Preparation method of patch for skin surface metabolite extraction and in situ detection

Abstract

The present invention provides a method for preparing a patch for skin surface metabolite extraction and in-situ detection, and relates to the technical field of skin surface metabolite detection. The method comprises: preparing a porous polyvinyl alcohol hydrogel; designing and preparing a portable small biochemical sensor; laser cutting out micro-chamber components required for metabolite extraction and detection patches; assembling the micro-chamber components, the portable small biochemical sensor and the hydrogel to obtain a patch; attaching the patch to a target object to extract and in-situ detect skin surface metabolites. In the present invention, the hydrogel can quickly and efficiently extract skin surface metabolites, while providing a liquid environment. The sensor attached to the surface of the hydrogel can perform in-situ detection of metabolites diffused into the hydrogel, thereby realizing on-site rapid detection of skin surface metabolites. In order to prevent excessive water loss in the hydrogel, a micro-chamber is designed to isolate the hydrogel from the external environment.

Description

Technical Field

[0001] The present invention relates to the technical field of skin surface metabolite detection, and in particular to a method for preparing a patch for skin surface metabolite extraction and in-situ detection. Background Art

[0002] Metabolites on the skin surface usually have specific physiological functions, and changes in their content can indicate the occurrence of disease or changes in physiological state. The collection, detection and quantitative analysis of small molecule compounds on the skin surface can be used to evaluate the integrity of skin barrier function, and has certain potential in disease diagnosis and health assessment.

[0003] In order to study small molecule compounds on the skin surface, there are different methods for collecting and identifying skin specimens. Conventional sampling techniques for skin metabolites mainly include skin biopsy, microdialysis, and microperfusion. These methods are generally considered to be time-consuming and have potential complications. In recent years, some non-invasive in situ sampling techniques have been gradually developed, including sweat sampling, tape stripping sampling, hydrogel patch sampling, etc., which can be used for micro-collection of metabolites on the skin surface and the next step of metabolite detection. At present, the main detection platforms for skin surface metabolites are nuclear magnetic resonance and mass spectrometry, which mainly use nuclear magnetic resonance, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and capillary electrophoresis-mass spectrometry to identify metabolites. These detection methods require sample purification, extraction, and analyte pre-concentration of the collected samples before metabolite analysis in order to convert the biological samples into a state more suitable for detection by analytical instruments. They are time-consuming, expensive, and require professional operation. They cannot be miniaturized or portable.

[0004] Therefore, there is a need to develop a key technology to achieve effective, non-invasive extraction of metabolites on the skin surface and to conduct in situ, rapid detection of these substances, in order to avoid the discomfort caused by traditional invasive sampling methods. At the same time, the in situ detection method can avoid the tedious steps and risk of contamination during pretreatment, storage and transportation after sample extraction, thereby achieving a simple daily assessment of the biochemical state of the skin. Summary of the invention

[0005] The present invention provides a method for preparing a patch for the extraction and in-situ detection of metabolites on the skin surface. A porous polyvinyl alcohol hydrogel is prepared to achieve efficient sampling of metabolites on the skin surface. To avoid complicated extraction and further storage and transportation for traditional laboratory metabolite detection, the present invention can directly use a portable small biochemical sensor to perform in-situ, non-invasive detection of skin surface metabolites extracted instantly from the hydrogel. At the same time, a micro-chamber component is designed to place the hydrogel to prevent excessive water loss in the air environment, thereby ensuring the accuracy of metabolite detection.

[0006] To achieve the above-mentioned purpose, the present invention provides a method for preparing a patch for extracting and in-situ detecting metabolites on the skin surface, comprising:

[0007] Step S1, preparing a porous polyvinyl alcohol hydrogel;

[0008] Step S2, designing and preparing a portable small biochemical sensor;

[0009] Step S3, laser cutting out the micro-chamber components required for the metabolite extraction and detection patch;

[0010] Step S4, assembling the microchamber component, the portable small biochemical sensor and the hydrogel to obtain a patch;

[0011] Step S5, attaching the patch to the target object to extract and in-situ detect metabolites on the skin surface.

