CN108745428B - Multi-channel three-dimensional microfluidic paper chip and preparation method thereof - Google Patents
Multi-channel three-dimensional microfluidic paper chip and preparation method thereof Download PDFInfo
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- CN108745428B CN108745428B CN201810568835.5A CN201810568835A CN108745428B CN 108745428 B CN108745428 B CN 108745428B CN 201810568835 A CN201810568835 A CN 201810568835A CN 108745428 B CN108745428 B CN 108745428B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
Abstract
The invention discloses a multi-channel three-dimensional microfluidic paper chip and a preparation method thereof. The multi-channel three-dimensional microfluidic paper chip provided by the invention realizes simultaneous, rapid and sensitive determination of different substances, and can be used in the fields of biochemical analysis, immunoassay, sensor, instant detection device research and the like.
Description
Technical Field
The invention relates to a multi-channel three-dimensional microfluidic paper chip and a preparation method thereof, and belongs to the technical field of microfluidic paper chip manufacturing.
Background
The Whitesides group first proposed the concept of paper chips in 2007, where paper chips are used as a reaction carrier and corresponding hydrophilic and hydrophobic regions are constructed on the surface of paper by various methods, mainly including photolithography, inkjet printing, paraffin printing, screen printing, drawing, laser processing, and cutting. The sample and the reaction solution are driven by the capillary force of the paper, so that the directional flow of the liquid in the hydrophilic channel is realized, and the novel paper chip detection technology integrating a series of operations such as sample introduction, reaction and detection is finally realized by combining specific detection reaction. The paper chip has the characteristics of low cost, good biocompatibility, less sample consumption, flexible design, easy miniaturization, portability, integration and the like. As a new research field, the paper chip is rapidly developed in recent years, and provides a novel cheap detection platform for medical diagnosis, environmental monitoring and food safety monitoring. At present, the manufacturing method of the paper chip is mature day by day, and the research focus is shifted to the realization of the rapid, high-sensitivity and simultaneous detection of multiple substances by using the paper chip.
Among the paper chip detection methods, the colorimetric method is the most common method, and the paper chip colorimetric analysis method determines the content of a sample by changing the color on a paper chip, such as a Human Chorionic Gonadotropin (HCG) detection test strip, a urine test strip, and the like. However, the traditional paper chip colorimetric method only can give a yes/no result or a semi-quantitative result by using naked eyes for distinguishing, has low detection sensitivity and is not beneficial to accurate quantitative detection of low-content samples. In addition, the conventional paper chip colorimetric analysis method has the disadvantages of uneven color development, poor sensitivity and poor reproducibility due to the serious capillary diffusion phenomenon of the color development reagent.
In recent years, the incidence of diabetes, gout and cardiovascular diseases has increased dramatically, seriously harming human health. The detection of small molecules such as glucose, uric acid, cholesterol, choline and the like in human body fluid is particularly important for the early discovery and early diagnosis of the diseases. Although many methods for detecting the above markers have been developed, there is no research or patent report on a low-cost, portable paper-based detection method that can be used for simultaneously detecting the above four targets with high throughput and high sensitivity.
Disclosure of Invention
The invention aims to provide a multi-channel three-dimensional microfluidic paper chip and a preparation method thereof. The multi-channel three-dimensional microfluidic paper chip can be used for simultaneously detecting glucose, uric acid, cholesterol, choline and the like, realizes the simultaneous, rapid and sensitive determination of different substances, and can be used in the fields of biochemical analysis, immunoassay, sensors, instant detection device research and the like.
The invention relates to a multi-channel three-dimensional microfluidic paper chip, which is characterized in that two pieces of upper filter paper A and lower filter paper B printed with different microfluidic channels are correspondingly superposed to form a three-dimensional microfluidic channel system;
the central point of upper filter paper A puts and is provided with into appearance district, with it outwards extends many elongated circulation passageways as the center to advance the appearance district the epitaxial end of elongated circulation passageway is provided with the inspection hole respectively, be provided with hydrophobic interval between the epitaxial end of elongated circulation passageway and the inspection hole that corresponds.
