blood separation paper chip and application thereof. Background Art

[0002] As the end product of purine metabolism, uric acid is mainly excreted from the body through the kidneys under normal physiological conditions, thus maintaining the balance of uric acid in the body. Hyperuricemia is a common pathological condition. With the improvement and change of people's living standards and lifestyles, the number of hyperuricemia patients in the world continues to increase. Epidemiological observations in the past 10 years have shown that hyperuricemia has become the "fourth highest" after diabetes, hypertension, and hyperlipidemia. Abnormal uric acid levels are closely related to a variety of diseases and may indicate the occurrence of a variety of diseases, such as chronic arthritis, joint deformities, tophi, characteristic acute arthritis, cardiovascular disease, diabetes, etc. Therefore, regular testing of uric acid levels and maintaining them within the normal range are crucial for the prevention and treatment of related diseases.

[0003] Whole blood is composed of red blood cells, white blood cells, platelets and other blood cells and plasma. Blood cells account for 40% to 45% of whole blood, and plasma accounts for 55% to 60%. Many components in plasma are directly related to various life activities of the human body. Since blood cells, hemoglobin, heme, enzymes and other substances often interfere with the analysis of plasma components, macromolecules such as blood cells need to be removed in advance in related tests. Therefore, rapid separation of blood is of great significance in medical research and clinical practice. At present, whole blood separation mainly adopts centrifugation, ultrasonic method, dielectrophoresis, membrane filtration and emerging microfluidics technology. In traditional laboratory analysis methods, the most common way to separate plasma is to centrifuge whole blood, and plasma can be obtained by taking the supernatant. This centrifuge-based method mainly uses the difference in sedimentation speed of different components under the action of centrifugal force to achieve separation. The ultrasonic method mainly uses the cavitation effect, mechanical vibration and thermal energy effect generated by ultrasound in the liquid to separate cells, plasma and its components in whole blood. The dielectrophoresis method is based on the difference in migration speed of cells or plasma components under the action of electric field for separation. During the dielectrophoresis process, a whole blood sample is placed in a non-uniform electric field. Since different components in the blood have different dielectric properties, they will be affected by dielectrophoretic forces to varying degrees. By adjusting the electric field parameters, specific types of cells or particles can be moved to areas with higher or lower electric field strengths, thereby achieving separation. As point-of-care testing (POCT) equipment is increasingly widely used, the traditional plasma preparation methods reported so far are difficult to meet actual testing requirements due to the large size of the equipment required, high cost, large blood sample consumption, low separation efficiency, cumbersome operation, long processing time, and high professionalism.

[0004] Microfluidic paper-based analytical devices (μPADs) is a major branch of microfluidic technology. Due to its advantages such as small size, low cost, high throughput, low reagent consumption, and fast reaction, it has been widely used in various POCT devices and has shown great potential in the medical industry. However, microfluidic paper chip technology is currently mostly used to detect the content of components in blood, and there has been no report on its use in whole blood separation. Summary of the invention

[0005] The purpose of the present invention is to provide a whole blood separation paper chip and its application to solve the problems existing in the above-mentioned prior art. A channel bridge type whole blood separation color development system is established through the whole blood separation paper chip, serum is separated from the whole blood and connected to the color development system, a sample pretreatment method that can be used for whole blood uric acid detection is established, and an experimental foundation is laid for the use of microfluidic paper chips for real-time detection of uric acid.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a whole blood separation paper chip, the paper chip comprising a uric acid color development paper chip and a plasma separation module;

[0008] The uric acid colorimetric paper chip is a rectangular paper chip, with a hydrophilic circular structure set on both sides of the surface of the rectangular paper chip, and other areas are set as hydrophobic structures; a paper tape is attached to the bottom of the circular structure;

[0009] The plasma separation module is a rectangular blood filter membrane, which is horizontally fixed between the two circular structures, with two ends of the rectangular blood filter membrane contacting the two circular structures respectively.

[0010] Preferably, the hydrophobic structure is made by printing the designed paper chip document on chromatography paper using a wax jet printer and then placing it on a heat press and heating it at 176° C. for 2 minutes.

[0011] Preferably, the rectangular paper chip has a specification of 20 mm×10 mm, the circular structure has a diameter of 4 mm, and the rectangular blood filter membrane has a specification of 10 mm×2.0 mm.

[0012] Preferably, the two circular structures are the sample loading area and the color development area respectively.

