CN110975952B - Paper-based microfluid chip and preparation method and application thereof - Google Patents
Paper-based microfluid chip and preparation method and application thereof Download PDFInfo
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- CN110975952B CN110975952B CN201911260934.8A CN201911260934A CN110975952B CN 110975952 B CN110975952 B CN 110975952B CN 201911260934 A CN201911260934 A CN 201911260934A CN 110975952 B CN110975952 B CN 110975952B
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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
- B01L3/502707—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 characterised by the manufacture of the container or its components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
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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
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
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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
- B01L2300/00—Additional constructional details
- B01L2300/12—Specific details about materials
- B01L2300/126—Paper
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00099—Characterised by type of test elements
- G01N2035/00158—Elements containing microarrays, i.e. "biochip"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
Abstract
The invention relates to a paper-based microfluid chip and a preparation method and application thereof, belonging to the technical field of paper-based chips. The substrate of the chip is a hydrophilic substrate, a part of the surface of the hydrophilic substrate is solidified with hydrophobic materials, and the hydrophobic materials are used for surrounding hydrophilic areas which are not covered by the hydrophobic materials on the hydrophilic substrate and are exposed so as to enable the hydrophilic areas to have hydrophobic boundaries; the hydrophilic area is provided with a cellulose suspension which is dehydrated and deposited to form a paper-based microfluidic channel. Preparing a hydrophobic seal, paving one side of the seal with the recess on a paraffin film, and heating the paraffin film to convert the paraffin film into a molten state; and placing the stamp on a hydrophilic substrate, solidifying molten paraffin on the surface of the hydrophilic substrate, and dripping the cellulose suspension on the surface of the substrate to obtain the chip. The chip has the advantages of simple manufacture, low cost, diversity of manufactured substrates, high channel resolution and the like, can be used as a biosensing platform, and is widely applied to instant detection under the condition of limited resources.
Description
Technical Field
The invention belongs to the technical field of paper-based chips, and particularly relates to a paper-based microfluid chip and a preparation method and application thereof.
Background
Mu PADs have attracted considerable interest to researchers since the first time Whitesids group proposed the concept of Microfluidic Paper-based analytical devices (mu PADs) in 2007. Compared with microfluidic devices manufactured on glass, silicon wafers and polymers, the micro-PADs have the advantages of low cost, convenience in use, convenience, degradability and no need of external force driving. The above features make μ PADs extremely suitable for point-of-care diagnostics, food analysis, and environmental monitoring, especially in less developed countries and resource-limited areas.
To date, many methods have been proposed for making μ PADs by patterning hydrophilic celluloses, including photolithography, plotting, cutting, wax impregnation, ink jet printing, screen printing, flexography, etcPlasma treatment, laser treatment and wet etching. However, the above methods have respective drawbacks. With photolithography, multiple materials, multiple time-consuming processing steps, and highly trained personnel are required. The drawing and cutting methods require a custom drawing machine and a computer controlled knife, respectively. The processes of bating, plasma treating, laser treating, ink jet printing, etc. each rely on specific and expensive equipment, such as wax printers, plasma oxidants, CO2Laser, ink jet printer, etc. Other methods, such as wax dipping, screen printing require a metal mask or die, which is typically cut by a linear cutter or laser cutter. Furthermore, the above methods are all top-down methods, in which a fluid channel is formed in a piece of paper by selectively delivering hydrophobic materials (wax, SU-8, polydimethylsiloxane, etc.), energy, or both to the paper. One limitation of the top-down approach is that the channel lateral resolution is higher than 200 μm. Additionally, Liuhong task group describes a bottom-up method for making μ PADs by knife coating a cellulose solution onto a patterned surface. In their work, the lateral resolution of the channels was 10 μm, however, wax jet printers were relied upon. In addition, their methods are hardly applicable to various substrates such as glass substrates widely used in biochemical experiments. Therefore, how to manufacture high resolution μ PADs in a simple and general method has yet to be realized.