[0012] Optionally, the step S1 specifically includes: mixing a polyvinyl alcohol solution with an alkaline solution, adding water-soluble sucrose as a template molecule, and soaking the polyvinyl alcohol gel in deionized water after cross-linking to remove the water-soluble sucrose and excess alkali to form the porous polyvinyl alcohol hydrogel.

[0013] Optionally, after step S1, the method further includes: cutting the porous polyvinyl alcohol hydrogel into a desired shape, and soaking it in a specific solvent for storage for later use, wherein the specific solvent includes deionized water, phosphate buffer or potassium chloride solution.

[0014] Optionally, the step S2 includes: selecting polyethylene terephthalate or polyimide as the substrate.

[0015] Optionally, the step S2 further includes: preparing the electrode in a predetermined manner, wherein the predetermined manner includes any one of the following: spin coating carbon ink, laser etching graphene, thermal evaporation of chromium / gold on the substrate, and screen printing carbon ink on the substrate.

[0016] Optionally, the step S2 further includes: designing the sensor according to a detection target, wherein the sensor includes an amino acid sensor, a urea sensor, a sodium ion sensor or a potassium ion sensor.

[0017] Optionally, in step S3, the microchamber component includes: a base layer for supporting the sensor; a cavity layer for storing the hydrogel, wherein the height of the cavity layer is greater than that of the hydrogel; and a covering layer for preventing the hydrogel from losing water.

[0018] Optionally, in step S5, before attaching the patch to the target object, the method further includes: tearing off the covering layer.

[0019] Optionally, step S5 includes: after the patch contacts the skin, the skin, the hydrogel and the sensor form a sandwich structure, wherein the hydrogel serves as an intermediate layer, and its two sides are respectively in contact with the skin and the sensor; the side of the hydrogel in contact with the skin can realize the extraction of metabolites, and the side of the hydrogel in contact with the sensor can provide a liquid environment containing metabolites for the electrode, so that the sensor can simultaneously measure the metabolite level.

[0020] Optionally, the step S5 further comprises: when extracting and in-situ detecting metabolites on the skin surface, the sampling time is greater than 3 minutes.

[0021] Compared with the prior art, the preparation method of the patch for skin surface metabolite extraction and in situ detection provided by the present invention has at least the following beneficial effects:

[0022] (1) Compared with invasive and minimally invasive skin metabolite extraction methods such as biopsy and tape stripping, the porous hydrogel extraction method of the present invention has high extraction efficiency and does not cause discomfort to the target and the risk of complications;

[0023] (2) Compared with detection methods based on nuclear magnetic resonance, mass spectrometry and other equipment, the patch is simple to operate, low cost, and easy to miniaturize;

[0024] (3) Samples collected from porous hydrogels can be directly used for analysis without any pretreatment, including tedious steps such as purification and preconcentration;

[0025] (4) The detection target is flexible and different sensors can be designed according to needs for the detection of skin metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and other objects, features and advantages of the present invention will become more apparent through the following description of the embodiments of the present invention with reference to the accompanying drawings, in which:

[0027] Figure 1 A flow chart schematically shows a method for preparing a patch for skin surface metabolite extraction and in-situ detection according to an embodiment of the present invention;

[0028] Figure 2 The schematic diagram shows the principle diagram of patch assembly according to an embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The terms "comprise", "include", etc. used herein indicate the existence of the features, steps, operations and / or components, but do not exclude the existence or addition of one or more other features, steps, operations or components.

[0031] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification, and should not be interpreted in an idealized or overly rigid manner.

[0032] Figure 1 The flowchart of the method for preparing a patch for extracting and in-situ detecting metabolites on the skin surface according to an embodiment of the present invention is schematically shown.

[0033] like Figure 1 As shown, the method for preparing a patch for extracting and in-situ detecting metabolites on the skin surface according to this embodiment may include steps S1 to S4.