[0013] The principle of separating red blood cells using the above-mentioned whole blood separation paper chip is as follows: superimpose the SZ0883 whole blood separation membrane on the Whatman NO.1 filter paper of the paper chip platform, and then add the whole blood sample. By utilizing the blocking of cells by the microscopic pores of the whole blood filter membrane and the capillary action of the paper, the cellular components in the whole blood sample can be filtered and adsorbed, thereby quickly separating the serum.

[0014] The present invention also provides application of the whole blood separation paper chip in preparing a product for detecting the uric acid content in blood.

[0015] Preferably, the method for detecting the uric acid content in blood by the whole blood separation paper chip comprises: adding the whole blood sample to be tested and physiological saline into the sample loading area, separating the serum by intercepting red blood cells through a rectangular blood filter membrane, allowing the serum to enter the color development area under the capillary adsorption action of the rectangular blood filter membrane, observing the color development of the color development area, and calculating the uric acid content in the whole blood sample to be tested according to the RGB value of the color development area.

[0016] Preferably, the sample loading volume in the sample loading area is 10 μL of the whole blood sample to be tested and 5 μL of physiological saline.

[0017] Preferably, the color developer in the color development zone includes the following components: 26 μL of 10% FeCl3, 104 μL of o-phenanthroline and 470 μL of 0.05% chitosan.

[0018] The principle of using the whole blood separation paper chip to separate serum for uric acid detection is as follows: the red blood cells are intercepted by the SZ0883 whole blood separation membrane to filter and adsorb the cell components in the whole blood sample, thereby quickly separating the serum; the reducing property of uric acid is used to react with FeCl3 to generate Fe 2+ It complexes with o-phenanthroline to develop color, and the color intensity RGB value is positively correlated with the uric acid concentration, thereby detecting the uric acid content.

[0019] The present invention discloses the following technical effects:

[0020] The present invention uses chitosan clarified liquid as a diluent of a color developer for the first time, and utilizes the polyhydroxyl characteristics of chitosan to enhance the metal ion complexing degree, thereby deepening the color development, improving the detection accuracy and the slope of the standard curve.

[0021] The present invention establishes a whole blood bridge channel separation and color development system, organically combines uric acid color development with whole blood separation, and realizes the seamless connection between whole blood separation and the previous uric acid color development system of the research group (CN111443082A a microfluidic paper chip dedicated to uric acid detection and detection and analysis method), laying the premise and foundation for the subsequent development of uric acid analyzers for whole blood detection.

[0022] The present invention uses a paper engraving machine for the first time to directly engrave the designed graphics on a blank paper chip, which simplifies the production process of the paper chip compared to the previous method of making paper chips using wax spray printing and hot pressing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Create a flow chart for the uric acid colorimetric paper chip;

[0025] Figure 2 It is a double-hole single-channel paper chip structure; A.: paper chip structure, B: whole blood separation module, C: combination diagram;

[0026] Figure 3 It is a gourd-shaped paper chip structure; A.: paper chip structure, B: whole blood separation module, C: combination diagram;

[0027] Figure 4 It is a paper chip structure with elongated channels; A.: paper chip structure, B: whole blood separation module, C: combination diagram;

[0028] Figure 5 It is a single-hole stacked permeation paper chip structure; A.: paper chip structure, B: whole blood separation module, C: combination diagram;

[0029] Figure 6 It is a channel bridge type separation paper chip structure; A.: paper chip structure, B: construction diagram;

[0030] Figure 7 This is the experimental flow chart for whole blood uric acid detection;

[0031] Figure 8 This is the investigation result of the total liquid loading volume of the double-hole single-channel paper chip;

[0032] Fig. 9 This is the result of the investigation on the sample volume of whole blood and diluent on the double-hole single-pass paper chip;

[0033] Fig.10 This is a diagram of the separation effect of the gourd-shaped paper chip separation system; A: whole blood separation result, B: sample color development result;

[0034] Fig.11 The separation effect diagram of the slender channel separation system; A: the result of the diluent color developer dispersion, B: the sample color development result;

[0035] Fig.12 The results of whole blood separation and color development of a single-hole stacked permeable paper chip; A: structure of a single-hole stacked permeable paper chip; B: adding sample and diluent, C: whole blood separation results, D: paper chip with color developer, E: color development results of the paper chip;

[0036] Fig.13 Whole blood separation and color development results for the long and narrow channel separation system;

[0037] Fig.14 is the standard curve;

[0038] Fig.15 The sample test results;

[0039] Fig.16 It is intra-day (first day) and inter-day (first to fifth day) repeatability. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Some experimental materials and reagents involved in the following examples:

