CN115808390A - Microfluidic device for instantly detecting epinephrine and preparation method and use method thereof - Google Patents
Microfluidic device for instantly detecting epinephrine and preparation method and use method thereof Download PDFInfo
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- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 title claims abstract description 80
- 229930182837 (R)-adrenaline Natural products 0.000 title claims abstract description 77
- 229960005139 epinephrine Drugs 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 24
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- 238000010438 heat treatment Methods 0.000 claims description 28
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- 229920003169 water-soluble polymer Polymers 0.000 claims description 15
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 10
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- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 claims description 6
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a microfluidic device for instantly detecting epinephrine and a preparation method and a use method thereof. The invention utilizes the foldability of filter paper and the visualization of fluorescence color, wherein polyamines generate yellow fluorescent materials through the interaction with epinephrine, thereby determining the concentration range of epinephrine. The microfluidic device provided by the invention can effectively solve the problems of long detection period, complex operation and the like of epinephrine in the prior art, can effectively detect epinephrine, is convenient and rapid to detect, is easy to carry, and has a wide detection range.
Description
Technical Field
The application relates to the field of chemical detection, in particular to a micro-fluidic device for instantly detecting epinephrine and a preparation method and a using method thereof.
Background
Epinephrine (Ep) is a hormone and neurotransmitter secreted by the human body and released by the adrenal gland, which has been studied for many years as an important neurotransmitter and hormone due to its strong physiological properties and pharmacological functions. It is currently approved for use in a variety of conditions, including anaphylactic shock, the induction and maintenance of eye contraction during intraocular surgery, and hypotension from septic shock. Abnormal levels of Ep concentrations in urine and plasma have been used to diagnose and evaluate the therapeutic and pharmacological effects of neurological, psychiatric and cardiovascular diseases. Currently, many methods for detecting epinephrine in biological samples have been proposed, such as capillary electrochromatography, HPLC-MS, UPLC-MS/MS, and hydrophilic interaction chromatography, as well as electrochemical detection. But the method has weak specificity and complex operation; meanwhile, no simple device capable of realizing the instant detection of epinephrine is available on the market. Therefore, there is an urgent need to develop a simple device capable of realizing immediate detection of epinephrine.
Disclosure of Invention
Based on the above problems, the invention provides a micro-fluidic device for instantly detecting epinephrine and a preparation method and a use method thereof.
The technical scheme is as follows: in order to achieve the above object, the method for preparing a microfluidic device for instantly detecting epinephrine according to the present invention comprises the following steps:
s101: the filter paper provides a buffer layer and a detection layer in a laminating mode;
s102: defining closed sub-buffer regions and closed sub-detection regions on the buffer layer and the detection layer using a hydrophobic material;
s103: adding a first solution to the sub-buffer region, wherein the first solution is obtained by dispersing a water-soluble polymer in a buffer solution;
s104: adding a polyamine substance solution to the sub-detection region;
s105: and integrally heating the microfluidic device for immediately detecting the epinephrine.
In one embodiment, the filter paper is selected from the filter paper model 3Chr, 3MMChr, 2668Chr and 2727Chr for whatman chromatography.
In one embodiment, the hydrophobic material is black oil marker.
In one embodiment, the water-soluble polymer is selected from any one of polyethylene oxide, polyvinyl alcohol, and polyvinyl pyrrolidone.
In one embodiment, the buffer is selected from any one of birutan-robinson, phosphate buffer solution and HEPES buffer solution, the pH value of the buffer is 7.0, and the concentration of the buffer is 10mM.
In one embodiment, the concentration of the aqueous polymer in the buffer is from 5mg/mL to 10mg/mL.
In one embodiment, the polyamine is any one of putrescine, spermidine, spermine and cadaverine.
In one embodiment, the concentration of the polyamine substance solution is 0.1M.
In one embodiment, the heating temperature of step S105 is 60-80 ℃, and the heating time of step S105 is not more than 2min.
The invention relates to a micro-fluidic device prepared by the preparation method of the micro-fluidic device.
The invention relates to a use method of a microfluidic device in the instant detection of epinephrine.
The using method comprises the following steps:
s201: adding an object to be tested into the sub-buffer area;
s202: completely attaching the sub-buffer regions to the sub-detection regions;
s203: integrally heating the microfluidic device for instantly detecting epinephrine;
s204: and collecting a fluorescence image of the sub-detection area under the excitation of a 365nm ultraviolet lamp by using a spectrometer, analyzing RGB values of the colors of the fluorescence image through an intelligent terminal, and determining the concentration range of epinephrine according to the RGB values.