Disclosure of Invention
The invention solves the technical problems that the paper-based microfluid chip in the prior art needs to depend on an ink-jet printer in the preparation process and has complicated process, provides a simple, low-cost and universal preparation method for preparing the paper-based microfluid chip by coating cellulose suspension on the patterned surface of paraffin, does not need any special equipment, is used for forming a hydrophobic seal of a paraffin film pattern on a target substrate, can be repeatedly used, and reduces the preparation cost.
According to a first aspect of the present invention, there is provided a paper-based microfluidic chip, the substrate of which is a hydrophilic substrate, on a partial area of which a hydrophobic material is cured, the hydrophobic material being used to surround hydrophilic areas on the hydrophilic substrate that are not covered by the hydrophobic material and are thus exposed, the hydrophilic areas being provided with hydrophobic boundaries; and, the cellulose suspension on the hydrophilic areas forms paper-based micro-fluid channels through dehydration deposition.
Preferably, the hydrophilic substrate is glass or a flexible polyester film with a surface hydrophilically treated; the flexible polyester film has a thickness of 50 μm to 100 μm.
Preferably, the hydrophilic region is in the shape of a circle, square, triangle, strip or flower.
According to another aspect of the present invention, there is provided a method of making any of the paper-based microfluidic chips, comprising the steps of:
(1) preparing a hydrophobic seal, wherein the seal is provided with a sunken structure;
(2) flatly paving the side, with the recess, of the stamp in the step (1) on a paraffin film, heating the paraffin film to enable the paraffin film to be converted into a molten state, and sticking molten paraffin to the protruding part of the stamp;
(3) placing the stamp with the protruding part adhered with the molten paraffin on the hydrophilic substrate, so that the molten paraffin on the stamp is solidified on the surface of the hydrophilic substrate, forming a hydrophobic area at the corresponding position of the protruding part of the stamp on the hydrophilic substrate, and tearing off the hydrophilic substrate;
(4) dropwise adding the cellulose suspension to the surface of the substrate formed with the hydrophobic areas obtained in the step (3), wherein the hydrophilic areas of the hydrophilic substrate form droplets of the cellulose suspension due to the hydrophilic-hydrophobic difference of the surface of the hydrophilic substrate; and after the solvent is evaporated, stacking the cellulose microfibrils in the liquid drop to form a paper-like structure, and obtaining the paper-based microfluid chip.
Preferably, the hydrophobic seal is a polydimethylsiloxane seal; the shape of the concave structure is round, square, triangular, strip-shaped or flower-shaped; the hydrophilic substrate is glass or a flexible polyester film with a surface subjected to hydrophilic treatment.
Preferably, the mass fraction of the cellulose suspension is 10% to 25%.
Preferably, the cellulose suspension is acidified, in particular: adding cellulose powder into acid solution, heating, centrifuging, dialyzing, and dissolving in water to obtain cellulose suspension.
According to another aspect of the present invention, there is provided the use of any one of the described paper-based microfluidic chips as a biosensing detection platform.
According to another aspect of the present invention, there is provided the use of a paper-based microfluidic chip comprising a paper-based microfluidic channel formed by dehydrating deposition of a cellulose suspension for the preparation of a microfluidic chip for determining blood glucose content.
Preferably, the application comprises the steps of:
s1: respectively dripping KI solutions with the same concentration and the same volume into a plurality of micro-fluid channels of the paper-based micro-fluid chip, or dripping KI solutions with the same concentration and the same volume into a certain pattern-type channel of the paper-based micro-fluid chip;
s2: after the KI solution dripped in the step S1 is dried, respectively dripping mixed solution of glucose oxidase and horseradish peroxidase into the microfluidic channels of the paper-based microfluidic chip, or dripping mixed solution of glucose oxidase and horseradish peroxidase into the pattern channel of the paper-based microfluidic chip; and drying the enzyme solution to obtain the biosensing detection platform for measuring the blood sugar content.
Generally, compared with the prior art, the technical scheme of the invention mainly has the following beneficial effects:
(1) the invention has simple manufacture and low cost, and the whole process only leads the cellulose water solution to pass through the surface of the substrate patterned by the paraffin film without any special equipment. In addition, the hydrophobic seal for forming the paraffin film pattern on the target substrate can be repeatedly used, so that the manufacturing cost is reduced.