[0046] (1) Experimental instruments: electronic balance (1 / 100,000, Precisa); oil filter membrane; whole blood filter membrane (SZ0883, Xiamen Quick Diagnosis New Material Technology Co., Ltd.); NO.1 qualitative filter paper (Whatman No.1); wax printer (Xerox Phaser8570, Fuji Xerox); paper cutting machine (MT4030, Shenzhen Meitu Intelligent Equipment Co., Ltd.); pipette gun; preheating table (Luxianzi CNC constant temperature preheating table); laboratory self-made camera box (disclosed in the utility model patent "CN220438174U A paper chip detection image acquisition device"); heat press (G311, Fujian Applex Electric Co., Ltd.); transparent tape; masking tape; aluminum foil tape; pH meter (PB-10, Sartorius); wide range pH test paper; Android mobile phone (vivo iQOO U5, vivo Guangdong Mobile Communications Co., Ltd.), mobile phone external camera (Vishinsgae 1600W, USB-16M-SV01).

[0047] (2) Reagents: uric acid (≧99, Shanghai Aladdin Reagent Co., Ltd.); o-phenanthroline monohydrate (AR, Shanghai Aladdin Reagent Co., Ltd.); anhydrous FeCl3 (AR, Shanghai Shanpu Chemical Co., Ltd.); FeCl3·6H2O (analytical grade, Shanghai Shanpu Chemical Co., Ltd.); 95% ethanol (AR, Hongyan Reagent Factory, Hedong District, Tianjin); NaOH (AR, Hongyan Reagent Factory, Hedong District, Tianjin); deionized water (Xianyang Xiquan Pure Water Co., Ltd.); sodium chloride (AR, Tianjin Komeo Fine Chemicals Co., Ltd.); PBS used in the present invention is 0.1 mmol·L -1 (pH 7.4-7.5, Beijing Lanjieke Technology Co., Ltd.); chitosan (AR, Shanghai Aladdin Reagent Co., Ltd.); concentrated hydrochloric acid (AR, Tianjin Hedong District Hongyan Reagent Factory).

[0048] Example 1

[0049] 1. Effect of uric acid chromogenic signal amplifier

[0050] The present invention uses a clear solution of chitosan as a diluent, and the other conditions are all prepared using the formula studied by the research group in the early stage to prepare the color developer of uric acid, which will make the uric acid color development deepen. The color developer formula is 10% FeCl326μL, 10% o-phenanthroline 104μL and diluent 470μL. The present invention investigates the effect of chitosan solution as a diluent on the color intensity, prepares three color developers according to the formula shown in Table 1, and uses 0.3, 0.45, 0.60, 0.75, 0.90mmol / L uric acid standard solutions to perform dry uric acid color development experiments, the color developer and uric acid loading amounts are 2.5 and 3μL respectively, the color development time is 1.5min, and the paper chip used is a 6mm single-hole paper chip.

[0051] Table 1: Color developer formula with different concentrations of signal amplifier added

[0052] Color developer number\formula <![CDATA[10%FeCl3(μL)]]> O-phenanthroline(μL) Dilution volume / type 0 control group 26 104 470 μL PBS 1 26 104 470 μL 0.025% chitosan 2 26 104 470 μL 0.050% chitosan

[0053] 2. Microfluidic whole blood separation paper chip

[0054] The microfluidic whole blood separation paper chip is mainly composed of two parts: the uric acid colorimetric paper chip and the whole blood separation module, which together constitute the whole blood separation colorimetric system. The uric acid colorimetric paper chip uses Whatman No.1 qualitative paper as the reaction platform. Based on the previous laboratory research results on uric acid colorimetric conditions, it is used to carry and colorimetric uric acid samples separated from whole blood. The whole blood separation module is composed of two porous separation materials: ink paper and whole blood filter membrane. One or two of them are used in an organic combination with the uric acid colorimetric paper chip to form a microfluidic whole blood separation paper chip.

[0055] A variety of different separation and color development systems were constructed in the experiment (Table 2). The separation system was evaluated from all aspects, including whole blood separation effect, blood loading amount, color development uniformity, and reaction time, to establish the optimal whole blood separation uric acid color development system.

