CN114113024B - Microneedle for detecting marker in interstitial fluid of breast cancer tissue and preparation method thereof - Google Patents
Microneedle for detecting marker in interstitial fluid of breast cancer tissue and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a microneedle for detecting markers in interstitial fluid of breast cancer tissues and a preparation method thereof, wherein the method comprises the following steps: methyl group isMixing the acrylic acid hyaluronic acid solution and the methacrylic acid gelatin solution according to a preset volume ratio to prepare a prepolymer solution; providing an H1, H2 solution of the amino carbon quantum dots and CHA reaction with target miRNA in breast cancer; adding the amino carbon quantum dots, the H1, H2 solution and the photo-curing agent into the prepolymer solution and mixing to obtain a composite solution; and adding the composite solution into a prefabricated polydimethylsiloxane microneedle female die, and performing light curing treatment to obtain the microneedle for detecting the marker in the breast cancer interstitial fluid. The micro needle prepared by the invention improves the mechanical property and the expansion rate, and simultaneously, the micro RNA and Cu in the breast cancer tissue fluid 2+ And the in-situ synchronous detection is realized.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a microneedle for detecting markers in interstitial fluid of breast cancer tissues and a preparation method thereof.
Background
Point-of-care testing (POCT) technology is well suited for public screening and personalized healthcare longitudinal monitoring, and development of minimally invasive, low-cost and highly automated body fluid sampling methods is critical for POCT testing. Currently, blood is a routine means of diagnosing and tracking disease progression due to its abundant biological information, but venous blood collection causes tissue damage, infection, and discomfort to the patient, and thus, it is becoming more and more interesting to find alternative blood sampling methods that can provide information about personal health.
Skin interstitial fluid (ISF) is obtained from blood by capillary filtration, where the amount of small molecules, electrolytes and proteins is similar to the components in plasma, while ISF acts as an intermediate between cells/cancer cells and the circulatory system, so some biomarkers in ISF are unique. The advantage of using microneedles for ISF sampling is that the penetration depth is greatly reduced while avoiding activation of pain-sensing neurons, an emerging method for noninvasively monitoring important parameters related to health. Many health-related information is carried by skin and interstitial fluid of skin tissue, and Microneedles (MNs) have important features for acquiring such information in a non-invasive manner. Breast cancer is the malignant tumor with highest female mortality worldwide, and for breast cancer examination, interstitial fluid around the breast can reflect the state of cancer cells in real time. Epidemiological and laboratory studies have demonstrated that mirnas are aberrantly expressed in breast cancer. Metal ions are essential for health, and are essential for cancer formation and metastasisThe process is often accompanied by angiogenesis, while Cu 2+ Play an important role in the formation of blood vessels. Therefore, development of a method capable of synchronously detecting miRNA and Cu in ISF 2+ Has important clinical value for breast cancer monitoring.
The hydrogel microneedle prepared at present is mainly used for drug delivery, can overcome the barrier effect of skin stratum corneum, greatly improves the drug delivery efficiency, but the microneedle used for extracting the biomarker is few. Document 1 (Al suiman, d.hydrogel-Coated Microneedle Arrays for Minimally Invasive Sampling and Sensing of Specific Circulating Nucleic Acids from Skin Interstitial fluid.acs Nano 2019,13,9620-9628.) reports that with hydrogel coated microneedles, specific miRNA biomarkers can be taken and isolated from the skin ISF while being able to detect captured mirnas in situ, but the captured biomarkers still require specific isolation steps and detection instrumentation later. Document 2 (Philip R, miller. Extraction and biomolecular analysis of dermal interstitial fluid collected with hollow micro devices. Commun Biol 2018,1,173.) reports that separation and characterization of exosomes in ISF was achieved for the first time using hollow microneedles, but the material of the microneedles is stainless steel, the manufacturing process is cumbersome and requires expensive instrumentation, and can cause biomaterial medical waste. Document 3 (Chang, H.A Swellable Microneedle Patch to Rapidly Extract Skin Interstitial Fluid for Timely Metabolic analysis.adv. Mater.2017,29 (37): 1702243.) reports that glucose in ISF can be extracted by using a polymer hydrogel microneedle to realize personal healthcare monitoring, but has the disadvantages of small sampling amount, insufficient mechanical properties, and the like. The prepared micro needle has the advantages of insufficient mechanical property, less sampling amount, expensive and complex required equipment, and causes the problems of extra medical waste and the like, besides drug administration, even if ISF sampling is carried out, the subsequent separation monitoring step is also required, the micro needle is not suitable for POCT application, and the synchronous detection of the biomarker of breast cancer is not realized.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a microneedle for detecting a marker in interstitial fluid of breast cancer tissues and a preparation method thereof, and aims to solve the problems that the existing microneedle is insufficient in mechanical strength, low in water absorption expansion rate and incapable of detecting the marker of breast cancer in real time.
