CN113985034A - Coding microneedle patch and preparation method and application thereof - Google Patents

Coding microneedle patch and preparation method and application thereof Download PDF

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Publication number
CN113985034A
CN113985034A CN202111259822.8A CN202111259822A CN113985034A CN 113985034 A CN113985034 A CN 113985034A CN 202111259822 A CN202111259822 A CN 202111259822A CN 113985034 A CN113985034 A CN 113985034A
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microneedle
coded
microneedle patch
biomarkers
micro
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杨圆
胥玲玲
赵超超
李舟
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Zhao Chaochao
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Zhao Chaochao
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Abstract

The invention discloses a coding microneedle patch and a preparation method and application thereof, wherein the coding microneedle patch comprises a microneedle base and a microneedle body; the micro-needle body is positioned on the micro-needle base and is used for encapsulating protein molecules for identifying biomarkers; the protein molecule is one or more of an antibody, an antigen or a biological enzyme; the types of the protein molecules are identified through the difference of the signal molecules or the micro-needle body arrays, and the quantitative detection of the biomarkers is realized through the specific combination of the biomarkers and the protein molecules, so that the simultaneous qualitative and quantitative detection of various biomarkers is realized, and the method has wide application prospect.

Description

Coding microneedle patch and preparation method and application thereof
Technical Field
The invention belongs to the field of biomolecule detection, and particularly relates to a coding microneedle patch as well as a preparation method and application thereof.
Background
At present, the blood examination or the urine examination is usually adopted to determine the biomarkers in the human body, but the blood examination damages the skin and causes pain, and the blood examination or the urine examination needs to be operated by professional personnel and has the risk of infection, so that patients have certain psychological conflict.
The skin interstitial fluid is a fluid existing in intercellular substance, is a medium for exchanging substances between blood and surrounding tissues, has substantially the same components as those in plasma except that it does not contain macromolecular proteins, and includes small molecules such as glucose, cholesterol and ascorbic acid, and various proteins such as growth factors, inflammatory factors and antibodies. In recent years, a certain amount of research is carried out on the detection of biomarkers by using microneedle technology to absorb a relatively large amount of skin interstitial fluid, and the detection of biomarkers is gradually an important mode due to low invasiveness, no pain and no blood cell interference. Patent literature (CN111544756A) reports a photosensitizer-loaded painless soluble microneedle, a microneedle array and a preparation method thereof, so as to solve the problem of unreasonable administration mode in photodynamic therapy; patent literature (CN104117137A) reports a capsule hollow drug-loaded microneedle array and a preparation method thereof, so as to solve the problem of fusion of delivered drugs and biological materials. After careful retrieval, the inventor finds that most researches adopt the micro-needle to extract interstitial fluid at present, and then a series of complex reactions are carried out in vitro through a complex extraction process to finally determine the content of the substance to be detected, so that the operation has the problems of complex flow and low efficiency; in addition, the micro needles are also arranged in a multi-way positive mode, and the loaded substance is single.
In order to promote the application of the micro-needle in the detection of the biological molecules and meet the requirement of instant detection, the invention provides the coded micro-needle patch which can rapidly distinguish the protein molecular species encapsulated by the micro-needle and realize the simultaneous rapid detection of various biological markers.
Disclosure of Invention
Aiming at the defects in the prior art, one of the purposes of the invention is to provide a coding microneedle patch, which realizes the rapid distinguishing of protein molecule types carried by microneedles and the simultaneous quantitative detection of various biomarkers, and has the advantages of simple operation and high efficiency.
Another object of the present invention is to provide a method for preparing the coded microneedle patch, which has the advantages of simple preparation process, high efficiency and convenience.
The last purpose of the invention is to provide the application of the coded microneedle patch in biomarker detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a coding microneedle patch comprises a microneedle base and a microneedle body; the micro-needle body is positioned on the micro-needle base and is used for encapsulating signal molecules and protein molecules for identifying biomarkers, the types of the protein molecules are identified through the difference of the signal molecules or the micro-needle body array, and the quantitative detection of the biomarkers is realized through the specific combination of the biomarkers and the protein molecules, so that the simultaneous qualitative and quantitative detection of various biomarkers is realized;
wherein, the protein molecule is one or more of antigen antibody and biological enzyme.