[0056] Table 2 Main parameters of porous separation materials

[0057] System\Module Uric acid colorimetric paper chip Whole blood separation system Double hole single channel barbell system Double hole single channel paper chip Circular ink paper and whole blood filter membrane stack Channel-free gourd system Gourd Shape Paper Chips Circular ink paper and whole blood filter membrane stack Slim channel system Double hole slim single channel Circular ink paper and whole blood filter membrane stack Single hole superposition permeability system Single hole round paper chip Circular ink paper and whole blood filter membrane stack Channel bridge system Double hole non-channel paper chip Rectangular whole blood filter

[0058] 2.1 Preparation of uric acid colorimetric paper chip

[0059] First, the shape of the paper chip was designed in Inkscape, and then the paper chip was treated to prevent leakage in the longitudinal direction, and paper tape was pasted on it. The treated paper chip was fixed on the cutting plane of the paper cutter, and the blank paper chip design was imported into the software supporting the paper cutter. Then the color developer was dripped into the color development area of ​​the paper chip, and after drying on the preheating table, the uric acid color development paper chip ( Figure 1 ).

[0060] The uric acid color paper chip changes as needed according to the shape of the whole blood separation module. This experiment will follow Figure 1 The process is to produce uric acid colorimetric paper chips in various shapes.

[0061] 2.2 Whole blood separation module

[0062] The main function of the whole blood separation module is to intercept the red blood cell components of whole blood, such as red blood cells, hemoglobin, heme, etc. It is mainly composed of porous adsorption materials. In addition, the module also needs to be combined with the uric acid color development paper chip to flow the separated liquid back to the color development area. The porous adsorption materials used in the present invention are ink paper and whole blood filter membrane. Two types of ink paper with thickness of 0.6mm and 1mm were used. SZ0883 whole blood filter membrane was used in the experiment, and the relevant parameters and models are shown in Table 3.

[0063] Table 3 Main parameters of porous separation materials

[0064]

[0065] 2.3 Double-hole single-channel barbell system

[0066] The color paper chip of this system is a double-hole single-channel ( Figure 2 A), the whole blood separation module uses a circular blood filter membrane and a circular ink paper double layer stack ( Figure 2 In B), the two parts are Figure 2 You can get it by superimposing and combining as shown in C.

[0067] 2.3.1 Investigation of paper chip channel length and total liquid loading volume

[0068] After the whole blood separation microfluidic paper chip structure with different channel lengths was built, the diluent saline was added to the sample loading area according to the volume shown in Table 4, and it was observed whether the liquid could flow from the sample loading area through the microfluidic paper chip structure and cover the color development area within a period of time. The results were recorded and analyzed by taking photos to investigate the optimal double-hole single-channel paper chip channel length and the total liquid loading volume.

[0069] Table 4 Investigation table of paper chip channel length and total liquid loading volume

[0070]

[0071] 2.3.2 Investigation of ink filter paper thickness

[0072] Cut the ink filter papers of different thicknesses into round paper chips with a diameter of 6 mm. Figure 2 The method shown in C in the middle is spliced ​​with a paper chip with a channel length of 8 mm to form a double-hole single-channel whole blood separation system. According to the effect of the ink filter paper thickness on the total sample loading volume as shown in Table 5, the appropriate ink filter paper thickness is selected.

[0073] Table 5 Ink filter paper thickness investigation

[0074]

[0075] 2.3.3 Blood Filter Membrane Investigation

[0076] The present invention uses a whole blood filter membrane as the plasma separation part. The micropores in the membrane can intercept blood cells in the whole blood and provide capillary driving force to achieve the separation of blood cells and serum. Various blood filter membranes with different parameters are investigated to observe whether they can effectively intercept red blood cells. The final filter membrane selected is the blood filter membrane SZ0883. The membrane climbing speed, water absorption rate, etc. are more in line with the requirements of this experiment. Its main model parameters are shown in Table 3.

[0077] 2.3.4 Investigation of the loading amount of whole blood samples and diluents

[0078] After the whole blood separation microfluidic paper chip structure is built, different volumes of blood samples and diluent saline are added to the sample loading area as shown in Table 6. Observe within a period of time whether the whole blood sample can be separated into plasma by the microfluidic paper chip structure, eliminate the interference of red blood cells, flow into and cover the color development area without red, and examine the appropriate loading volume of whole blood samples and diluents.

[0079] Table 6 Whole blood sample and diluent loading amount inspection table

[0080]

[0081] 2.4 Channelless gourd-type system

[0082] like Figure 3 As shown, all the color-developing paper chips in this system are gourd-shaped paper chips without channels ( Figure 3 A), which is composed of three circular areas connected. The whole blood separation module uses a circular blood filter membrane and a circular ink paper double layer stack ( Figure 3 In B), the two parts are Figure 3 It can be obtained by superimposing and combining as shown in Figure C.