The technical scheme of the invention is as follows:
a preparation method of a microneedle for detecting markers in interstitial fluid of breast cancer tissue comprises the following steps:
mixing the methacrylic acid solution and the methacrylic acid gelatin solution according to a preset volume ratio to prepare a prepolymer solution;
providing an H1, H2 solution of the amino carbon quantum dots and CHA reaction with target miRNA in breast cancer;
adding the amino carbon quantum dots, the H1, H2 solution and the photo-curing agent into the prepolymer solution and mixing to obtain a composite solution;
and adding the composite solution into a prefabricated polydimethylsiloxane microneedle female die, and performing light curing treatment to obtain the microneedle for detecting the marker in the breast cancer interstitial fluid.
The preparation method of the microneedle for detecting the marker in the interstitial fluid of the breast cancer tissue comprises the following steps:
adding methacrylic anhydride into the hyaluronic acid aqueous solution, regulating the pH to 8-10, and reacting for 12-24 hours at room temperature to obtain the methacrylic acid hyaluronic acid solution.
The preparation method of the microneedle for detecting the marker in the breast cancer interstitial fluid comprises the following steps:
adding methacrylic anhydride into the gelatin water solution, reacting for 2-4h, and then adding PBS to stop the reaction, thus obtaining the methacrylic gelatin solution.
The preparation method of the microneedle for detecting the marker in the breast cancer interstitial fluid comprises the steps of preparing a methacrylic hyaluronic acid solution and a methacrylic gelatin solution, wherein the volume ratio of the methacrylic hyaluronic acid solution to the methacrylic gelatin solution is 1:1-10:3.
The preparation method of the microneedle for detecting the marker in the breast cancer interstitial fluid comprises the following steps:
dissolving citric acid and polyethyleneimine in deionized water, and performing ultrasonic treatment to form a uniform mixed solution;
and (3) placing the mixed solution into a hydrothermal reaction kettle, heating for 2-4 hours at 180-250 ℃, and naturally cooling to room temperature to obtain the amino carbon quantum dots.
The preparation method of the microneedle for detecting the marker in the breast cancer interstitial fluid comprises the following steps:
mixing polydimethylsiloxane and a curing agent, pouring the mixed solution on the surface of a metal commercial microneedle template, and vacuumizing to remove bubbles in the gas;
and (3) placing the metal commercial microneedle mould plate poured with the mixed solution into an oven for heating and solidifying, and separating the solidified polydimethylsiloxane from the metal commercial microneedle mould plate after cooling to obtain the polydimethylsiloxane microneedle female mould.
The preparation method of the microneedle for detecting the marker in the breast cancer interstitial fluid comprises the step of preparing a photo-curing agent, wherein the photo-curing agent is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonite.
The preparation method of the microneedle for detecting the marker in the breast cancer interstitial fluid comprises the steps of adding the composite solution into a prefabricated polydimethylsiloxane microneedle female die, and carrying out light curing treatment, wherein the light curing treatment is blue light irradiation for 5-15s.
The preparation method of the microneedle for detecting the marker in the interstitial fluid of the breast cancer tissue comprises the steps of 2+ 。
The invention discloses a microneedle for detecting markers in interstitial fluid of breast cancer tissues, which is prepared by the preparation method.