(2) The invention has the advantages of multiple substrates, transparent non-paper pattern area and paper-based pattern which can be made on various substrates, and preferably a rigid glass substrate and a flexible polyester film. In addition, because the adopted manufacturing substrate is transparent, and the paraffin film is also transparent, the manufactured paper-based microfluidic chip is transparent except for the paper pattern.
(3) The invention has higher manufacturing precision. Due to the higher resolution of the photoetching technology, in the scheme, the minimum resolution of the manufactured paper chip channel can be 40 microns, and the minimum hydrophobic resolution between adjacent paper-based micro-channels can be 40 microns.
(4) The invention provides a method for manufacturing a microfluid paper chip from bottom to top by coating cellulose on the surface of a substrate patterned by a paraffin film. The manufacturing method is simple, low in cost and universal, and the manufactured paper chip device can be used as a biosensing detection platform and has the potential to be widely applied to instant detection under the condition of limited resources.
Drawings
FIG. 1 is a schematic diagram of the preparation of a paper-based microfluidic chip according to the present invention, including a three-dimensional schematic diagram of hydrophilic and hydrophobic surface preparation with paraffin film patterning and a schematic diagram of a paper-based microfluidic chip prepared by cellulose self-assembly, in which 1-PDMS stamp, 2-sealing film, 3-tin foil paper, 4-liquid paraffin at high temperature, 5-surface hydrophilic substrate, 6-solidified paraffin film, 7-water-soluble suspension of cellulose, 8-glass rod, and 9-paper-based pattern formed by dehydrated and deposited cellulose.
FIG. 2 is an SEM image of the boundary line of the paraffin film on the surface of the glass substrate, wherein the scale bar is 10 μm.
FIG. 3 is an SEM image of a cross section of a paraffin film on a glass substrate, wherein the scale bar is 20 μm.
FIG. 4 is an SEM image of a cross section of a paraffin film on a glass substrate, wherein the scale bar is 5 μm.
Fig. 5 is an SEM image of hydrophilic areas of different widths shaped paraffin film, with a scale of 500 μm.
FIG. 6 is a plot of the designed channel width versus the width of the hydrophilic region actually shaped by the paraffin film.
Fig. 7 is the water contact angle on a paraffin film.
Fig. 8 is a water contact angle on a glass substrate.
FIG. 9 is a droplet array of rhodamine solution molded from a paraffin film on a glass substrate, illustrating good uniformity, with a scale bar of 2 mm.
FIG. 10 is a timing diagram of the flow of an aqueous solution of Coomassie Brilliant blue in a paper-based pattern on a glass substrate, with a scale bar of 2 mm.
FIG. 11 is a scanning electron micrograph of a paper-based channel having a width of about 40 μm, with a scale bar of 10 μm.
FIG. 12 is a scanning electron micrograph of a paper-based channel having a width of about 80 μm, with a scale bar of 20 μm.
FIG. 13 is a micrograph of different widths of paper-based channels formed by drawing down a cellulose solution, with a scale bar of 500 μm.
FIG. 14 is a diagram of a liquid drop array formed by drawing a rhodamine water solution across a circular paper-based hydrophilic array and dehydrating the liquid drop array, wherein the scale is 2 mm.
Fig. 15 is a statistical distribution diagram of the gray scale intensity of the circular lattice in fig. 14.
Fig. 16 is a physical representation of a paper-based pattern formed on a flexible PET film.
FIG. 17 is a graph of the wetting of paper-based channels of different widths with a drop of Coomassie Brilliant blue solution at one end;
FIG. 18 is a graph of the wetting of hydrophobic barriers of different widths between adjacent channels with a drop of Coomassie Brilliant blue solution at one end; the scale bar in the figure is 2 mm.
FIG. 19 is a graph of a colorimetric reaction-based determination of glucose concentration in serum on a patterned paper-based microfluidic chip; the scale bar in the figure is 2 mm.
FIG. 20 is a calibration curve for glucose content.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides a microfluidic paper-based chip, which comprises a paper-based chip and a supporting substrate, wherein the paper-based chip is provided with a microfluidic channel, and the supporting substrate is positioned at the bottom of the paper-based chip; the paper-based chip is formed by dehydrating and depositing a cellulose suspension; the surface of the support substrate is modified by a paraffin film pattern, and the surface area has obvious hydrophilic-hydrophobic difference.