[0083] 2.4.1 Investigation of total liquid loading volume

[0084] After the whole blood separation microfluidic paper chip structure was built, 15, 20, 25, 30, and 35 μL of diluted saline solution were added to the sample loading area respectively. It was observed over a period of time whether the liquid could flow from the sample loading area through the microfluidic paper chip structure and cover the color development area. The results were recorded and analyzed by photographing. The optimal total sample loading volume was 30 μL.

[0085] 2.4.2 Investigation of the loading amount of whole blood samples and diluents

[0086] After the whole blood separation microfluidic paper chip structure is completed, different volumes of blood samples and diluent saline are added to the gourd-shaped paper chip loading area as shown in Table 7, and it is observed over a period of time whether the whole blood sample can be separated into plasma by the microfluidic paper chip structure, eliminate the interference of red blood cells, flow into and cover the color development area without showing red, and examine the appropriate loading volume of whole blood samples and diluents.

[0087] Table 7 Whole blood sample and diluent loading amount inspection table

[0088]

[0089]

[0090] 2.5 Slender channel system

[0091] like Figure 4 As shown, the color developing paper chip of the system is a double-hole elongated channel paper chip ( Figure 4 The whole blood separation module uses a circular blood filter membrane and a circular ink paper double layer stack ( Figure 4 In B), the two parts are Figure 4 It can be obtained by superimposing and combining as shown in Figure C.

[0092] 2.5.1 Investigation of total liquid loading volume

[0093] After the whole blood separation microfluidic paper chip structure was built, 15, 20, 25, 30, and 35 μL of diluted saline solution were added to the sample loading area respectively. It was observed over a period of time whether the liquid could flow from the sample loading area through the microfluidic paper chip structure and cover the color development area. The results were recorded and analyzed by photographing. The optimal total sample loading volume was 30 μL.

[0094] 2.5.2 Investigation of the loading amount of whole blood samples and diluents

[0095] After the whole blood separation microfluidic paper chip structure is completed, different volumes of blood samples and diluent saline are added to the sample loading area of ​​the slender channel paper chip as shown in Table 8, and the whole blood sample is observed over a period of time to see whether the plasma can be separated by the microfluidic paper chip structure, the interference of red blood cells is eliminated, and the whole blood sample flows into and covers the color development area without showing red, and the appropriate loading volume of the whole blood sample and the diluent is examined.

[0096] Table 8 Whole blood sample and diluent loading amount inspection table

[0097]

[0098] 2.6 Single-hole superimposed permeability system

[0099] like Figure 5 As shown, the color developing paper chip of this system is a circular paper chip ( Figure 5 The outer ring is a 1mm wide circular hydrophobic enclosure formed by heating and melting the wax printer. The whole blood separation module uses a circular blood filter membrane and a circular ink paper double layer stack ( Figure 5 In B), the two parts are Figure 5 It can be obtained by superimposing and combining as shown in Figure C.

[0100] 2.6.1 Investigation of total liquid loading volume

[0101] After the whole blood separation microfluidic paper chip structure was built, 15, 20, 25, 30, and 35 μL of diluent were added to the sample loading area to observe whether the liquid could flow from the sample loading area through the microfluidic paper chip structure and cover the color development area within a period of time. The results were recorded and analyzed by taking photos. The optimal total sample loading volume was 35 μL.

[0102] 2.6.2 Investigation of the loading amount of whole blood samples and diluents

[0103] After the whole blood separation microfluidic paper chip structure is built, different volumes of blood samples and diluent saline are added as shown in Table 9 to observe whether the whole blood sample can be separated into plasma by the microfluidic paper chip structure within a period of time, eliminating the interference of red blood cells, flowing into and covering the color development area without showing red, and examining the appropriate loading volume of whole blood samples and diluents.