The beneficial effects are that: the invention provides a preparation method of a microneedle for detecting markers in interstitial fluid of breast cancer tissuesThe method is characterized in that the microneedle is prepared by mixing the methacrylic acid hyaluronic acid solution and the methacrylic acid gelatin solution together in different volume ratios, compared with the existing single hydrogel microneedle, the method not only solves the problem of insufficient mechanical strength of the existing microneedle, but also solves the problem of low water absorption expansion rate of the existing microneedle, and the water absorption expansion rate can reach 700%; the invention also provides H1, H2 solutions that react with target miRNA in breast cancer CHA and respond to Cu 2+ The aminated carbon quantum dots of the (2) are loaded into the micro needle, so that the micro ribonucleic acid (miRNA) and copper (Cu) in breast cancer tissue fluid are realized 2+ Is performed in-situ synchronous detection of (a).
Drawings
FIG. 1 is a flowchart of a method for preparing a microneedle for detecting markers in interstitial fluid of breast cancer tissue according to a preferred embodiment of the present invention.
FIG. 2 is a graph showing the comparison of the water swelling ratios of composite microneedles made by mixing two hydrogels in different volume ratios.
FIG. 3 is a graph showing the mechanical properties of composite microneedles made by mixing two hydrogels in different volume ratios.
FIG. 4 is a volume ratio MeHA; gelma=7: skin insertion depth map at 3 (v/v).
Fig. 5 is a graph of fluorescence change after microneedle sampling using a skin model.
Detailed Description
The invention provides a microneedle for detecting markers in interstitial fluid of breast cancer tissues and a preparation method thereof, and the invention is further described in detail below for the purpose, the technical scheme and the effect of the invention to be clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Referring to fig. 1, fig. 1 is a flowchart of a preferred embodiment of a method for preparing a microneedle for detecting a marker in interstitial fluid of breast cancer tissue, according to the present invention, as shown in the drawing, comprising the steps of:
s10, mixing the methacrylic acid hyaluronic acid solution and the methacrylic acid gelatin solution according to a preset volume ratio to prepare a prepolymer solution;
s20, providing an H1 and H2 solution of the amino carbon quantum dot and the CHA reaction with the target miRNA in the breast cancer;
s30, adding the aminated carbon quantum dots, the H1, H2 solution and the photo-curing agent into the prepolymer solution, and mixing to obtain a composite solution;
and S40, adding the composite solution into a prefabricated polydimethylsiloxane microneedle female die, and performing light curing treatment to obtain the microneedle for detecting the marker in the breast cancer interstitial fluid.
In particular, hydrogel microneedles prepared from single methacrylated gelatin (GelMA) are strong in mechanical properties, but brittle, easy to break, and low in water swelling rate, so that biomarkers of tumors in ISF are not easily detected for small volumes of samples; hydrogel microneedles prepared from single methacrylated hyaluronic acid (MeHA) have a large water swelling rate, can absorb 10 times more solution than their own volume, but are soft and have low mechanical properties.
Based on the method, the microneedle is prepared by mixing the methacrylic acid hyaluronic acid solution and the methacrylic acid gelatin solution together in different volume ratios, and the two are mixed together to form the composite hydrogel with a double-network structure after curing treatment. Compared with the existing single hydrogel microneedle, the prepared microneedle not only overcomes the problem of insufficient mechanical strength of the existing microneedle, but also solves the problem of low water absorption expansion rate of the existing microneedle, and the water absorption expansion rate can reach 700%; the invention also provides H1, H2 solutions that react with target miRNA in breast cancer CHA and respond to Cu 2+ The aminated carbon quantum dots of the (2) are loaded into the micro needle, so that the micro ribonucleic acid (miRNA) and copper (Cu) in breast cancer tissue fluid are realized 2+ Is performed in-situ synchronous detection of (a). That is, the invention can reduce the subsequent steps of centrifugation, detection and the like after the sampling of the micro-needle, and load miRNA and Cu on the micro-needle by using a mask method 2+ The detection system realizes in-situ synchronous detection, saves time, improves the accuracy of diagnosis and prognosis, and is suitable for POCT.