Preferably, the signal molecule is one or more of dye, fluorescence, photonic crystal and quantum dot.
Preferably, the dye is one or more of red, orange, yellow, green, cyan, blue and violet in color.
Preferably, the antigen antibody is one or more of TNF-alpha, IL-1, IL-6, CD80 or CD86, and the biological enzyme is one or more of glucose oxidase, cholesterol oxidase or ascorbate oxidase. The specific combination of corresponding components to be detected in the biomarker can be realized by using protein molecules, and the principle is that the antigen is specifically combined with the antibody, the cholesterol oxidase is specifically combined with cholesterol, the ascorbic acid oxidase is specifically combined with ascorbic acid, and if the glucose oxidase and glucose are based on enzyme-linked color development. Further, based on the specific combination of protein molecules and biomarkers, biological signals are amplified by dropping or soaking in fluorescent labeled antibodies, enzyme linked color development or electrochemical reaction, and finally, the quantitative detection of the corresponding biomarkers is realized by detecting the fluorescence intensity, color intensity, absorbance or Raman scattering intensity and other modes, so that the simultaneous qualitative or quantitative detection of various biomarkers is realized.
As a further preference of the technical scheme of the invention, the signal molecule for the protein molecule can be visible color or fluorescence with different colors, but the color is matched with the specificity of the protein molecule; as an example of visible color, microneedles carrying TNF antibodies can be labeled with a red base; marking the microneedle carrying the IL-1 antibody by using a blue base, and marking the microneedle carrying the TL-6 antibody by using a yellow base; when the protein antigen enters the micro-needle body to be specifically combined with the entrapped antibody, and under the action of a fluorescence-labeled secondary antibody, the content of the protein molecule is quantitatively judged according to the fluorescence intensity.
As a further preferred embodiment of the present invention, antibodies labeled with fluorescence of different colors can also be used, for example, TNF can be labeled with green-emitting FITC, IL-6 can be labeled with red-emitting PE, and quantitative and qualitative detection of protein molecules can be realized under the dual effects of fluorescence color and fluorescence intensity.
In a further preferred embodiment of the present invention, the microneedle body may include glucose oxidase and Tetramethylbenzidine (TMB) for small molecules such as glucose, and glucose in interstitial fluid reacts with the glucose oxidase to generate H2O2，H2O2Further reacts with TMB to make TMB develop color, so that the content of glucose can be determined according to the shade of color.
Preferably, the polymer material is one or more of collagen, cross-linked hyaluronic acid, chitosan, pectin, gelatin, polyethylene glycol diacrylate, polyvinyl alcohol, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, polyol, alginate, cyclodextrin, dextrin, fructose, starch, trehalose, maltose, lactose, lactulose, turanose, raffinose, melezitose, dextran, xylitol, polylactic acid, polyglycolic acid, polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid or polymaleic acid.
Preferably, the height of the microneedle body is 50 to 1500 μm.
The invention also provides a preparation method of the coding microneedle patch, which comprises the following steps:
(1) preparing a PDMS microneedle template: engraving a cavity of the microneedle body on the polydimethylsiloxane membrane by using a laser engraving machine to prepare a PDMS microneedle template;
(2) uniformly mixing a high molecular material, a signal molecule and a protein molecule to obtain a mixture;
(3) filling the cavity of the microneedle body: covering a mask on the PDMS microneedle template obtained in the step (1), coating the mixture obtained in the step (2) on the PDMS microneedle template, and uniformly filling the mixture into cavities of microneedle bodies;
(4) curing and demolding treatment: and (4) solidifying the microneedle filled with the mixture in the step (3), and demolding to obtain the coding microneedle patch.
Preferably, the filling in step (3) is performed by one or more of vacuum pumping, centrifugation or extrusion.
Preferably, the curing in step (4) is performed by one or more of vacuum drying, photo-curing and freeze-drying.
Meanwhile, the invention also claims the application of the coded microneedle patch in the detection of the biomarkers.