The lengths of the plurality of long flow channels are the same; the plurality of long-type circulation channels extend outwards by taking the sample injection area as a circle center, and included angles formed between adjacent long-type circulation channels are the same.
Chitosan, specific oxidase, horseradish peroxidase and a color reagent are sequentially arranged in the detection holes, and the proportion has no specific requirement.
The specific oxidase is adjusted according to different detection objects, and comprises glucose oxidase, urate oxidase, cholesterol oxidase, choline oxidase, heme oxidase, lactate oxidase, glycerophosphate oxidase and the like.
The color reagent is selected from a mixed solution of No. 1-4-aminoantipyrine and 3, 5-dichloro sodium o-hydroxybenzenesulfonate or a mixed solution of No. 2-4-aminoantipyrine and N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt or No. 3-tetramethylbenzidine.
In the color reagent No. 1, the concentration of 4-aminoantipyrine is 4mmol/L, and the concentration of 3, 5-dichloro-o-hydroxyben-zenesulfonate is 8 mmol/L; in the color reagent No. 2, the concentration of 4-aminoantipyrine is 4mmol/L, and the concentration of N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium is 8 mmol/L; in the color reagent No. 3, the concentration of tetramethylbenzidine was 15 mmol/L.
Preferably, each detection object is provided with 2-3 detection holes, and the chromogenic reagents added into the 2-3 detection holes are respectively different, so that the combined quantification is carried out through chromogenic reactions with different colors respectively. For example, when glucose, uric acid, cholesterol and choline are required to be quantified jointly through two chromogenic reactions of different colors, eight long flow channels are required to be arranged, and eight corresponding detection holes are provided.
And a plurality of short type circulation channels are arranged on the lower layer filter paper B, and hydrophobic intervals are arranged among the short type circulation channels. The number of the short flow channels is adjusted according to the number of the long flow channels, and the short flow channels and the long flow channels are the same.
After the upper filter paper A and the lower filter paper B are correspondingly superposed, each short circulation channel respectively covers the hydrophobic interval between the extension end of each long circulation channel and the corresponding detection hole and is partially overlapped with the extension end of each long circulation channel and the corresponding detection hole, so that a three-dimensional circulation channel is constructed between the extension end of each long circulation channel and the corresponding detection hole.
The filter paper used for the upper filter paper a and the lower filter paper B is preferably Whatman No. 1 filter paper.
In the upper layer of filter paper A, a sample inlet area, a long flow channel and a detection hole are directly constructed through filter paper, and the rest parts are made of hydrophobic materials; in the lower filter paper B, the short flow channel is directly constructed through the filter paper, and the rest part is made of hydrophobic materials.
The hydrophobic material is preferably paraffin.
The preparation method of the multichannel three-dimensional microfluidic paper chip comprises the following steps:
step 1: firstly, designing a paper chip model by using drawing software to obtain a silk-screen printing template of the paper chip model, uniformly distributing hydrophobic materials on corresponding positions of filter paper in a silk-screen printing or printing mode to obtain upper filter paper A and lower filter paper B, then placing the upper filter paper A and the lower filter paper B into a constant-temperature drying box, and drying for 3 minutes at 120 ℃;
step 2: sequentially adding chitosan, a color developing reagent, specific oxidase and horseradish peroxidase into the detection holes of the upper filter paper A layer by layer, and drying at room temperature;
and step 3: and correspondingly overlapping and fixing the upper filter paper A and the lower filter paper B, wherein each short circulation channel covers the hydrophobic interval between the extension end of each long circulation channel and the corresponding detection hole, and is partially overlapped with the extension end of each long circulation channel and the corresponding detection hole, so that a three-dimensional circulation channel is constructed between the extension end of each long circulation channel and the corresponding detection hole, and the multi-channel three-dimensional microfluidic paper chip can be obtained.