[0034] Step S1, preparing porous polyvinyl alcohol (PVA) hydrogel.

[0035] Specifically, a PVA solution is mixed with an alkaline solution, and water-soluble sucrose is added as a template molecule. After cross-linking, the PVA gel is immersed in deionized water to remove the water-soluble sucrose and excess alkali, thereby forming a porous PVA hydrogel with rich pores and high permeability.

[0036] Optionally, after the porous hydrogel is prepared, the porous PVA hydrogel can be cut into a desired shape and soaked in a specific solvent for storage for later use, wherein the specific solvent includes deionized water, phosphate buffer or potassium chloride solution.

[0037] Step S2, designing and preparing a portable small biochemical sensor.

[0038] In this embodiment, when designing a portable small-sized biochemical sensor, it is first necessary to select a substrate material. The selection of the substrate has a certain flexibility, and it can be polyethylene terephthalate, polyimide, etc., for example.

[0039] Next, the electrode may be prepared in a predetermined manner, wherein the predetermined manner includes any one of the following: spin coating carbon ink, laser etching graphene, thermal evaporation of chromium / gold on a substrate, screen printing carbon ink on a substrate.

[0040] In addition, portable small-scale biochemical sensors can be designed according to the detection target, wherein the sensors include amino acid sensors, urea sensors, sodium ion sensors or potassium ion sensors.

[0041] Step S3, laser cutting out the micro-chamber components required for the metabolite extraction and detection patch.

[0042] In this embodiment, the microchamber components include:

[0043] A substrate layer for supporting a portable miniaturized biochemical sensor;

[0044] A cavity layer for storing porous PVA hydrogel, wherein the height of the cavity layer is greater than the porous PVA hydrogel; and

[0045] A covering layer used to prevent water loss from porous PVA hydrogel.

[0046] It should be noted that the height of the cavity layer storing the porous PVA hydrogel should be slightly larger than the porous PVA hydrogel. On the one hand, this is to prevent the overly large hydrogel from being unable to form a close contact with the sensor and the skin, resulting in unstable contact during detection; on the other hand, a hydrogel that is too small is prone to contact with the air environment, causing excessive water loss in the porous hydrogel.

[0047] Step S4, assembling the microchamber component, the portable small biochemical sensor and the hydrogel to obtain a patch.

[0048] The assembly steps of the patch will be described in detail later.

[0049] Step S5, attaching the patch to the target object to extract and in-situ detect metabolites on the skin surface.

[0050] Optionally, before attaching the patch to the target object, the method further includes: tearing off the covering layer that prevents the porous PVA hydrogel from losing water.

[0051] In this embodiment, in step S5, after the patch contacts the skin, the skin, the hydrogel and the sensor form a sandwich structure, wherein the hydrogel serves as an intermediate layer, and its two sides are respectively in contact with the skin and the sensor; the side of the hydrogel in contact with the skin can realize the extraction of metabolites, and the side of the hydrogel in contact with the sensor can provide a liquid environment containing metabolites for the electrode, so that the sensor can simultaneously measure the metabolite level.

[0052] Optionally, when performing the extraction and in situ detection of metabolites on the skin surface, the sampling time should be greater than 3 min to achieve a steady state of metabolite diffusion.

[0053] So far, the present invention provides a method for preparing a patch for extracting and in-situ detecting metabolites on the skin surface. The patch mainly includes three parts: a porous hydrogel for extracting metabolites on the skin surface, a chamber for preventing the hydrogel from losing water, and a portable small biochemical sensor. The highly permeable porous hydrogel is conducive to the efficient extraction of metabolites on the skin surface, and there is no need to perform complicated purification, concentration and other steps on the sampled samples. The biochemical sensor device in contact with the hydrogel can detect the substance to be tested in real time. At the same time, the patch structure is reasonably designed to obtain a microchamber for preventing the hydrogel from losing water, which effectively avoids excessive water loss of the hydrogel. The patch is attached to the skin surface to realize in-situ, non-invasive detection of skin surface metabolites, thereby simply and accurately evaluating skin biochemical indicators.