In one embodiment, the heating temperature of step S203 is 80-100 deg.C, and the heating time of step S203 is not less than 2min.
In one embodiment, before collecting the fluorescence image using the spectrometer, the method further includes the following step S203a: adding a reference solution to the sub-buffer region.
In one embodiment, the reference substance is selected from any one of fluorescent gold nanoclusters, fluorescent carbon quantum dots, fluorescent silicon quantum dots and fluorescent CdTe quantum dots, the reference substance shows red under ultraviolet illumination, and the concentration of the reference substance is 1mg/mL-5mg/mL.
The simple and easy-to-manufacture detection device formed by polyamine substances and water-soluble polymers on specific Woltmann chromatography filter paper can realize quick and instant detection of epinephrine, and has the advantages of low cost, high efficiency and wide application prospect.
Drawings
The following detailed description of embodiments of the invention is provided in conjunction with the appended drawings:
figure 1 shows a schematic view of a microfluidic device;
FIG. 2 shows the conditions (a) temperature and (b) reaction time for optimal instantaneous detection of epinephrine;
figure 3 shows a schematic of the process of microfluidic device for the immediate detection of epinephrine;
FIG. 4 shows a fluorescence image contrast before and after use of a buffer layer containing a water-soluble polymer;
figure 5 shows fluorescence images of microfluidic devices detecting different concentrations of epinephrine;
fig. 6 shows a variation curve of epinephrine concentration with respect to the corresponding fluorescence image RGB;
figure 7 shows a standard colorimetric card for the immediate measurement of epinephrine concentration.
Detailed Description
In order to clarify the invention in more detail, the technical solution of the invention is further elucidated below with reference to a preferred embodiment and the accompanying drawings.
In the present application, a portable, simple and easy-to-manufacture microfluidic device for real-time detection of epinephrine is proposed, which is prepared by the following steps:
the filter paper provides a buffer layer and a detection layer in a laminating mode; defining closed sub-buffer regions and closed sub-detection regions on the buffer layer and the detection layer by using a hydrophobic material; adding a first solution to the sub-buffer region, wherein the first solution is obtained by dispersing a water-soluble polymer in a buffer solution; adding a polyamine substance solution to the sub-detection region; and integrally heating the microfluidic device for instantly detecting the epinephrine. Fig. 1 is a schematic view of a microfluidic device manufactured by the above-described method of manufacturing a microfluidic device.
The application method of the microfluidic device for the real-time detection of epinephrine comprises the following steps:
adding an object to be tested into the sub-buffer area; completely attaching the sub-buffer regions to the sub-detection regions; integrally heating the microfluidic device for instantly detecting epinephrine; and collecting a fluorescence image of the sub-detection area under the excitation of a 365nm ultraviolet lamp by using a spectrometer, analyzing RGB values of the colors of the fluorescence image through an intelligent terminal, and determining the concentration range of epinephrine according to the RGB values.
In order to enable the instant detection of epinephrine on the microfluidic device to have the best fluorescence image color effect, the detection condition with the best fluorescence image color effect is determined by adjusting the heating reaction temperature and the heating reaction time in the reaction process of polyamine substances and epinephrine. In a number of exploratory experiments, referring to fig. 2a, when the optimal heating reaction temperature was explored, the heating reaction time was 2min; referring to fig. 2b, when the optimal heating reaction time was investigated, the heating reaction temperature was 90 ℃. Finally, the instant detection effect on the epinephrine is determined to be optimal under the conditions that the heating reaction temperature is 80-100 ℃ and the detection time is not less than 2min, wherein the instant detection effect is optimal when the heating reaction temperature is 90 ℃ and the detection time is 3 min.
Specifically, the microfluidic device and the real-time detection of epinephrine are prepared by combining the optimal results of the research experiment through the following steps: the model is 3MMChr Wlterman chromatography filter paper, and a buffer layer and a detection layer are provided in a laminating mode; defining closed sub-buffer regions and closed sub-detection regions on the buffer layer and the detection layer using a black oil marker; adding 30 mu L of a first solution to the sub-buffer region, wherein the first solution is obtained by adding 3.6mg of polyvinylpyrrolidone to 356.4 mu L of phosphate buffer solution with the pH value of 7; adding 20 mu L of spermine solution with the concentration of 0.1M to the sub-detection areas; and heating the whole micro-fluidic device for instantly detecting the epinephrine at 70 ℃ for 2min. Adding 60 μ L of epinephrine solution to the sub-buffer region; completely attaching the sub-buffer regions to the sub-detection regions; heating the whole micro-fluidic device for instantly detecting the epinephrine at 90 ℃ for 3min; adding 40 mu L of fluorescent gold nanocluster reference substance solution with the concentration of 5mg/mL into the sub buffer area; and collecting a fluorescence image of the sub-detection area under the excitation of a 365nm ultraviolet lamp by using a spectrometer, analyzing RGB values of the colors of the fluorescence image through an intelligent terminal, and determining the concentration range of epinephrine according to the RGB values. Fig. 3 is a schematic diagram of the whole process of preparing the microfluidic device and detecting epinephrine on-line.