In some embodiments, the preparation of the methacrylic hyaluronic acid solution comprises the steps of: adding methacrylic anhydride into the hyaluronic acid aqueous solution, regulating the pH to 8-10, and reacting for 12-24 hours at room temperature to obtain the methacrylic acid hyaluronic acid solution.
In some embodiments, the preparation of the methacrylated gelatin solution comprises the steps of: adding methacrylic anhydride into the gelatin water solution, reacting for 2-4h, and then adding PBS to stop the reaction, thus obtaining the methacrylic gelatin solution.
In some embodiments, the volume ratio of the methacrylic acid solution to the methacrylic acid gelatin solution is 1:1 to 10:3. In this ratio range, the prepared microneedle has both good mechanical strength and high water-absorbing expansion rate. Preferably, the volume ratio of the methacrylic acid solution to the methacrylic acid gelatin solution is 7:3.
In some embodiments, the preparation of the aminated carbon quantum dot comprises the steps of: dissolving citric acid and polyethyleneimine in deionized water, and performing ultrasonic treatment to form a uniform mixed solution; and (3) placing the mixed solution into a hydrothermal reaction kettle, heating for 2-4 hours at 180-250 ℃, and naturally cooling to room temperature to obtain the amino carbon quantum dots.
In some embodiments, the preparation of the polydimethylsiloxane microneedle negative mold comprises the steps of: mixing polydimethylsiloxane and a curing agent, pouring the mixed solution on the surface of a metal commercial microneedle template, and vacuumizing to remove bubbles in the gas; and (3) placing the metal commercial microneedle mould plate poured with the mixed solution into an oven for heating and solidifying, and separating the solidified polydimethylsiloxane from the metal commercial microneedle mould plate after cooling to obtain the polydimethylsiloxane microneedle female mould.
In some embodiments, when the photo-curing agent is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate, the composite solution is added to a preformed polydimethylsiloxane microneedle negative mold, after exposure to blue light for 5-15s, the composite solution is cured, and finally the prepared microneedle is dried at room temperature and stored at 4 ℃ before use.
In some embodimentsIn the formula, the micro-needle prepared by the invention is mainly used for detecting two biomarkers in breast cancer, namely target miRNA and Cu 2+ . The present invention utilizes H1, H2 solutions that react CHA with target miRNAs in breast cancer, and respond to Cu 2+ The aminated carbon quantum dot of (2) is used for detecting the same. The miRNA has various types, the concentration of the miRNA can be changed in a cancer patient, and when the CHA reaction is utilized for fluorescence detection, only H1 and H2 sequences in the CHA reaction are required to be replaced, and the H1 and H2 sequences can be prepared according to the prior art.
In some embodiments, a microneedle for detecting a marker in interstitial fluid of breast cancer tissue is also provided, which is prepared by the preparation method of the present invention.
The invention is further illustrated by the following examples:
example 1
Preparation of MeHA solution: 1g of hyaluronic acid was dissolved in water, methacrylic anhydride was slowly added dropwise, pH=8-10 was adjusted with 5M sodium hydroxide, and the reaction was carried out at room temperature for 24 hours. The solution was dialyzed against water at 4℃for 3 days to remove unreacted reagents (cut-off 12-14kDa dialysis bag), and the solid product was obtained by freeze-drying and stored at 4 ℃.
Preparation of gelma solution: 10 g of gelatin was dissolved in 100mL of water at 50℃and stirred until completely dissolved. 8mL of methacrylic anhydride was slowly added to the gelatin solution to react for 3 hours, followed by the addition of 300mL of BS to terminate the reaction. The solution was dialyzed against deionized water at 4 ℃ for one week to remove unreacted reagents (cut-off 12-14KDa dialysis bag).
And 3, uniformly mixing the solutions obtained in the step 1 and the step 2 according to different volume ratios of 1:0, 7:3, 5:5 and 3:7 respectively, adding 0.1% (w/v) of phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate (LAP), and reacting for 1h at 50 ℃. 1mL of the well-mixed prepolymer solution was added to the mold, and the solution was centrifuged (4000 rmp,5 min) to ensure that the solution entered the cavity and was cured by irradiation with blue light for 10 s. Finally, MNs were dried at room temperature and stored at 4 ℃ prior to use.