In the detection process, the solution to be detected is dripped into the sample inlet area and then shunted through the plurality of long flow channels, and the solution is transferred from the upper flow channel to the lower flow channel and then transferred back to the upper detection area.
The invention realizes the simultaneous detection of glucose, uric acid, cholesterol and choline in the following ways: fixing chitosan on a paper chip, further fixing a color reagent, specific oxidase and horseradish peroxidase on the paper chip modified by the chitosan, and protecting and stabilizing the color reagent and the enzyme on the paper chip by the chitosan on the paper chip. Glucose, uric acid, cholesterol and choline generate hydrogen peroxide under the catalysis of respective specific oxidases, the generated hydrogen peroxide oxidizes 4-aminoantipyrine under the catalysis of horseradish peroxidase, and the chromogenic reaction generates pink and purple substances respectively under the condition that 3, 5-dichloro-o-hydroxy-benzene sodium sulfonate and N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt exist. Glucose, uric acid, cholesterol and choline are subjected to joint quantification through two chromogenic reactions with different colors respectively.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the paper chip, the paper substrate of the detection area is modified by chitosan, and the chitosan protects and stabilizes the chromogenic reagent and enzyme in the detection area of the paper chip, so that the problem of uneven color development caused by the capillary diffusion phenomenon of the reagent in the detection area is solved; the paper chip transports the sample solution through the three-dimensional flow channel, so that the sample solution is not easy to migrate to the original flow channel after reaching the detection area, the problems of diffusion and uneven color development caused by migration of the reagent to the flow channel are solved, and the effects of improving the color development uniformity of the paper chip colorimetric analysis method and improving the detection sensitivity of the paper chip colorimetric analysis are achieved.
2. The paper chip of the invention respectively uses two or more chromogenic reactions with different colors to carry out combined quantification on the same target (glucose, uric acid, cholesterol, choline, bilirubin, lactic acid or triglyceride), thereby achieving the effects of improving the detection sensitivity and expanding the detection linear range.
3. The invention combines an enzyme detection system with a microfluidic technology to manufacture the three-dimensional microfluidic paper chip, the preparation method of the multi-channel three-dimensional microfluidic paper chip is simple, low in cost, green and environment-friendly, can realize shunting and multi-channel detection of samples, and is a new technology suitable for developing rapid detection and instant diagnosis and analysis devices.
Drawings
FIG. 1 is a structural diagram of a multi-channel three-dimensional microfluidic paper chip according to the present invention; wherein: a, a schematic diagram of upper-layer filter paper A of the three-dimensional microfluidic paper chip; b, a schematic diagram of lower-layer filter paper B of the three-dimensional detection paper chip.
Reference numbers in the figures: 1-8 are detection holes, wherein 1 and 2 are glucose detection holes, 3 and 4 are uric acid detection holes, 5 and 6 are cholesterol detection holes, and 7 and 8 are choline detection holes; and 9 is a sample injection area.
FIG. 2 is a schematic diagram of a three-dimensional microfluidic channel system formed by correspondingly overlapping an upper filter paper A and a lower filter paper B.
FIG. 3 is a graph of the total gray scale value of two detection zones for different concentrations of glucose in example 1.
FIG. 4 is a graph of the relationship between uric acid concentrations and total gray level values of two detection zones in example 1.
FIG. 5 is a graph of the total gray scale values for two detection zones for cholesterol at different concentrations in example 1.
FIG. 6 is a graph of the relationship between choline at different concentrations and the total gray value of two detection zones in example 1.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples. The embodiments of the present invention, and all other embodiments obtained by a person of ordinary skill in the art without any inventive work, belong to the scope of protection of the present invention.