[0104] Table 9 Investigation of the loading amount of whole blood samples and diluents

[0105]

[0106]

[0107] 2.7 Channel Bridge System

[0108] 2.7.1 Design and production of paper chips

[0109] The shape and structure of the paper chip were designed using inkscape software. The designed pattern was printed on Whatman No. 1 qualitative filter paper using a wax printer, and then heated at 175°C for 2 minutes in a hot press. The solid wax melted and penetrated into the paper to form a hydrophobic barrier. Figure 6 As shown in A, the overall symmetry mainly includes the uric acid color development part and the plasma separation part, which are combined in a superimposed manner ( Figure 6 (B). The uric acid color development part is mainly composed of a rectangular paper chip. The hydrophobic structure on the surface of the paper chip (20mm×10mm) is made by printing the designed paper chip document on the chromatography paper using a wax jet printer and then placing it on a hot press and heating it at 176℃ for 2min. The non-hydrophobic areas at both ends are circular with a diameter of 4mm, and paper tape is attached to the bottom to prevent penetration. The plasma separation and extraction part is mainly composed of a rectangular blood filter membrane. The rectangular blood filter membrane (10mm×2.0mm) is horizontally fixed in the middle of the two circular non-hydrophobic areas above the hydrophobic paper chip, and the two ends are in contact with the two circular non-hydrophobic structure areas, one end is the sample loading area, and the other end is the color development area.

[0110] 2.7.2 Blood Filter Membrane Investigation

[0111] Related studies have found that the red blood cell migration area is determined by the microscopic pores of the blood filter membrane and is not affected by the macroscopic appearance. Therefore, the present invention selects a 10mm×2mm rectangular blood filter membrane with a simple structure and easy processing as the core device for plasma separation. The blood filter membrane is selected as SZ0883 type.

[0112] 2.7.3 Investigation of the loading amount of whole blood samples and diluents

[0113] The whole blood separation microfluidic paper chip structure is Figure 6 After the construction shown in B is completed, different volumes of blood samples and diluent saline are added as shown in Table 10, and it is observed over a period of time whether the whole blood sample can be separated into serum by the microfluidic paper chip structure, eliminate the interference of red blood cells, flow into and cover the color development area without showing red, and examine the appropriate loading volume of whole blood samples and diluents.

[0114] Table 10 Investigation of the loading amount of whole blood samples and diluents

[0115] serial number 1 2 3 4 5 6 Whole blood (μL) 5 6 7 8 9 10 Dilution (μL) 10 9 8 7 6 5

[0116] 2.8 Whole blood uric acid test

[0117] The present invention finally selected the channel bridge system, based on which the uric acid in the actual sample was quantitatively analyzed. The specific operation process is as follows: Figure 7 As shown. First, a whole blood separation color paper chip was made according to the above method. The fingertip venous blood of 12 volunteers was obtained by acupuncture. 10 μL was transferred to the sample area. Driven by capillary force, the whole blood was sucked into the blood filter membrane. When the fingertip blood filled the sample area, 5 μL of physiological saline was added to the sample area. The diluted sample contacted the front end of the blood filter membrane from the circular edge of the sample area. The diluted blood in the sample area was transferred to the whole blood filter membrane under capillary action. The red blood cells were intercepted by the whole blood filter membrane, and the serum continued to flow forward. After 45 seconds, a clear separation line of red blood cells and serum appeared on the whole blood filter membrane. The serum flowed into the tail end of the whole blood filter membrane and gradually covered the color development area, and underwent redox and color development reaction with the FeCl3 color developer in the color development area. After 210 seconds, the color development area of ​​the paper chip was uniformly colored, which was used as the reaction time. Finally, the chromaticity value of the color development area was identified and data analysis was performed to obtain the test results.

[0118] 3. Results and analysis

[0119] 3.1 Factors affecting uric acid chromogenic signal amplifier

[0120] The effect of signal amplifier chitosan on color development was investigated. The results showed that the greater the chitosan concentration, the darker the color. Studies have shown that chitosan complexes with metal ions in a liquid state, which will intensify the complex aggregation of ions, thereby deepening the color intensity of the metal complex reaction. The color value of color developer No. 2 with 0.050% chitosan as the diluent increased the most, as shown in Table 11. However, when the chitosan solution was actually prepared, it was found that if the concentration of the prepared chitosan solution was greater than 0.050%, it was easy to precipitate at room temperature. Therefore, the subsequent experimental color development was prepared according to the formula of color developer No. 2.

[0121] Table 11 Color development results of different color developers

[0122]

[0123] 3.2 Double-hole single-channel barbell separation system

[0124] When using ink paper with a thickness of 1 mm, when the total liquid volume added is less than or equal to 30 μL, the liquid cannot completely cover the color development area in the double-hole single-channel paper chip structure with a channel length of 6 mm, 8 mm, and 10 mm ( Figure 8 ), the total volume should be 35μL. When using 0.6mm thick oil filter paper, the total volume should be 25μL ( Figure 8 ). Red blood cells can be successfully trapped through the middle rectangular channel.