[0054] In other words, porous hydrogels can quickly and efficiently extract metabolites from the skin surface while providing a liquid environment. Sensors attached to the surface of the hydrogel can perform in situ detection of metabolites that diffuse into the hydrogel, thereby achieving rapid on-site detection of metabolites on the skin surface. In order to prevent excessive water loss in the hydrogel, a microchamber is designed to isolate the hydrogel from the external environment.

[0055] The technical solution of the present invention will be described in detail below in conjunction with some specific embodiments of the present invention.

[0056] An example is the measurement of serine content on the front of the arm by skin surface metabolite extraction and in situ detection patch:

[0057] First, a porous PVA hydrogel is prepared, which may include the following steps (11) to (13).

[0058] (11): 10 g of 9% polyvinyl alcohol (PVA) solution, 15 g of 16.7% potassium hydroxide solution, and 2.6 g of sucrose were mixed and stirred to obtain a hydrogel precursor;

[0059] (12): The hydrogel precursor was poured into a culture dish and placed in a vacuum oven at 45 °C to dry and degas for 6 h to obtain a PVA gel. The thickness of the PVA gel was approximately 0.3 mm;

[0060] (13): The PVA gel was soaked in deionized water to remove the template and excess KOH until the pH of the PVA gel was neutral. The PVA hydrogel was cut into circular patches with a diameter of 9 mm and stored in a potassium chloride solution to obtain a porous hydrogel patch.

[0061] Next, the serine sensor is prepared, which may include the following steps (21) to (29).

[0062] (21): A PET (polyethylene terephthalate) substrate with a thickness of 0.08 mm was immersed in anhydrous ethanol and ultrasonically cleaned for 5 min, then immersed in deionized water and ultrasonically cleaned for 5 min, and finally rinsed with deionized water and dried with nitrogen gas;

[0063] (22): Spin-coat the carbon ink on the substrate at 3000 rpm for 30 seconds to obtain a carbon electrode of appropriate thickness. After drying, laser engrave the carbon ink to obtain an electrode array containing the target pattern.

[0064] (23): The working electrode portion of the electrode was activated by 60 cycles of cyclic voltammetry in 0.5 M sulfuric acid solution in the potential range of 0 to 1 V at a scan rate of 50 mV / s;

[0065] (24): A Prussian blue layer was deposited on the working electrode by cyclic voltammetry (scan rate of 50 mV / s, scan range of -0.2 to 0.6 V, scan 20 times). The Prussian blue deposition solution consisted of 3 mM ferric chloride, 3 mM potassium ferrocyanide, 0.1 M potassium chloride, and 0.1 M hydrochloric acid. After deposition, the electrode was rinsed with deionized water. The deposition step was repeated three times to deposit a Prussian blue layer on the working electrode area of ​​the carbon electrode. The Prussian blue electrode was then immersed in a mixed solution containing 0.1 M hydrochloric acid and 0.1 M potassium chloride. The CV scan was repeated until a stable response was obtained. The potential range was -0.2 V to 0.6 V, and the scan rate was 50 mV / s.

[0066] (25): The working electrode portion was placed in an electrochemical polymerization solution containing pyrrole, 3-aminophenylboronic acid, and serine for electropolymerization. The electrochemical polymerization solution used a phosphate buffer solution with a pH of 6.5 as a solvent. The concentrations of pyrrole, 3-aminophenylboronic acid, and serine were 37.5 mmol / L, 12.5 mmol / L, and 5 mmol / L, respectively. Cyclic voltammetry (scanning speed of 50 mV / s, scanning range of 0 to 1 V, and scanning for 10 cycles) was used to allow the working electrode region to continue to deposit a layer of electropolymerized film containing serine template molecules.