In order to reduce or avoid the coffee ring effect of the solution on the Woltmann 3MMChr chromatographic filter paper, namely a phenomenon that the color of a drop liquid gradually deepens a ring from inside to outside after the drop liquid is air-dried on the surface of a solid, and the generation of the phenomenon is not favorable for the color uniformity of a fluorescence image when epinephrine is detected immediately. When designing a microfluidic device for epinephrine real-time detection, a folded paper dropped with a water-soluble polymer is used as a buffer layer of the detection device to reduce or avoid the coffee ring effect in the detection process. The water-soluble polymer introduced during the detection process is selected from any one of polyethylene oxide, polyvinyl alcohol and polyvinylpyrrolidone, and the introduction of the water-soluble polymer increases the viscosity of the liquid drop, so that the resistance to the radially outward flow is significantly increased, and the amount of solid particle deposition at the edge of the liquid drop is reduced. As shown in fig. 4, the fluorescence image contrast of the detection layer of the microfluidic device before and after introducing the water-soluble polymer clearly shows that the "coffee ring effect" on the detection device is effectively alleviated.
In addition, in order to enlarge the detection range of epinephrine and realize semi-quantitative visual detection, the real-time detection result of epinephrine is presentedThe method is characterized in that at least one of a fluorescent gold nanocluster, a fluorescent carbon quantum dot, a fluorescent silicon quantum dot and a fluorescent CdTe quantum dot (all of which show red under 365nm purple light) is introduced as an internal reference in the detection process to construct a ratio fluorescence mechanism. As shown in fig. 5, after the gold nanocluster AuNCs reference substance is introduced, as expected, the color of the fluorescence image on the detection layer of the constructed ratio fluorescence detection device shows various change trends, and the purpose of directly and semi-quantitatively detecting the target analyte by naked eyes is achieved. In the presence of the red fluorescent gold nanoclusters, with the reduction of epinephrine concentration, the red fluorescence of the gold nanoclusters is continuously enhanced, the yellow fluorescence of a fluorescent material generated by the reaction of spermine and epinephrine is continuously weakened, and finally the color of a fluorescent image is gradually changed from yellow to red. As can be seen from FIG. 6, when the concentration of epinephrine is in the range of 0.001-0.1mM, the concentration and (G-B)/R have a certain linear relationship, and the regression of the linear equation is (G-B)/R =2.25X-0.17 (R) 2 = 0.98). The limit of detection for epinephrine was calculated as low as 7.2 μ M using the detection limit formula (LOD =3N/S, N represents the standard deviation of blank data and S represents the slope of the regression line). FIG. 7 is a color standard card of real-time fluorescence image for detecting epinephrine. The concentration of the epinephrine is in the range of 0-5mM, the RGB values of the fluorescence image colors corresponding to different epinephrine concentrations are different, and the concentration range of the epinephrine can be quickly obtained by comparing the fluorescence image collected by the intelligent terminal with the RGB values of the standard card.
According to the optimization of the microfluidic device, the invention provides a preparation method of the microfluidic device for instantly detecting epinephrine, which comprises the following steps of:
s101: the filter paper provides a buffer layer and a detection layer in a laminating mode;
s102: defining closed sub-buffer regions and closed sub-detection regions on the buffer layer and the detection layer by using a hydrophobic material;
s103: adding a first solution to the sub-buffer region, wherein the first solution is obtained by dispersing a water-soluble polymer in a buffer solution;
s104: adding a polyamine substance solution to the sub-detection region;
s105: and integrally heating the microfluidic device for instantly detecting the epinephrine.
According to the research on the experimental conditions for instantly detecting epinephrine by using the microfluidic device, the invention provides a using method of the microfluidic device for instantly detecting epinephrine, which comprises the following steps of:
s201: adding an object to be tested into the sub-buffer area;
s202: completely attaching the sub-buffer regions to the sub-detection regions;
s203: integrally heating the microfluidic device for instantly detecting epinephrine;
s204: and collecting a fluorescence image of the sub-detection area under the excitation of a 365nm ultraviolet lamp by using a spectrometer, analyzing RGB values of the colors of the fluorescence image through an intelligent terminal, and determining the concentration range of epinephrine according to the RGB values.