[0125] When using oil filter paper with a thickness of 1mm and 0.6mm, the total liquid loading volume should be 35μL and 25μL respectively. The analysis results show that 1mm thick oil filter paper has a better effect in intercepting red blood cells, so 1mm thick oil filter paper is selected, and the total loading volume is 35μL. The experimental results show ( Fig. 9 , Photo No. 12), the best red blood cell separation effect was achieved when the blood sample volume was 8 μL and the saline solution was 27 μL. The serum flowed from the channel to the color development area to achieve the separation of whole blood serum, but the required diluent saline solution volume was large and the blood dilution multiple was large, resulting in a decrease in uric acid concentration. At the same time, the color developer in the color development area was washed to the edge of the paper chip and no obvious color was developed.

[0126] 3.3 Gourd-shaped separation system

[0127] The gourd-shaped separation system is formed by connecting three circular areas, such as Fig.10 As shown, the experimental results show that ( Fig.10In the figure (A), the best red blood cell separation effect is achieved when the blood sample volume is 5 μL and the saline is 25 μL. Red blood cells can be successfully retained and not flow to the front color development area. The saline with water-soluble components quickly flows to the front color development area, which can achieve the separation of whole blood serum. Because there is no channel, the time for the sample to flow to the color development area is shortened. However, because the volume of the required diluent saline is large and the blood dilution multiple is large, the uric acid concentration is reduced and there is no obvious color development.

[0128] 3.4 Slender channel separation system

[0129] The elongated channel separation system can successfully intercept red blood cells through the middle rectangular channel, allowing serum to flow to the color development area to achieve whole blood separation. Fig.11 As shown, the color developer is washed to the edge of the paper chip, and finally the system cannot develop color uniformly.

[0130] 3.5 Single-pore superposition permeation separation system

[0131] The single-hole superposition permeation separation system combines ink paper and whole blood filter membrane Fig.12 As shown in A, it is superimposed on a circular paper chip, with saline as the diluent carrying uric acid ( Fig.12 B) penetrates to the point where the developer ( Fig.12 The experimental results showed that the best red blood cell separation effect was achieved when the blood sample volume was 8 μL and the saline solution was 27 μL ( Fig.12 Middle C). The middle whole blood filter can successfully intercept red blood cells, achieve the separation of whole blood serum, and evenly display color on the paper chip ( Fig.12 Middle E). The color development effect is good, but the color development time is more than 10 minutes, which is too long and does not meet the requirements of instant detection.

[0132] 3.6-channel bridge separation system

[0133] The channel bridge separation system can achieve red blood cell interception through a rectangular blood filter membrane ( Fig.13 ), the serum flows to the color development area through capillary action, and the hydrophobic structure between the two holes reduces the loss of water-soluble components such as uric acid. The blood and saline loading volumes are 10μL and 5μL respectively. The red blood cells are completely intercepted by the blood filter membrane, eliminating their interference with color development, and the color development effect is good ( Fig.13 ). At the same time, the color development time is shortened to 210s, which basically meets the requirements of instant detection. For practical application, the color development time needs to be further shortened.

[0134] 3.7 Whole blood uric acid test

[0135] Comparing the whole blood separation results of all the above paper chip structures, the channel bridge structure has the best separation effect and color development results. Therefore, it is determined to use the channel bridge separation structure for actual blood sample testing. The whole blood sample is tested, and the RGB value measured in the experiment is substituted into the serum uric acid standard curve obtained in the previous experiment ( Fig.14 ), the linear regression equation is: y = 0.0889x + 28.44, R 2 =0.9916, the uric acid concentration of the whole blood sample was obtained by the standard curve, and the results are shown in Table 12. The test results are in line with the provisions of the document No. 156 of the State Food and Drug Administration

[2010] . The portable uric acid test results are compared with the test results of the hospital's fully automated biochemical analyzer. The allowable error is: uric acid concentration ≤ 300 μmol·L -1 Absolute deviation does not exceed ±60%, uric acid concentration>300μmol·L -1 The relative deviation does not exceed ±20%. Compared with the test results of a uric acid detector on the market, the method established by the present invention has a smaller error and is more accurate than the hospital results.

[0136] Table 12 Whole blood uric acid concentration and RGB value

[0137]

[0138] The uric acid test results of the whole blood samples obtained by the present invention ( Fig.15 ) and the results of the hospital's fully automatic biochemical analyzer were analyzed for significance. The results are shown in Table 13. P-value>0.05 and F crit>F indicate that there is no significant difference in the test results of the two methods, and the test results of the method established by the present invention are reliable.