Preferably, the support substrate is a substrate such as glass, polyethylene terephthalate (PET) film, or the like.
Preferably, the thickness of the PET film is 50 to 100 μm.
Preferably, the cellulose solution is acidified by 64% sulfuric acid for 30min, and is prepared by centrifugation and dialysis.
Preferably, the mass fraction of the cellulose solution is 10% to 25%.
Preferably, the paraffin film pattern on the surface of the substrate is formed by lipophilic printing of Polydimethylsiloxane (PDMS).
According to another aspect of the present invention, there is provided a method of making a paper-based microfluidic chip, comprising the steps of:
(a) preparing a PDMS stamp with a pre-designed micro-pattern by using a standard photoetching technology and rapid forming;
(b) sequentially stacking the tinfoil paper, the commercial sealing film and the PDMS stamp obtained in the step (a) to form a sandwich structure, wherein the PDMS stamp is arranged on the uppermost layer, and the tinfoil paper is arranged on the lowermost layer. Then flatly placing the sandwich structure on a constant-temperature hot flat plate heated to 120 ℃, and pressing the PDMS stamp for about 30s by hands;
(c) due to the lipophilicity of PDMS and the melting of paraffin in the sealing film at high temperature, the PDMS stamp is torn off from the tin foil paper, so that the PDMS stamp attached with paraffin oil at high temperature is obtained, and the PDMS stamp is named as a PDMS-paraffin stamp;
(d) immediately placing the PDMS-paraffin seal obtained in the step (c) on a target substrate with a hydrophilic surface at normal temperature, and lightly pressing the PDMS-paraffin seal by hand for about 30 s. Because the high-temperature paraffin oil attached to the PDMS stamp is solidified at normal temperature, a layer of paraffin film is dyed on the surface of the target substrate, which is in contact with the PDMS-paraffin stamp, and the PDMS-paraffin stamp is torn away from the target substrate to obtain the hydrophilic and hydrophobic surface molded by the paraffin film;
(e) adding a large drop of a water-soluble solution of cellulose to the paraffin film patterned substrate surface obtained in step (d);
(f) drawing the cellulose suspension by a glass rod to pass through the surface of the substrate patterned by the paraffin film;
(g) since the paraffin is hydrophobic and the substrate surface is hydrophilic, the areas covered by the paraffin film are hydrophobic, while the areas not covered by the paraffin film are hydrophilic. When the cellulose is water-soluble across the paraffin film patterned substrate surface, the hydrophilic regions shaped according to the paraffin will retain the cellulose solution. After the water has evaporated, the cellulose microfibrils will pile up to form a paper-like structure.
Preferably, the target substrate in step (d) is glass or a PET film.
Preferably, the cellulose solution in the step (e) is acidified by 64% sulfuric acid for 30min, and is prepared by centrifugation and dialysis; the mass fraction of the cellulose solution is 10-25%.
According to another aspect of the present invention, there is provided a use of the paper-based microfluidic chip for preparing a biosensing test platform for measuring blood glucose level, comprising the steps of:
s1: respectively dripping KI solutions with the same concentration and the same volume into a plurality of micro-fluid channels of the paper-based micro-fluid chip, or dripping KI solutions with the same concentration and the same volume into a certain pattern-type channel of the paper-based micro-fluid chip;
s2: after the KI solution dripped in the step S1 is dried, respectively dripping mixed solution of glucose oxidase and horseradish peroxidase into the microfluidic channels of the paper-based microfluidic chip, or dripping mixed solution of glucose oxidase and horseradish peroxidase into the pattern channel of the paper-based microfluidic chip; and drying the enzyme solution to obtain the biosensing detection platform for measuring the blood sugar content.