The micro-fluidic device formed by polyamine substances and water-soluble polymers on specific Woltmann chromatography filter paper can realize rapid and instant detection of epinephrine. Meanwhile, according to the research experiment, in the process of immediately detecting epinephrine, the water-soluble polymer is selected from any one of polyethylene oxide, polyvinyl alcohol and polyvinyl pyrrolidone; the buffer solution is any one selected from birutan-robinson, phosphate buffer solution and HEPES buffer solution; the polyamine substance is any one of putrescine, spermidine, spermine and cadaverine; the concentration of the water-soluble polymer in the buffer solution is 5mg/mL-10mg/mL; further preferably, the closed pattern defined on the buffer layer and the detection layer by using a hydrophobic material is a circle; the filter paper is laminated with a buffer layer and a detection layer, and an isolation layer can be added at the same time.
Further, the instant detection of epinephrine proposed in the present invention may further include, after step S203, step S203a: adding a reference solution to the sub-buffer region. Preferably, the references are at least one of fluorescent gold nanoclusters, fluorescent carbon quantum dots, fluorescent silicon quantum dots and fluorescent CdTe quantum dots, and show red color under ultraviolet illumination, and the concentration of the references is 1mg/mL-5mg/mL.
The introduction of the reference substance can further improve the detection range of epinephrine, so that the detection range of epinephrine is expanded to 5mM, and the visual semi-quantitative detection of epinephrine can be realized.
The following describes the detection effect of the scheme proposed in the present application with reference to examples:
example 1:
detection of epinephrine in serum:
3MMChr Wlterman chromatography filter paper is laminated to obtain a buffer layer and a detection layer, an oily marker pen defines a closed circular sub-buffer area and a closed circular sub-detection area on the buffer layer and the detection layer, 30 mu L of polyvinylpyrrolidone phosphate buffer solution with the concentration of 10mg/mL and the pH =9 is respectively dripped into the sub-buffer area, 20 mu L of spermine solution with the concentration of 0.1M is respectively dripped into the sub-detection area, and then the whole prepared microfluidic device for instantly detecting adrenalin is placed in an oven at 70 ℃ and dried for 2min. And dripping 60 mu L of adrenaline solution with known concentration and prepared by serum into the sub-buffer area, completely attaching the sub-buffer area to the sub-detection area after dripping, placing the sub-buffer area in a 90 ℃ oven for heating for 3min, and dripping 40 mu L of fluorescent gold nanocluster reference substance solution with the concentration of 5mg/mL into the sub-buffer area after heating. The spectrometer captures a fluorescence image on the detection layer under 365nm ultraviolet lamp irradiation, and the RGB value of the fluorescence image color is analyzed through the intelligent terminal.
TABLE 1 detection of epinephrine in serum
The detection results are shown in Table 1, the recovery rate of the detection device of epinephrine in serum is in the range of 97.44-100.8%, and the relative standard deviation is less than or equal to 6.61%. This result demonstrates that the microfluidic device we developed has a higher accuracy in the detection of real samples.
Finally, it should be noted that: the above-mentioned embodiments are merely preferred examples for clearly illustrating the invention, but are not limited to the embodiments of the invention, and it should be understood by those skilled in the art that the technical features in the above-mentioned embodiments can be combined arbitrarily, and other modifications in different forms or equivalent replacements of part of the technical features can be made on the basis of the above-mentioned embodiments, and not all embodiments can be exhaustive, so that any modifications, improvements, equivalents and the like which are included in the technical solution of the present invention are within the technical scope of the claims of the present invention.
Claims (10)
1. Micro-fluidic device of real-time detection adrenaline, its characterized in that includes through buffer layer, the detection layer that stacks gradually the setting:
the buffer layer comprises a first solution, and the first solution is obtained by dispersing a water-soluble polymer in a buffer solution;
the detection layer comprises polyamine substance solution;
the buffer layer is provided with a plurality of closed sub-buffer areas which are regularly distributed and are limited by hydrophobic substances, and the first solution is distributed in each sub-buffer area;
the detection layer is provided with a plurality of closed sub-detection areas which are regularly distributed and defined by hydrophobic substances, and the polyamine substance solution is distributed in each sub-detection area;
the positions of the sub-buffer regions correspond to the positions of the sub-detection regions, and each sub-detection region completely covers the area of the corresponding sub-buffer region.