Example 1:
the specific manufacturing, assembling and detecting processes are as follows:
1. firstly, a paper chip model is designed by utilizing Computer Aided Design (CAD) drawing software, wherein the paper chip model has two sides and comprises an upper layer filter paper A and a lower layer filter paper B (shown in figure 1). The design parameters of the upper filter paper A and the lower filter paper B are as follows:
upper filter paper A has a diameter and is 10 mm's introduction area 9, introduction area 9 uses the same contained angle to extend eight elongated circulation passageways to eight directions, the width of elongated circulation passageway is 3mm, length is 8mm, the epitaxial end of eight elongated circulation passageways is provided with the circular shape inspection hole respectively, the hydrophobic clearance of interval 2mm between the epitaxial end of elongated circulation passageway and the inspection hole that corresponds thereof, the diameter of eight inspection holes is 6 mm. Eight detection holes are numbered from 1 to 8: wherein, No. 1 and No. 2 are glucose detection holes; no. 3 and No. 4 are uric acid detection holes; no. 5 and No. 6 are cholesterol test holes; no. 7 and 8 are choline detection wells.
The lower filter paper B is provided with eight short-type flow channels with the width of 4mm and the length of 7mm, and hydrophobic intervals are arranged among the short-type flow channels. After the upper filter paper a and the lower filter paper B are aligned and overlapped (as shown in fig. 2), each short flow channel covers the hydrophobic space between the extension end of each long flow channel and the corresponding detection hole, and is partially overlapped with the extension end of each long flow channel and the corresponding detection hole, so that a three-dimensional flow channel is constructed between the extension end of each long flow channel and the corresponding detection hole. The overlapping length of each short flow channel and the extension end of the long flow channel is 2mm, and the overlapping length of each short flow channel and the detection hole is 3 mm.
2. According to the paper chip model, a silk-screen printing template is prepared, Whatman No. 1 filter paper is used as a paper base material, paraffin is used as a hydrophobic material, hydrophobic areas and hydrophilic channels are printed on the upper layer filter paper A and the lower layer filter paper B respectively through silk-screen printing, then the paper is placed into a constant-temperature drying oven and dried for 3 minutes at 120 ℃, so that the paraffin is melted and permeates the thickness of the whole paper, and a hydrophilic-hydrophobic microchannel network is formed.
3. In the invention, the construction process of the detection system of the three-dimensional microfluidic paper chip is as follows:
3a, dissolving chitosan in 0.25% (v/v) glacial acetic acid to prepare a 1mg/mL chitosan solution, respectively dropping 5.0. mu.L of the 1mg/mL chitosan solution into eight detection holes labeled 1-8 on the upper filter paper A, and drying at room temperature for 3 minutes.
3b, respectively dripping 3.0 mu L of No. 1 color reagent into four detection holes with the labels of 1, 3,5 and 7 on the upper layer A of the paper chip; 3.0 μ L of the color reagent No. 2 was added dropwise to four detection wells of the upper layer A of the paper chip, labeled 2, 4, 6, and 8, respectively, and dried at room temperature for 3 minutes.
Wherein, the No. 1 color reagent is: the mixed solution of 4-aminoantipyrine and 3, 5-dichloro sodium ortho-hydroxybenzenesulfonate, wherein the concentration of the 4-aminoantipyrine is 4mmol/L, and the concentration of the 3, 5-dichloro sodium ortho-hydroxybenzenesulfonate is 8 mmol/L; no. 2 of the color reagent is as follows: the mixed solution of 4-aminoantipyrine and N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt is prepared, wherein the concentration of the 4-aminoantipyrine is 4mmol/L, and the concentration of the N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt is 8 mmol/L.
And 3c, preparing a detection solution of glucose, uric acid, cholesterol and choline, wherein the detection solution comprises specific oxidase and horseradish peroxidase.
The glucose detection solution comprises: 170U/mL glucose oxidase dissolved in phosphate buffered saline at pH 7.0 and 0.15mg/mL horseradish peroxidase dissolved in phosphate buffered saline at pH 5.6.
The uric acid detection solution comprises: 80U/mL urate oxidase dissolved in phosphate buffer solution of pH 8.5 and 0.15mg/mL horseradish peroxidase dissolved in phosphate buffer solution of pH 5.6.