Step 4, carrying out a water absorption expansion rate test on the microneedle obtained in the step 3: first, M in dry state is recordedThe weight of Ns, denoted M 0 Then MN is carried out S The patch was immersed in phosphate buffered saline (PBS, ph=7.4) for 2 hours. Removing all excessive water on MNs surface and recording MN S Weight of patch (M t ). Likewise, the weights of MNs patches after different soak times (1, 5, 7, 10 and 20 minutes) were recorded. In addition, the swelling properties of microneedles of different volume ratios were also tested. As a result, as shown in FIG. 2, the single MeHA microneedle showed the highest water swelling rate, and the water swelling rate was decreased to various degrees with the addition of GelMA. This is because GelMA binds to hydroxyl groups in MeHA, resulting in a decrease in the hydrophilicity of MeHA.
Step 6, analyzing the test results of the step 4 and the step 5, and finally selecting MeHA: gelma=7: 3 as a microneedle for minimally invasive monitoring of breast cancer by using biomarkers in interstitial fluid;
example 2
Preparation of MeHA solution: 1g of hyaluronic acid was dissolved in water, methacrylic anhydride was slowly added dropwise, pH=8-10 was adjusted with 5M sodium hydroxide, and the reaction was carried out at room temperature for 24 hours. The solution was dialyzed against water at 4℃for 3 days to remove unreacted reagents (cut-off 12-14kDa dialysis bag), and the solid product was obtained by freeze-drying and stored at 4 ℃.
Preparation of gelma solution: 10 g of gelatin was dissolved in 100mL of water at 50℃and stirred until completely dissolved. 8mL of methacrylic anhydride was slowly added to the gelatin solution to react for 3 hours, followed by the addition of 300mL of BS to terminate the reaction. The solution was dialyzed against deionized water at 4 ℃ for one week to remove unreacted reagents (cut-off 12-14KDa dialysis bag).
And 3, uniformly mixing the solutions obtained in the step 1 and the step 2 according to the volume ratio of 7:3, adding 0.1% (w/v) LAP, and reacting for 1h at 50 ℃. 1mL of the well-mixed prepolymer solution was added to the mold, and the solution was centrifuged (4000 rmp,5 min) to ensure that the solution entered the cavity and was cured by irradiation with blue light for 10 s. Finally, MNs were dried at room temperature and stored at 4 ℃ prior to use.
Step 4, selecting 8-week-old BALB/c female mice, removing abdominal hair of the mice by using depilatory cream, pressing the microneedle patch of step 3 on the abdomen of the treated mice for about 5 minutes, then taking out the corresponding skin area, fixing with 4% paraformaldehyde, and finally staining with hematoxylin & eosin (H & E). As a result, as shown in FIG. 4, the microneedles were inserted into the skin to a depth of about 350 μm, which is greater than the typical microneedle penetration depth, sufficient to penetrate the stratum corneum to reach the dermis layer for ISF sampling.
Example 3
Preparation of MeHA solution: 1g of hyaluronic acid was dissolved in water, methacrylic anhydride was slowly added dropwise, pH=8-10 was adjusted with 5M sodium hydroxide, and the reaction was carried out at room temperature for 24 hours. The solution was dialyzed against water at 4℃for 3 days to remove unreacted reagents (cut-off 12-14kDa dialysis bag), and the solid product was obtained by freeze-drying and stored at 4 ℃.
Preparation of gelma solution: 10 g of gelatin was dissolved in 100mL of water at 50℃and stirred until completely dissolved. 8mL of methacrylic anhydride was slowly added to the gelatin solution to react for 3 hours, followed by the addition of 300mL of BS to terminate the reaction. The solution was dialyzed against deionized water at 4 ℃ for one week to remove unreacted reagents (cut-off 12-14KDa dialysis bag).