The method for detecting the blood sugar content by the biosensing detection platform for determining the blood sugar content comprises the following steps:
(1) respectively dripping glucose solutions with different concentrations and samples to be detected into the microfluidic channels of the paper-based microfluidic chip, or respectively dripping glucose solutions with different concentrations and samples to be detected into different branches of the pattern channel of the paper-based microfluidic chip; the glucose is catalyzed and oxidized into H by the glucose oxidase2O2Then under the catalysis of horseradish peroxidase, the H2O2Oxidation of KI to I2(ii) a The brown yellow color is different in shade due to different glucose concentrations;
(2) and (2) constructing a standard curve according to the concentration of the glucose solution in the step (1) and the gray value in the corresponding microfluidic channel, or constructing a standard curve according to the concentration of the glucose solution in the step S3 and the gray value in different branches of the flower-type channel, and calculating to obtain the concentration of glucose in the sample to be detected.
Example 1
In this embodiment, a paper-based microfluidic chip is composed of two parts, a paper-based chip and a supporting substrate. As shown in fig. 1, the paper-based chip is formed by evaporating and depositing a cellulose aqueous solution. The supporting substrate is a substrate with obvious hydrophilic-hydrophobic difference on the patterned surface of the paraffin film and is positioned at the bottom of the paper-based chip.
Example 2
The preparation method of the microfluidic paper-based chip comprises the following steps:
(1) the method is characterized in that a PDMS (polydimethylsiloxane) fat-soluble printing is used for manufacturing a patterned hydrophilic and hydrophobic surface, as shown in the first four steps of FIG. 1, the specific manufacturing process can be divided into the following steps:
(1-1) preparing a PDMS stamp with a pre-designed micro-pattern by using a standard photoetching technology and rapid prototyping.
And (1-2) sequentially stacking the tinfoil paper, the commercial sealing film and the PDMS stamp to form a sandwich structure, wherein the PDMS stamp is arranged on the uppermost layer, and the tinfoil paper is arranged on the lowermost layer. The sandwich was then placed flat on a thermostatically heated plate heated to 120 ℃ and the PDMS stamp was pressed by hand for about 30 seconds.
(1-3) tearing off the PDMS stamp from the tin foil paper due to the lipophilicity of the PDMS and the melting of paraffin in the sealing film at high temperature to obtain the PDMS stamp attached with paraffin oil at high temperature, which is named as PDMS-paraffin stamp.
(1-4) immediately placing the PDMS-paraffin seal on a target substrate with a hydrophilic surface at the normal temperature, and lightly pressing the PDMS-paraffin seal by hand for about 30 s. Because the high-temperature paraffin oil attached to the PDMS stamp is solidified at normal temperature, a layer of paraffin film is dyed on the surface of the target substrate, which is in contact with the PDMS-paraffin stamp, and the PDMS-paraffin stamp is torn away from the target substrate, so that the hydrophilic and hydrophobic surface molded by the paraffin film is obtained.
(2) The method comprises the following steps of (1) preparing a microfluid paper-based pattern by coating a cellulose suspension on a patterned hydrophilic and hydrophobic surface in a scraping way, wherein the specific operation process can be divided into the following steps:
(2-1) A large drop of an aqueous solution of cellulose is applied to the surface of the substrate patterned with the paraffin film.
(2-2) drawing the cellulose suspension across the surface of the paraffin film patterned substrate with a glass rod.
(2-3) since the paraffin is hydrophobic and the substrate surface is hydrophilic, the area covered by the paraffin film is hydrophobic, and the area not covered by the paraffin film is hydrophilic. When the cellulose is water-soluble across the paraffin film patterned substrate surface, the hydrophilic regions shaped according to the paraffin will retain the cellulose solution. After the water has evaporated, the cellulose microfibrils will pile up to form a paper-like structure.