The cholesterol detection solution comprises: 50U/mL cholesterol oxidase dissolved in phosphate buffered saline at pH 8.0 and 0.15mg/mL horseradish peroxidase dissolved in phosphate buffered saline at pH 5.6.
The choline detection solution comprises: 50U/mL choline oxidase dissolved in phosphate buffered saline at pH 8.0 and horseradish peroxidase dissolved in phosphate buffered saline at pH 5.6 at 0.15 mg/mL.
3d, respectively dripping 3.0 mu L of detection solution of glucose, uric acid, cholesterol and choline into the detection hole of the upper filter paper A, wherein the No. 1 detection hole contains a chromogenic reagent 1, and dripping the glucose detection solution; the No. 2 detection hole contains a chromogenic reagent 2, and a glucose detection solution is dripped; the No. 3 detection hole contains a color reagent 1, and a uric acid detection solution is dripped; the No. 4 detection hole contains a color reagent 2, and a uric acid detection solution is dripped; the No. 5 detection hole contains a chromogenic reagent 1, and a cholesterol detection solution is dripped; the No. 6 detection hole contains a color development reagent 2, and a cholesterol detection solution is dripped; the No. 7 detection hole contains a chromogenic reagent 1, and a choline detection solution is dropwise added; and (3) adding a choline detection solution dropwise into the detection hole No. 8 containing the chromogenic reagent 2, and drying at room temperature for 3 minutes.
And 3e, aligning and overlapping the upper filter paper A and the lower filter paper B, and fixing the overlapped upper filter paper A and the lower filter paper B by using a clamp (as shown in figure 2), so that the three-dimensional microfluidic paper chip for simultaneously detecting the glucose, the uric acid, the cholesterol and the choline is obtained.
4. The use method of the three-dimensional microfluidic paper chip comprises the following steps:
and 4a, transferring 120 mu L of liquid to be detected by using a liquid transfer gun, adding the liquid to be detected into the sample injection region 9 of the three-dimensional microfluidic paper chip, standing for 10min, allowing the liquid to be detected to directionally migrate to the eight long flow channels through the sample injection region 9, migrating to the hydrophobic region at the top end of the long flow channel, allowing the liquid to be detected to downwards migrate to the short flow channel at the lower layer, and then directionally migrating to the detection hole region at the upper layer through the short flow channel to form a three-dimensional flow path.
4b, different coupled color developers show different colors. If the liquid to be detected only contains glucose, the detection hole with the label 1 of the microfluidic paper chip is pink, and the detection hole with the label 2 is purple; if the liquid to be detected only contains uric acid, the detection hole with the label 3 of the microfluidic paper chip is pink, and the detection hole with the label 4 is purple; if the liquid to be detected only contains cholesterol, the detection hole with the label 5 of the microfluidic paper chip is pink, and the detection hole with the label 6 is purple; if the liquid to be detected only contains choline, the detection hole with the label of 7 of the microfluidic paper chip is pink, and the detection hole with the label of 8 is purple. If the liquid to be detected contains glucose, uric acid, cholesterol and choline at the same time, the detection holes with the labels of 1, 3,5 and 7 of the microfluidic paper chip are pink, and the detection holes with the labels of 2, 4, 6 and 8 are purple.
And 4c, placing the three-dimensional microfluidic paper chip above the LED lamp panel, using a mobile phone, a camera and other equipment to obtain a microfluidic paper chip color development picture, reading the gray value of the detection area by using Image J software, processing experimental data by using origin software, and obtaining a relation graph between the concentrations of glucose, uric acid, cholesterol and choline and the total gray value of the gray value of each of 2 detection holes. The relationship between the glucose, uric acid, cholesterol and choline at different concentrations and the total value of the gray values of the 2 detection holes is shown in fig. 3, 4, 5 and 6. Therefore, the three-dimensional microfluidic paper chip can realize the simultaneous detection of glucose, uric acid, cholesterol and choline.