2. The microfluidic device for real-time epinephrine detection according to claim 1, wherein the aqueous polymer is at least one selected from polyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone; the buffer solution is at least one of Bertany-Robinson, phosphate buffer solution and HEPES buffer solution, the pH value of the buffer solution is 7.0, and the concentration of the buffer solution is 10mM; the concentration of the aqueous polymer in the buffer solution is 5mg/mL-10mg/mL; the polyamine substance is any one of putrescine, spermidine, spermine and cadaverine, and the concentration of the polyamine substance solution is 0.1M.
3. The microfluidic device for instantly detecting epinephrine according to claim 1, wherein the detection layer and the buffer layer are separated by folding filter paper at least once, and the filter paper is selected from the group consisting of whatman chromatography filter paper models 3Chr, 3MMChr, 2668Chr and 2727 Chr.
4. The microfluidic device for instantly detecting epinephrine according to claim 1, further comprising a reference in the buffer layer, wherein the reference is selected from any one of fluorescent gold nanoclusters, fluorescent carbon quantum dots, fluorescent silicon quantum dots and fluorescent CdTe quantum dots, and the reference shows red under ultraviolet light, and the concentration of the reference is 1mg/mL-5mg/mL.
5. The preparation method of the microfluidic device for instantly detecting epinephrine is characterized by comprising the following steps of:
s101: the filter paper provides a buffer layer and a detection layer in a laminating mode;
s102: defining closed sub-buffer regions and closed sub-detection regions on the buffer layer and the detection layer using a hydrophobic material;
s103: adding a first solution to the sub-buffer region, wherein the first solution is obtained by dispersing a water-soluble polymer in a buffer solution;
s104: adding a polyamine substance solution to the sub-detection region;
s105: and integrally heating the microfluidic device for instantly detecting the epinephrine.
6. The method for preparing a microfluidic device for immediately detecting epinephrine according to claim 5, wherein the heating temperature in the step S105 is 60-80 ℃; the heating time of the step S105 is not more than 2min.
7. The method of using the microfluidic device for the on-line detection of epinephrine according to any of claims 1 to 4, comprising the steps of:
s201: adding an object to be tested into the sub-buffer area;
s202: completely attaching the sub-buffer regions to the sub-detection regions;
s203: integrally heating the microfluidic device for instantly detecting epinephrine;
s204: and collecting a fluorescence image of the sub-detection area under the excitation of a 365nm ultraviolet lamp by using a spectrometer, analyzing RGB values of the colors of the fluorescence image through an intelligent terminal, and determining the concentration range of epinephrine according to the RGB values.
8. The use of the microfluidic device for the real-time detection of epinephrine according to claim 7, wherein the heating temperature of the step S203 is 80-100 ℃; the heating time of the step S203 is not less than 2min.
9. The method for using a microfluidic device for real-time epinephrine detection according to claim 7, further comprising the following step S203a before collecting the fluorescence image using a spectrometer: adding a reference solution to the sub-buffer region.
10. The use method of the microfluidic device for the real-time detection of epinephrine according to claim 9, wherein the reference is selected from any one of fluorescent gold nanoclusters, fluorescent carbon quantum dots, fluorescent silicon quantum dots and fluorescent CdTe quantum dots, the reference shows red under ultraviolet light, and the concentration of the reference is 1mg/mL-5mg/mL.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101978272A (en) * | 2008-03-27 | 2011-02-16 | 哈佛学院院长等 | Paper-based cellular arrays |
| WO2022034102A1 (en) * | 2020-08-14 | 2022-02-17 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Aqueous particle dispersion and process for forming an aqueous particle dispersion |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101978272A (en) * | 2008-03-27 | 2011-02-16 | 哈佛学院院长等 | Paper-based cellular arrays |
| WO2022034102A1 (en) * | 2020-08-14 | 2022-02-17 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Aqueous particle dispersion and process for forming an aqueous particle dispersion |
Non-Patent Citations (2)
| Title |
|---|
| JIA AN等: "Multichannel ratiometric fluorescence sensor arrays for rapid visual monitoring of epinephrine, norepinephrine, and levodopa", 《CHEMICAL ENGINEERING JOURNAL》, 9 June 2021 (2021-06-09), pages 1 - 7 * |
| SHENG-QUAN JIN ET AL: "A cost-effective Z-folding controlled liquid handling microfluidic paper analysis device for pathogen detection via ATP quantification", 《BIOSENSORS AND BIOELECTRONICS》, 2 August 2014 (2014-08-02), pages 379 - 383 * |
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