Example 3
In this example, we characterized the paraffin thin film patterned substrate surface with glass as the substrate. The areas covered and uncovered by the paraffin film showed distinct boundary lines, as shown in fig. 2. The paraffin thin film coated on the glass substrate exhibited a thickness of about 4 μm as measured by SEM, as shown in fig. 3 and 4. Due to the high resolution of the lithography technique, the minimum resolution of the micro-channels molded by the PDMS-wax stamp is about 20 μm, as shown in FIG. 5. We measured the actual dimensions of the pre-designed microchannels of different widths and plotted the results as shown in fig. 6. The micro-channel width for molding the paraffin film and the pre-designed micro-channel width keep better relevance and consistency. To show whether the patterned surface of the paraffin film can be used for shaping the liquid drop, we first measured the water contact angles on the paraffin film and on the clean glass surface, and add 5 μ L of water drop to the paraffin film and the clean glass surface respectively in the experiment, as shown in fig. 7 and fig. 8. The water contact angle on the paraffin film was about 113 ° (third-order average), and the water contact angle on the glass surface was about 25.9 ° (third-order average), both of which exhibited greater differences in water contact angle. We further fabricated a circular array substrate surface shaped with paraffin film. When 0.1% (w/v) rhodamine water soluble solution was drawn across the arrayed surface by a glass rod, an array of droplets was formed as shown in FIG. 9. The above results show that the PDMS-paraffin stamp can accurately form hydrophilic and hydrophobic patterns on a substrate with a hydrophilic surface.
Example 4
In this example, we show a paper-based chip fabricated on a paraffin film patterned substrate. To demonstrate the hydrophilicity of the produced μ PADs, we prepared a pattern-type paper-like chip using a 15% (w/w) cellulose solution. When 10 μ L of 0.1% (w/v) Coomassie brilliant blue aqueous solution was added to the center area of the pattern paper chip, the aqueous solution would penetrate inside the paper-based chip due to capillary effect throughout the entire paper-based pattern within 40s, as shown in FIG. 10. Scanning electron microscope observation shows that the paper-based micro-channel is composed of cellulose piles, no obvious cellulose is observed outside the channel, as shown in figures 11 and 12. Cellulose microfibers are preferred to fill the hydrophilic regions of the paraffin film shapes as shown in fig. 11-13. The minimum width of the paper-like passage produced by this scheme is about 40 μm, as shown in fig. 13. We further prepared a circular array of paper-based patterns. When 0.1% (w/v) rhodamine water-soluble solution was drawn across the circular paper-based array by a glass rod, only the paper-based pattern region retained the solution, and the paper-based pattern region and the non-paper-based pattern region exhibited significant color differences, as shown in fig. 14. The above results confirm the feasibility of preparing paper-based chips by coating cellulose on the surface of a paraffin film patterned hydrophilic substrate. We measured the intensity of the gray scale after dehydration of the rhodamine droplet array formed in fig. 14, and the statistical results are shown in fig. 15. The liquid drop array shows better consistency, can be applied to high-flux and consistent liquid drop arrays, and has potential application to paper chip-based drug screening. In addition to glass substrates, our method also enables the fabrication of paper-based chips on flexible substrates, such as flexible PET films, as shown in fig. 16.
Example 5
In this example, we investigated the resolution of the paper-based channel under wet conditions. To determine the minimum width of the wetted paper channel, we made paper-based micro-channels of different widths and dropped a coomassie aqueous solution at one end of the paper-based channel. As shown in fig. 17, the minimum width of the paper-based channel to be fully wetted is 400 μm; the narrower the channel width, the more difficult the solution flow channel. To determine the minimum width of the hydrophobic barrier between adjacent paper-based channels, we made paper-based patterns with different widths of hydrophobic barriers between adjacent paper-based channels and dropped a coomassie aqueous solution at one end of the paper-based channel. As shown in FIG. 18, the width of the smallest hydrophobic barrier between adjacent paper-based channels is about 40 μm.