[0067] (26): The serine in the electropolymerized membrane was removed by an eluent. The eluent composition was formaldehyde and acetic acid. The volume ratio of methanol to acetic acid was 3:7. The immersion time was 12 h. A molecularly imprinted serine sensing electrode that can specifically recognize serine was obtained. After template extraction, an IT scan was performed at 1 V in 0.5 M hydrochloric acid for 600 s, and then a CV scan was performed in a 0.1 M potassium chloride solution until it stabilized (scanning potential was -0.2 to 0.6 V, and the scanning rate was 50 mV / s) to activate the Prussian blue sensitive layer.

[0068] (27): The electrode area can be used as a counter electrode without any treatment;

[0069] (28): using an electroplating solution containing 0.25 M nitrate, 0.75 M sodium thiosulfate, and 0.5 M sodium sulfite to electroplate silver on the reference electrode region, wherein the procedure for electroplating silver is a multi-current deposition mode, which is sequentially deposited at a current of -0.01 mA for 150 s, at a current of -0.02 mA for 50 s, at a current of -0.05 mA for 50 s, at a current of -0.08 mA for 50 s, and at a current of -0.1 mA for 350 s;

[0070] (29): The reference electrode after silver deposition was immersed in 0.1 M potassium chloride solution for 20 s to chlorinate the silver deposited in the reference electrode area, thereby obtaining a silver / silver chloride electrode. 1 μL of a potential stabilizing mixture was repeatedly dripped on its surface three times. The components of the potential stabilizing mixture were sodium chloride, polyvinyl alcohol formaldehyde, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) and multi-walled carbon nanotubes. After drying, a silver / silver chloride reference electrode was obtained. The reference electrode, molecularly imprinted serine sensing electrode and counter electrode together formed a sensitive electrode array for electrochemical detection of serine.

[0071] Then, the patch is designed and assembled. Figure 2 The schematic diagram shows the principle diagram of patch assembly according to an embodiment of the present invention.

[0072] like Figure 2 As shown, the design and assembly of the patch may include the following steps (31) to (36), for example.

[0073] (31): The first layer selects PET with a thickness of 0.5 mm as the base structure and covers its surface with a layer of double-sided tape to facilitate the fixation of the electrochemical sensor on the PET base. Through laser cutting, it is cut into a 35 mm × 7 mm rounded rectangle with double-sided tape;

[0074] (32): The second layer is black single-sided adhesive, which is cut into a 30mm×7mm rounded rectangle by laser, and has a 5mm diameter through hole in the middle, which serves as the insulating layer of the sensor;

[0075] (33): The third layer is a sandwich structure with a thickness of 1 mm, with PET as the middle layer and double-sided tape on the upper and lower sides. It is cut into a 15 mm × 7 mm rounded rectangle using a laser marking machine, and there is a 5 mm diameter through-hole in the middle as the storage cavity for the hydrogel;

[0076] (34): The fourth layer is a waterproof silver single-sided adhesive, which is cut into a 15 mm × 7 mm rounded rectangle by laser to prevent the hydrogel in the patch from losing water;

[0077] (35): The patch is assembled by first attaching the sensor to the double-sided adhesive side of the first substrate structure, then aligning the second layer of the patch to the first substrate, and allowing the working area of ​​the biochemical sensor to be exposed through the through-hole, aligning the third hydrogel cavity layer with the second layer, placing the hydrogel in the cavity, and covering the molecular imprinting-based serine sensor at the same time, and finally sealing the hydrogel with a silver single-sided adhesive;

[0078] (36): When using, tear off the silver one-sided adhesive and stick the patch on the front side of the target arm. Finally, the concentrations of serine in different target arms were measured as shown in Table 1.

[0079] Table 1

[0080] Target arm area Serine concentration Target object 1 2.85mM Target object 2 1.67mM Target audience 3 4.35mM

[0081] It can be seen from Table 1 that by attaching the patch to the skin surface, in situ, non-invasive detection of metabolites on the skin surface can be achieved, thereby simply and accurately evaluating skin biochemical indicators.