Example 2:
to prove the beneficial effects of the invention, the inventor uses the paper chip of example 1 to detect the concentrations of glucose, uric acid, cholesterol and choline in the actual blood sample by the following specific method:
the "Computer Aid Design (CAD) drawing software" in example 1 was changed to "Photoshop CS4 software"; the "screen printing" in example 1 was changed to "wax-jet printing"; preparing a three-dimensional microfluidic paper chip for simultaneously detecting glucose, uric acid, cholesterol and choline according to the steps 1, 2 and 3 in the embodiment 1; dropping 120 mu L of ultrafiltered serum sample in a sample entering area in the center of a paper chip, reacting for 10min, photographing by using a mobile phone to obtain a color development picture, acquiring gray value data of each detection area by using Adobe Photoshop software, judging whether glucose, uric acid, cholesterol and choline exist according to the color development conditions of eight detection holes of the paper chip, calculating the contents of the glucose, the uric acid, the cholesterol and the choline in the serum sample according to a linear regression equation of a standard curve obtained by an experiment if the contents of the glucose, the uric acid, the cholesterol and the choline exist, wherein the detection result is shown in table 1, and the detection result shows that the serum sample contains 4.54mmol/L of glucose, 0.42mmol/L of uric acid and 0.28mmol/L of free cholesterol, and the concentration of the free choline is lower than the detection limit of 0.06 mmol/L. The detection result is consistent with the contents of glucose, uric acid, free cholesterol and free choline in a normal human serum sample. In this example, the detection accuracy of the paper chip of this example was further examined by using a spiked recovery experiment, and a standard solution of 1mmol/L glucose, 0.1mmol/L uric acid, 0.2mmol/L cholesterol and 0.2mmol/L choline was quantitatively added to the above blood sample, so as to detect the contents of glucose, uric acid, cholesterol and choline in the spiked blood sample, and calculate the standard deviation between the recovery rate and the detection data, and the detection results are shown in Table 1. As can be seen from Table 1, the recovery rate is between 90% and 110%, the standard deviation is less than 5%, and the standard addition recovery experiment data is ideal, and the data show that the paper chip of the embodiment can realize the simultaneous detection of glucose, uric acid, cholesterol and choline in an actual blood sample, and the detection result is accurate.
Table 1 the paper chip of this example detects glucose, uric acid, cholesterol and choline in serum.
Example 3:
the protection scope of the invention is not limited to the simultaneous detection of glucose, uric acid, cholesterol and choline, and the three-dimensional microfluidic paper chip for the simultaneous detection of heme, lactic acid and triglyceride is prepared according to the method for preparing the multi-channel three-dimensional microfluidic paper chip in step 1 and step 2 of the embodiment 1, and the specific method is as follows:
1. "Computer Aid Design (CAD) drawing software" in example 1 was changed to "vector drawing software Adobe Illustrator"; the "screen printing" in example 1 was changed to "wax-jet printing"; the 'eight flow channels' in the embodiment 1 is changed into 'nine flow channels'; the "eight detection wells" in example 1 were changed to "nine detection wells". The preparation of three-dimensional microfluidic paper channels was carried out as described in example 1, step 1) and step 2).
2. In the invention, the construction process of the detection system of the three-dimensional microfluidic paper chip for simultaneously detecting the heme, the lactic acid and the triglyceride is as follows:
a. chitosan was dissolved in 0.25% (v/v) glacial acetic acid to prepare a 1mg/mL chitosan solution, 5.0. mu.L of the 1mg/mL chitosan solution was added dropwise to nine detection wells in the upper layer of the paper chip, and the mixture was dried at room temperature for 3 minutes.
b. Dripping 3.0 mu L of No. 1 color reagent into three detection holes with the upper layer labels of 1, 4 and 7 on the paper chip respectively; respectively dripping 3.0 mu L of No. 2 color reagent into three detection holes with the upper layer labels of 2, 5 and 8 on the paper chip; 3.0 mu L of the chromogenic reagent No. 3 is respectively dripped into three detection holes with the upper layer numbers of 3, 6 and 9 on the paper chip. Dried at room temperature for 3 minutes.