Example 6
In this example we used a patterned paper chip for colorimetric analysis of the glucose content in serum. A flower-type paper-based pattern was made on the PET film, consisting of one central unit and eight detection areas. First, 10. mu.L of 0.6M KI solution was dropped into the central area of the flower-shaped paper chip. After the paper chip is dried, 0.3 mu L of mixed solution of glucose oxidase and horseradish peroxidase is respectively dripped into the eight detection areas. After drying the enzyme solution, we drop samples to eight detection zones, respectively. In the reaction, glucose is catalytically oxidized into H by glucose oxidase2O2Then under the catalysis of horseradish peroxidase, H2O2Oxidation of KI to I2And according to the difference of the glucose concentration, the displayed brown yellow color is different in shade, so that the quantitative analysis of the glucose in the sample is realized. FIG. 19 shows the use of a patterned paper chip on grapes in serumPhysical map of the quantitative analysis of sugars, where 6 detection zones were used for the standard sample and 2 zones were used for the serum sample. We measured the gray scale intensities of eight detection regions in three independent experiments. Within the range of 0-16mM, the gray scale intensity of the brown-yellow color and the concentration of glucose were linearly related, as shown in FIG. 20. The concentration of glucose in the sample determined by this method was 4.7. + -. 0.4 and 9.0. + -. 0.7mmol L-1And (4.9. + -. 0.2 and 9.8. + -. 0.3mmol L) determined by standard methods-1) Can be compared. The above results indicate that the designed flower type of μ PADs can be used as an advantageous platform for quantitative analysis of glucose content in serum samples.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (5)
1. The preparation method of the paper-based microfluid chip is characterized by comprising the following steps:
(1) preparing a hydrophobic seal, wherein the seal is provided with a sunken structure;
(2) flatly paving the side, with the recess, of the stamp in the step (1) on a paraffin film, heating the paraffin film to enable the paraffin film to be converted into a molten state, and sticking molten paraffin to the protruding part of the stamp;
(3) placing the stamp with the protruding part adhered with the molten paraffin on the hydrophilic substrate, so that the molten paraffin on the stamp is solidified on the surface of the hydrophilic substrate, forming a hydrophobic area at the corresponding position of the protruding part of the stamp on the hydrophilic substrate, and tearing off the hydrophilic substrate;
(4) dropwise adding the cellulose suspension to the surface of the substrate formed with the hydrophobic areas obtained in the step (3), wherein the hydrophilic areas of the hydrophilic substrate form droplets of the cellulose suspension due to the hydrophilic-hydrophobic difference of the surface of the hydrophilic substrate; after the solvent is evaporated, stacking the cellulose microfibrils in the liquid drop to form a paper-like structure, and obtaining the paper-based microfluid chip;
the substrate of the paper-based microfluidic chip is a hydrophilic substrate, hydrophobic materials are solidified on partial areas of the surface of the hydrophilic substrate, and the hydrophobic materials are used for surrounding hydrophilic areas which are not covered by the hydrophobic materials on the hydrophilic substrate and are exposed, so that the hydrophilic areas have hydrophobic boundaries; and, the cellulose suspension on the hydrophilic areas forms paper-based micro-fluid channels through dehydration deposition.
2. The method for preparing the paper-based microfluidic chip according to claim 1, wherein the hydrophobic stamp is a polydimethylsiloxane stamp; the shape of the concave structure is round, square, triangular, strip-shaped or flower-shaped; the hydrophilic substrate is glass or a flexible polyester film with a surface subjected to hydrophilic treatment.
3. The method for preparing a paper-based microfluidic chip according to claim 1, wherein the cellulose suspension has a mass fraction of 10% to 25%.
4. The method for preparing a paper-based microfluidic chip according to claim 1, wherein the cellulose suspension is subjected to an acidification treatment, specifically: adding cellulose powder into acid solution, heating, centrifuging, dialyzing, and dissolving in water to obtain cellulose suspension.
5. The application of the paper-based microfluidic chip prepared by the method according to any one of claims 1 to 4 in preparing a biosensing detection platform for measuring blood glucose content, which is characterized by comprising the following steps:
s1: respectively dripping KI solutions with the same concentration and the same volume into a plurality of micro-fluid channels of the paper-based micro-fluid chip, or dripping KI solutions with the same concentration and the same volume into a certain pattern-type channel of the paper-based micro-fluid chip;
s2: after the KI solution dripped in the step S1 is dried, respectively dripping mixed solution of glucose oxidase and horseradish peroxidase into the microfluidic channels of the paper-based microfluidic chip, or dripping mixed solution of glucose oxidase and horseradish peroxidase into the pattern channel of the paper-based microfluidic chip; and drying the enzyme solution to obtain the biosensing detection platform for measuring the blood sugar content.
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| CN112851821B (en) * | 2021-01-12 | 2022-07-05 | 山东省科学院生物研究所 | Fusion enzyme and application of fusion enzyme in paper-based biosensor |
| CN112973816B (en) * | 2021-04-15 | 2022-10-04 | 上海理工大学 | Differential shunt device and preparation method and application thereof |
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