Compared with the prior art, the invention has the following beneficial effects:
(1) different protein molecules are loaded on the coding microneedle patch, and the different protein molecules are distinguished based on the arrangement characteristics or signal molecules of the microneedles, so that the types of the protein molecules loaded in different areas of the coding microneedle patch are discriminated; meanwhile, based on the specific combination of protein molecules and biomarkers, quantitative detection of the biomarkers is realized by adopting analysis means such as fluorescence analysis and Raman analysis, and finally, the simultaneous qualitative and quantitative detection of various biomarkers is realized.
(2) The invention is beneficial to distinguishing the categories of the microneedles in actual detection by introducing signal molecules into the microneedles or identifying the types of protein molecules carried by the microneedles through different microneedle arrays, and is simple and practical.
(3) The invention firstly proposes that different protein molecules are loaded in the micro-needle so as to achieve the purpose of simultaneously detecting various biological molecules, and the micro-needle is convenient and efficient.
(4) The coded microneedle patch prepared by the invention can realize amplification of biological signals through specific combination of protein molecules and biomarkers, and further carry out quantitative detection through fluorescence intensity, absorbance, color intensity and other modes, and has unique conception and simple method.
(5) The coded microneedle patch prepared by the invention has good application prospect, for example, a data acquisition and recognition system and an image processing system can be used together, a database combined with corresponding antibody and antigen is established, the coded microneedle patch of a corresponding patient is scanned, the coded microneedle patch is compared with the database after being processed by the image processing system, and the content of the biomarker can be obtained, so that the coded microneedle patch has good application prospect in disease diagnosis.
Drawings
Fig. 1 is a front view of the coded microneedle patch of example 1;
FIG. 2 is a top view of the color barcode on the base of the coded microneedle patch of example 1;
fig. 3 is a perspective view of the coded microneedle patch of example 1;
fig. 4 is a schematic view of the coded microneedle patch of example 2;
fig. 5 a schematic view of a coded microneedle patch of example 3;
fig. 6a schematic view of a coded microneedle patch of example 4;
figure 7a schematic view of a coded microneedle patch of example 5.
Wherein, 1, the microneedle base; 2. a color bar code; 21. a first color barcode; 22. a second color bar code; 23. a third color barcode; 3. a microneedle body; 31. a first microneedle body; 32. a second microneedle body; 33. a third microneedle body.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following 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.
Example 1
Referring to fig. 1 to 3, a coded microneedle patch comprises a microneedle base 1 and a microneedle body 3, wherein the microneedle body is located on the microneedle base, the microneedle body 5, the microneedle body 6 and the microneedle body 7 are used for encapsulating and identifying protein molecules of three inflammation factors TNF-alpha, IL-1 and IL-6, the coded microneedle patch identifies the types of the protein molecules through different colors of dyes, and the dyes form corresponding color bar codes 2 on the microneedle base at the bottom of the microneedle body.
In this embodiment, the color bar codes include a first color bar code 21, a second color bar code 22, and a third color bar code 23; the microneedle body includes a first microneedle body 31, a second microneedle body 32, and a third microneedle body 33.
In this embodiment, the material of the microneedle body is poly (ethylene glycol) diacrylate (PEGDA) with good biocompatibility, the size of the microneedle array is 6 × 10, and the microneedle array is divided into three groups, 2 × 10 microneedle arrays are one group, and each group of microneedle body arrays contains antibodies corresponding to three inflammation factors, i.e., TNF- α, IL-1 and IL-6.
In this embodiment, the PEGDA mixed with the red, blue, and yellow dyes is used to prepare barcodes of different colors for labeling different antibodies encapsulated by different microneedles. Specifically, microneedles encapsulating TNF- α antibodies, labeled with red barcodes; microneedles encapsulating IL-1 antibodies, labeled with blue barcodes; the micro-needle carrying the TL-6 antibody is marked by a yellow bar code, and the types of protein molecules in the micro-needles can be rapidly distinguished through the difference of the colors of the bar codes.