[0139] Table 13 Significance analysis results

[0140] Source of Difference SS df MS F P-value Fcrit Between groups 703.7148309 1 703.7148309 0.115257415 0.740104055 4.747225347 Within group 73267.11207 12 6105.592672 total 73970.8269 13

[0141] 3.8 Precision experiment

[0142] The precision of this experimental method was investigated using 10 μL whole blood as a sample. The experimental results are shown in Table 14 and Fig.16 The intra-day precision of RGB mean value was 1.3%-1.9% (n=5), and the inter-day precision was 2.0% (n=5), with good precision.

[0143] Table 14 Repeatability data table

[0144]

[0145] It can be seen from the above experimental results that the present invention has established a whole blood separation-dry method uric acid detection system based on microfluidic paper chips. A systematic investigation was conducted on the preparation of solutions, image acquisition, paper chip design and production, design and production of different separation systems, blood and diluent loading, and optimization of serum uric acid color development system. A stable whole blood uric acid detection system has been established, laying the foundation for the development of whole blood uric acid on-site instant detection and intelligent and convenient uric acid analyzers.

[0146] The paper chip structure design includes double-hole single-channel barbell type, gourd type without channel, slender channel type, single-hole superimposed osmosis type and channel bridge type. Whole blood separation is achieved by changing the shape of the paper chip and the length and presence of the channel. Finally, the channel bridge type paper chip is determined based on the separation results. (1) Whole blood sample pretreatment. The present invention tried 17 different types of whole blood filter membranes produced by different companies. According to factors such as red blood cell retention effect and cost, the SZ0883 whole blood filter membrane was finally selected as the core part of blood separation. The loading amount of normal saline for whole blood and diluted blood samples under different separation structures was investigated. Finally, it was determined that the loading amount of blood for the channel bridge type was 10μL and normal saline was 5μL, the loading amount of blood for the barbell type and single-hole superimposed osmosis type was 8μL and normal saline was 27μL, and the loading amount of blood for the gourd type and slender channel type was 5μL and normal saline was 25μL. (2) Color development results. The problem that the double-hole single-channel type cannot develop color evenly and the single-hole superposition penetration type takes too long to develop color is solved. The channel bridge separation structure is used to shorten the detection time to 3.5 minutes. (3) Whole blood sample testing. The whole blood RGB value obtained in the experiment is substituted into the serum uric acid standard curve established in the early stage to calculate the whole blood uric acid content. The test results are compared with the test results of the hospital's fully automatic biochemical analyzer. The error is within 20%, which is in line with the provisions of the National Food and Drug Administration

[2010] No. 156 document. The results of the significance analysis show that there is no significant difference between this experimental method and the hospital test, and the method is reliable.

[0147] The present invention performs online pretreatment of whole blood through a SZ0883 whole blood separation membrane, establishes a sample pretreatment method that can be used for whole blood uric acid detection, and makes it possible to use μPADs for uric acid POCT.

[0148] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a whole blood separation paper chip in the preparation of a product for detecting uric acid content in blood, characterized in that: The method for detecting the uric acid content in blood using the whole blood separation paper chip comprises: Adding the whole blood sample to be tested and physiological saline to the sample loading area, observing the color development of the color development area, and calculating the content of uric acid in the whole blood sample to be tested according to the RGB value of the color development area; The loading volume of the loading area is 10 μL of the whole blood sample to be tested and 5 μL of physiological saline; The color developer in the color development area includes the following components: 26 μL of 10% FeCl3, 104 μL of o-phenanthroline and 470 μL of 0.05% chitosan; The whole blood separation paper chip includes a uric acid color development paper chip and a plasma separation module; The uric acid colorimetric paper chip is a rectangular paper chip, with a hydrophilic circular structure set on both sides of the surface of the rectangular paper chip, and other areas are set as hydrophobic structures; a paper tape is attached to the bottom of the circular structure; The plasma separation module is a rectangular blood filter membrane, which is horizontally fixed between the two circular structures, with two ends of the rectangular blood filter membrane contacting the two circular structures respectively; The specification of the rectangular paper chip is 20 mm×10 mm, the diameter of the circular structure is 4 mm, and the specification of the rectangular blood filter membrane is 10 mm×2.0 mm; The two circular structures are respectively a sample loading area and a color development area.

2. The use according to claim 1, characterized in that The hydrophobic structure is made by printing a designed paper chip document on chromatography paper using a wax jet printer and then placing the paper chip document on a heat press and heating it at 176° C. for 2 minutes.

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