[0082] From the above description, it can be seen that the above embodiments of the present invention achieve at least the following technical effects:

[0083] (1) Compared with invasive and minimally invasive skin metabolite extraction methods such as biopsy and tape stripping, the porous hydrogel extraction method of the present invention has high extraction efficiency and does not cause discomfort to the target and the risk of complications;

[0084] (2) Compared with detection methods based on nuclear magnetic resonance, mass spectrometry and other equipment, the patch is simple to operate, low cost, and easy to miniaturize;

[0085] (3) Samples collected from porous hydrogels can be directly used for analysis without any pretreatment, including tedious steps such as purification and preconcentration;

[0086] (4) The detection target is flexible and different sensors can be designed according to needs for the detection of skin metabolites.

[0087] Some block diagrams and / or flow charts are shown in the accompanying drawings. It should be understood that some blocks or combinations thereof in the block diagrams and / or flow charts may be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device, so that these instructions, when executed by the processor, may create a device for implementing the functions / operations described in these block diagrams and / or flow charts.

[0088] In addition, the terms "first" and "second" are used only for descriptive purposes and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the feature. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In addition, the word "one" or "an" preceding an element does not exclude the presence of multiple such elements.

[0089] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a patch for extracting and in-situ detecting metabolites on the skin surface, characterized in that: include: Step S1, preparing a porous polyvinyl alcohol hydrogel; Step S2, designing and preparing a portable small biochemical sensor; Step S3, laser cutting out the micro-chamber components required for the metabolite extraction and detection patch; Step S4, assembling the microchamber component, the portable small biochemical sensor and the hydrogel to obtain a patch; Step S5, attaching the patch to the target object to extract and in-situ detect metabolites on the skin surface; The step S2 comprises: designing the sensor according to the detection target, wherein the sensor comprises an amino acid sensor, a urea sensor, a sodium ion sensor or a potassium ion sensor; Wherein, the microchamber component comprises: A substrate layer for supporting the sensor; a cavity layer for storing the hydrogel, wherein the height of the cavity layer is greater than the hydrogel; and A covering layer is used to prevent the hydrogel from losing water.

2. The preparation method according to claim 1, characterized in that: The step S1 specifically includes: The polyvinyl alcohol solution is mixed with the alkaline solution, and water-soluble sucrose is added as a template molecule. After cross-linking, the polyvinyl alcohol gel is immersed in deionized water to remove the water-soluble sucrose and excess alkali to form the porous polyvinyl alcohol hydrogel.

3. The preparation method according to claim 1, characterized in that: After step S1, the method further includes: The porous polyvinyl alcohol hydrogel is cut into a desired shape and soaked in a specific solvent for storage for later use, wherein the specific solvent includes deionized water, phosphate buffer or potassium chloride solution.

4. The preparation method according to claim 1, characterized in that: The step S2 further comprises: Polyethylene terephthalate or polyimide was selected as the substrate.

5. The preparation method according to claim 4, characterized in that: The step S2 further comprises: The electrode is prepared in a predetermined manner, wherein the predetermined manner includes any one of the following: spin coating carbon ink, laser etching graphene, thermal evaporation of chromium / gold on the substrate, and screen printing carbon ink on the substrate.

6. The preparation method according to claim 1, characterized in that: In step S5, before attaching the patch to the target object, the method further includes: The cover layer is peeled off.

7. The preparation method according to claim 1, characterized in that: The step S5 comprises: After the patch comes into contact with the skin, the skin, the hydrogel and the sensor form a sandwich structure, wherein the hydrogel serves as an intermediate layer, and its two sides are respectively in contact with the skin and the sensor; the side of the hydrogel in contact with the skin can realize the extraction of metabolites, and the side of the hydrogel in contact with the sensor can provide a liquid environment containing metabolites for the electrode, so that the sensor can simultaneously measure the metabolite level.

8. The preparation method according to claim 1, characterized in that: The step S5 further comprises: When extracting and in situ detecting metabolites on the skin surface, the sampling time is greater than 3 minutes.

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