Step 4, skin model preparation: fresh pigskin was washed with water (3 times) And transferred to a petri dish, 100 μlpbs (control), 500nM miRNA and 100 μΜ Cu were added, respectively 2+ Solutions (experiments). The skin samples were incubated overnight. The MN patch prepared in step 3 was pressed onto the skin for sampling for about 20 minutes and left at 37 ℃ for 2 hours. As shown in FIG. 5, the experimental groups showed different fluorescence changes compared with the control group, indicating that the prepared microneedles can realize miRNA and Cu in breast cancer 2+ Qualitative and semi-quantitative detection of (c).
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (2)
1. The preparation method of the microneedle for detecting the marker in the interstitial fluid of the breast cancer tissue is characterized by comprising the following steps:
mixing the methacrylic acid solution and the methacrylic acid gelatin solution according to a preset volume ratio to prepare a prepolymer solution;
providing an H1, H2 solution of the amino carbon quantum dots and CHA reaction with target miRNA in breast cancer;
adding the amino carbon quantum dots, the H1, H2 solution and the photo-curing agent into the prepolymer solution and mixing to obtain a composite solution;
adding the composite solution into a prefabricated polydimethylsiloxane microneedle female die, and preparing the microneedle for detecting the marker in the breast cancer interstitial fluid after light curing treatment;
the preparation of the methacrylic hyaluronic acid solution comprises the following steps:
adding methacrylic anhydride into the hyaluronic acid aqueous solution, regulating the pH to 8-10, and reacting for 12-24 hours at room temperature to obtain the methacrylic acid hyaluronic acid solution;
the preparation of the methacrylic acid gelatin solution comprises the following steps:
adding methacrylic anhydride into the gelatin water solution, reacting for 2-4h, and then adding PBS to terminate the reaction to obtain the methacrylic gelatin solution;
the volume ratio of the methacrylic acid hyaluronic acid solution to the methacrylic acid gelatin solution is 1:1-10:3;
the preparation of the aminated carbon quantum dot comprises the following steps:
dissolving citric acid and polyethyleneimine in deionized water, and performing ultrasonic treatment to form a uniform mixed solution;
placing the mixed solution into a hydrothermal reaction kettle, heating for 2-4 hours at 180-250 ℃, and naturally cooling to room temperature to obtain the aminated carbon quantum dots;
the preparation of the polydimethylsiloxane microneedle female mold comprises the following steps:
mixing polydimethylsiloxane and a curing agent, pouring the mixed solution on the surface of a metal commercial microneedle template, and vacuumizing to remove bubbles in the gas;
placing the metal commercial microneedle mould plate poured with the mixed solution into an oven for heating and solidifying, and separating solidified polydimethylsiloxane from the metal commercial microneedle mould plate after cooling to obtain a polydimethylsiloxane microneedle female mould;
the photocuring agent adopted by the light curing is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphonite;
adding the composite solution into a prefabricated polydimethylsiloxane microneedle female die, and performing light curing treatment, wherein the light curing treatment is blue light irradiation for 5-15s;
the breast cancer marker is target miRNA and Cu 2+ 。
2. A microneedle for detecting a marker in interstitial fluid of breast cancer tissue, which is prepared by the preparation method of claim 1.
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| WO2016141307A1 (en) * | 2015-03-05 | 2016-09-09 | The Trustees Of Columbia University In The City Of New York | Devices and systems for optically determining a concentration of an analyte in a living subject using hydrogel-based, fluorescent microneedles and methods of manufacture thereof |
| WO2020092229A1 (en) * | 2018-10-31 | 2020-05-07 | The Regents Of The University Of California | Biodegradable microneedles for transdermal therapeutic agent delivery |
| US12018315B2 (en) * | 2019-05-30 | 2024-06-25 | Massachusetts Institute Of Technology | Peptide nucleic acid functionalized hydrogel microneedles for sampling and detection of interstitial fluid nucleic acids |
| WO2021142160A1 (en) * | 2020-01-10 | 2021-07-15 | The Regents Of The University Of California | Gelatin-based microneedle patch for minimally-invasive extraction of bodily fluids |
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