Wherein, the No. 1 color reagent is: 4 mmol/L4-aminoantipyrine and 8 mmol/L3, 5 dichloro-sodium ortho-hydroxybenzenesulfonate mixed solution; no. 2 of the color reagent is as follows: 4mmol/L of 4-aminoantipyrine and 8mmol/L N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt mixed solution; no. 3 of the color reagent is as follows: 15mmol/L tetramethylbenzidine.
c. Preparing a detection solution of heme, lactic acid and triglyceride, which comprises specific oxidase, horseradish peroxidase and a chromogenic reagent.
The heme detection solution comprises: 50U/mL heme oxidase and 0.15mg/mL horseradish peroxidase.
The lactic acid detection solution comprises: lactate oxidase 50U/mL and horseradish peroxidase 0.15 mg/mL.
The triglyceride detection solution comprises: 50U/mL glycerophosphate oxidase and 0.15mg/mL horseradish peroxidase.
d. Dripping 3.0 μ L of detection solution of heme, lactic acid and triglyceride into nine detection wells, dripping detection solution of heme into detection wells No. 1, No. 2 and No. 3, wherein detection well No. 1 contains chromogenic reagent No. 1, and detection well No. 2 contains chromogenic reagent No. 2; the No. 3 detection hole contains a chromogenic reagent 3; 4. dropwise adding a detection solution of lactic acid into the detection holes No. 5 and No. 6, wherein the detection hole No. 4 contains a chromogenic reagent No. 1, and the detection hole No. 5 contains a chromogenic reagent No. 2; no. 6 detection hole contains a chromogenic reagent 3; 7. dropping detection solution of triglyceride into the detection holes 8 and 9, wherein the detection hole 7 contains a chromogenic reagent No. 1, and the detection hole 8 contains a chromogenic reagent No. 2; the No. 9 detection hole contains a color reagent 3. Dried at room temperature for 3 minutes.
e. And aligning and overlapping the upper layer of the paper chip and the lower layer of the paper chip, and fixing the paper chip and the lower layer of the paper chip by using a clamp to obtain the three-dimensional microfluidic paper chip for simultaneously detecting the heme, the lactic acid and the triglyceride.
3. The use method of the three-dimensional microfluidic paper chip comprises the following steps:
120 μ L of sample was dropped on the sample entry area in the center of the paper chip. After the reaction is carried out for 10min, a developed picture is obtained by photographing with a mobile phone, and gray value data of a detection area is collected by Adobe Photoshop software. And processing experimental data by using origin software to obtain a relational graph between the concentrations of the heme, the lactic acid and the triglyceride and the total gray value of the 3 detection holes. And calculating the contents of the heme, the lactic acid and the triglyceride in the sample according to a linear regression equation of the standard curve obtained by the experiment.
Claims (9)
1. A multichannel three-dimensional microfluidic paper chip is characterized in that: the multi-channel three-dimensional microfluidic paper chip is formed by correspondingly superposing an upper layer filter paper A and a lower layer filter paper B printed with different microfluidic channels to form a three-dimensional microfluidic channel system;
a sample inlet area is arranged at the central position of the upper filter paper A, a plurality of long circulating channels extend outwards by taking the sample inlet area as the center, detection holes are respectively arranged at the extension ends of the long circulating channels, and a drainage interval is arranged between the extension end of each long circulating channel and the corresponding detection hole;
chitosan, specific oxidase, horseradish peroxidase and a color developing reagent are sequentially arranged in the detection holes;
a plurality of short-type flow channels are arranged on the lower layer filter paper B, and hydrophobic intervals are arranged among the short-type flow channels; the number of the short flow channels is adjusted according to the number of the long flow channels, and the short flow channels and the long flow channels are the same;
after the upper filter paper A and the lower filter paper B are correspondingly superposed, each short circulation channel respectively covers the hydrophobic interval between the extension end of each long circulation channel and the corresponding detection hole and is partially overlapped with the extension end of each long circulation channel and the corresponding detection hole, so that a three-dimensional circulation channel is constructed between the extension end of each long circulation channel and the corresponding detection hole;
the multichannel three-dimensional microfluidic paper chip can realize simultaneous detection of glucose, uric acid, cholesterol and choline.