The coded microneedle patch in the present example is prepared by the following method:
(1) preparing a PDMS microneedle template: engraving a cavity of a microneedle body on a Polydimethylsiloxane (PDMS) membrane by using a laser engraving machine to prepare a PDMS microneedle template, wherein the size of a microneedle array is 6 x 10 and is divided into three groups, 2 x 10 microneedle arrays are one group, the height of the microneedles is 800 mu m, and the three groups of microneedle arrays contain antibodies corresponding to TNF-alpha, IL-1 and IL-6 inflammatory factors;
(2) directly mixing the three antibodies, the dye and the PEGDA material to obtain a mixture;
(3) filling the cavity of the microneedle body: covering a mask on the PDMS microneedle template obtained in the step (2), coating the mixture obtained in the step (2) on the PDMS microneedle template, and filling the mixture into the cavity of the microneedle body by adopting a vacuumizing method;
(4) curing and demolding treatment: and (4) solidifying the microneedle body filled in the step (3) by adopting a vacuum drying method, and obtaining the coding microneedle patch after demolding treatment.
The coded microneedle patch was used as follows: according to the principle of antibody antigen specific binding, after a microneedle penetrates into skin, an antibody carried by the microneedle body can specifically capture inflammatory factors, namely a TNF-alpha antibody is bound with the TNF-alpha inflammatory factors, an IL-1 antibody is bound with the IL-1 inflammatory factors, an IL-6 antibody is bound with the IL-6 inflammatory factors, and the types of the antibodies contained in the microneedle patch can be identified through the difference of colors of patch bases.
After sampling the encoded microneedle patch, quantitative analysis of both TNF- α antibodies and TNF- α inflammatory factors can be performed simultaneously. In this embodiment, the signal molecule is a dye, and according to the principle of antibody-antigen specific binding, under a specific fluorescence wavelength, the antibody or antigen binding content is different, and the fluorescence intensity is different. By establishing a linear relationship between the concentration of the antigen or antibody and the fluorescence intensity, the concentration of the corresponding antibody or antigen can be calculated with the known fluorescence intensity, and at the same time, the specific type of inflammatory agent can be identified with the color-coded marker, as described above. Therefore, the coded microneedle patch of the embodiment can realize simultaneous qualitative and quantitative analysis of three inflammatory factors.
As an extension of the application of the coded microneedle patch, the coded microneedle patch can be used together with a data acquisition and recognition system and an image processing system, the coded microneedle patch of a corresponding patient is scanned by establishing a database combined with corresponding antibody and antigen, and the coded microneedle patch is compared with the database after being processed by the image processing system, so that the content of the biomarker can be obtained. The application mode is simple and convenient, and accords with the development direction of intelligent medical treatment.
Example 2
Referring to fig. 4, another embodiment of a coded microneedle patch of the present invention is shown. In this embodiment, the coded microneedle patch also includes a microneedle base 1 and a microneedle body (not shown), the microneedle body is made of polymethacrylic acid, and the microneedle body has a height of 500 μm, except that the size of the microneedle array is adjusted. As can be seen from the figure, there are 10 color barcodes 2, and accordingly, 10 different protein molecules can be encapsulated, and the types of the protein molecules encapsulated by the microneedle bodies can be rapidly distinguished through the difference of colors.
In this example, the method of manufacturing the coding microneedle patch was the same as in example 1. Quantitative analysis of 10 different biomarkers can be achieved by using the corresponding detection methods.
Therefore, in this embodiment, the detection number of the biomarkers can be flexibly adjusted by changing the size of the microneedle patch array, the types of the protein molecules are discriminated by the color bar codes, and quantitative analysis is performed by adopting a corresponding detection means after specific binding between the protein molecules and the biomarkers, so that qualitative and quantitative analysis of up to 10 biomarkers is realized.
Example 3
Referring to fig. 5, another embodiment of a coded microneedle patch of the present invention is shown. In this embodiment, the coded microneedle patch also comprises a microneedle base 1 and a microneedle body 2, wherein the microneedle body 2 is made of sodium carboxymethylcellulose, and the height of the microneedle is 600 μm, but the difference is that the dye is not arranged at the bottom of the microneedle body, but is arranged on the microneedle body.
The types of the protein molecules carried by the microneedle bodies can be rapidly distinguished through the difference of the colors of the dyes.
As for the preparation method of the microneedle body and the specific implementation of the quantitative analysis, refer to example 1.