2. The multi-channel three-dimensional microfluidic paper chip of claim 1, wherein:
the lengths of the plurality of long flow channels are the same; the plurality of long-type circulation channels extend outwards by taking the sample injection area as a circle center, and included angles formed between adjacent long-type circulation channels are the same.
3. The multi-channel three-dimensional microfluidic paper chip of claim 1, wherein:
the specific oxidase is adjusted according to different detection objects, and comprises glucose oxidase, urate oxidase, cholesterol oxidase, choline oxidase, heme oxidase, lactate oxidase or glycerophosphate oxidase.
4. The multi-channel three-dimensional microfluidic paper chip of claim 3, wherein:
each detection object is provided with 2-3 detection holes, and the chromogenic reagents added in the 2-3 detection holes are respectively different, so that the combined quantification is carried out through chromogenic reactions of different colors.
5. The multi-channel three-dimensional microfluidic paper chip of claim 1, wherein:
the color reagent is selected from a mixed solution of No. 1-4-aminoantipyrine and 3, 5-dichloro sodium o-hydroxybenzenesulfonate or a mixed solution of No. 2-4-aminoantipyrine and N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt or No. 3-tetramethylbenzidine.
6. The multi-channel three-dimensional microfluidic paper chip of claim 5, wherein:
in the color reagent No. 1, the concentration of 4-aminoantipyrine is 4mmol/L, and the concentration of 3, 5-dichloro-o-hydroxyben-zenesulfonate is 8 mmol/L; in the color reagent No. 2, the concentration of 4-aminoantipyrine is 4mmol/L, and the concentration of N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium is 8 mmol/L; in the color reagent No. 3, the concentration of tetramethylbenzidine was 15 mmol/L.
7. The multi-channel three-dimensional microfluidic paper chip of claim 1, wherein:
the filter paper used for the upper filter paper A and the lower filter paper B is Whatman No. 1 filter paper.
8. The multi-channel three-dimensional microfluidic paper chip of claim 1, wherein:
in the upper layer of filter paper A, a sample inlet area, a long flow channel and a detection hole are directly constructed through filter paper, and the rest parts are made of hydrophobic materials; in the lower filter paper B, the short flow channel is directly constructed through the filter paper, and the rest part is made of hydrophobic materials.
9. A preparation method of the multi-channel three-dimensional microfluidic paper chip as claimed in any one of claims 1 to 8, which is characterized by comprising the following steps:
step 1: firstly, designing a paper chip model by using drawing software to obtain a silk-screen printing template of the paper chip model, uniformly distributing hydrophobic materials on corresponding positions of filter paper in a silk-screen printing or printing mode to obtain upper filter paper A and lower filter paper B, then placing the upper filter paper A and the lower filter paper B into a constant-temperature drying box, and drying for 3 minutes at 120 ℃;
step 2: sequentially adding chitosan, a color developing reagent, specific oxidase and horseradish peroxidase into the detection holes of the upper filter paper A layer by layer, and drying at room temperature;
and step 3: and correspondingly overlapping and fixing the upper filter paper A and the lower filter paper B, wherein each short circulation channel covers the hydrophobic interval between the extension end of each long circulation channel and the corresponding detection hole, and is partially overlapped with the extension end of each long circulation channel and the corresponding detection hole, so that a three-dimensional circulation channel is constructed between the extension end of each long circulation channel and the corresponding detection hole, and the multi-channel three-dimensional microfluidic paper chip can be obtained.
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