Example 4
Referring to fig. 6, another embodiment of a coded microneedle patch of the present invention is shown. In this embodiment, the coded microneedle patch also comprises a microneedle base 1 and a microneedle body 2, the microneedle body is prepared from hydroxyethyl cellulose, and the microneedle body is 1000 μm high, and the difference is that the microneedle body 2 is in a two-section type, one section near the microneedle base is coated with a dye, the length is 400 μm, and the other section is coated with a corresponding protein molecule. The types of the protein molecules carried by the microneedle bodies can be rapidly distinguished through the difference of the colors of the dyes.
As for the preparation method of the microneedle body and the specific implementation of the quantitative analysis, refer to example 1.
Example 5
Referring to fig. 7, another embodiment of a coded microneedle patch of the present invention is shown. In this embodiment, the coded microneedle patch also includes a microneedle base 1 and a microneedle body (not shown in the figure), the microneedle body is made of polyethylene glycol diacrylate, the microneedle body has a height of 500 μm, the difference is that microneedle arrays with different sizes are formed on the microneedle base, and the types of protein molecules contained in the microneedle body can be rapidly distinguished through the difference in size of the microneedle arrays.
As for the preparation method of the microneedle body and the specific implementation of the quantitative analysis, refer to example 1.
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 (10)

1. The coded microneedle patch is characterized by comprising a microneedle base and a microneedle body; the micro-needle body is positioned on the micro-needle base and is used for encapsulating signal molecules and protein molecules for identifying biomarkers, the types of the protein molecules are identified through the difference of the signal molecules or the micro-needle body array, and the quantitative detection of the biomarkers is realized through the specific combination of the biomarkers and the protein molecules, so that the simultaneous qualitative and quantitative detection of various biomarkers is realized;
wherein, the protein molecule is one or more of antigen antibody and biological enzyme.
2. The coded microneedle patch according to claim 1, wherein said signal molecule is one or more of a dye, a fluorescent, a photonic crystal, a quantum dot.
3. A coded microneedle patch according to claim 2, wherein the dye is one or more of red, orange, yellow, green, cyan, blue and violet in color.
4. The microneedle patch according to claim 1, wherein said antigen-antibody is one or more of TNF-a, IL-1, IL-6, CD80 or CD86, and said biological enzyme is one or more of glucose oxidase, cholesterol oxidase or ascorbate oxidase.
5. A coded microneedle patch according to claim 1, wherein said polymeric material is one or more of collagen, cross-linked hyaluronic acid, chitosan, pectin, gelatin, polyethylene glycol diacrylate, polyvinyl alcohol, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, polyol, alginate, cyclodextrin, dextrin, fructose, starch, trehalose, maltose, lactose, lactulose, turanose, raffinose, melezitose, dextran, xylitol, polylactic acid, polyglycolic acid, polyethylene oxide, polyacrylic acid, polyacrylamide, polymethacrylic acid, or polymaleic acid.
6. The coded microneedle patch according to claim 1, wherein the height of said microneedle body is 50 to 1500 μm.
7. The method for preparing a coded microneedle patch according to any one of claims 1 to 6, comprising the steps of:
(1) preparing a PDMS microneedle template: engraving a cavity of the microneedle body on the polydimethylsiloxane membrane by using a laser engraving machine to prepare a PDMS microneedle template;
(2) uniformly mixing a high molecular material, a signal molecule and a protein molecule to obtain a mixture;
(3) filling the cavity of the microneedle body: covering a mask on the PDMS microneedle template obtained in the step (1), coating the mixture obtained in the step (2) on the PDMS microneedle template, and uniformly filling the mixture into cavities of microneedle bodies;
(4) curing and demolding treatment: and (4) solidifying the microneedle filled with the mixture in the step (3), and demolding to obtain the coding microneedle patch.
8. The method according to claim 7, wherein the filling in the step (3) is performed by one or more of evacuation, centrifugation, and extrusion.
9. The method according to claim 8, wherein the curing in step (4) is performed by one or more of vacuum drying, photo-curing, and freeze-drying.
10. The application of the coded microneedle patch prepared according to any one of claims 7 to 9 in biomarker detection.
CN202111259822.8A 2021-10-28 2021-10-28 Coding microneedle patch and preparation method and application thereof Pending CN113985034A (en)

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