Fluorescence detection microneedle for cytochrome c in biological membrane as well as preparation method and application of fluorescence detection microneedle

Abstract

The invention discloses a fluorescent detection microneedle for cytochrome c in a biological membrane. MeHA gel is used as a substrate filler, DNA / MeHA gel is used as a needle body filler, a microneedle PDMS mold is filled, cross-linking is conducted through an ultraviolet lamp, and demolding is conducted after sufficient drying is conducted in a drying oven. The fluorescence detection microneedle prepared by the invention can pierce into a biological membrane to realize in-situ rapid detection of the concentration of cytochrome c in the biological membrane. In addition, the microneedle has good specificity, and can specifically recognize and detect cytochrome c.

Description

Technical Field

[0001] The invention belongs to the field of fluorescence detection and biological analysis, and specifically relates to a microneedle for fluorescence detection of cytochrome c in a biological membrane and a preparation method and application thereof. Background Art

[0002] Cytochrome c, English name: Cytochrome C, is a single-chain heme protein, widely present in eukaryotic organisms and some bacteria. Cytochrome c is an indispensable element in the electron transfer process of the cell respiratory chain. It is slightly connected to the inner membrane of the mitochondria. In the process of oxidative phosphorylation, it carries electrons between the third complex and the fourth complex for transfer, helping to generate ATP. Cytochrome c is a highly soluble protein with a loose and tiny structure. It is composed of about 100 amino acids, with a molecular weight of about 12000da, and a solubility of 100g / L in water. It contains a heme c prosthetic group, which is covalently bonded to two cysteine ​​residues of the protein. Its amino acid sequence and three-dimensional structure are highly conserved among different species, ensuring the stability of its electron transfer function. Bacterial biofilm refers to a microbial community adhering to the surface of the feeder, relying on its own secretion of polysaccharides, proteins, nucleic acids, lipids, etc., to form a membrane-like microbial aggregate without a constant structure. Cytochrome c in the biofilm can help bacteria in the bacterial respiratory chain. Electron transfer and energy metabolism, can also help bacteria adjust metabolism to adapt to the complex ecosystem in the biofilm. When there are a large number of reactive oxygen species (ROS) in the biofilm, cytochrome c can help bacteria neutralize ROS and play an antioxidant defense role. At the same time, cytochrome c plays an important role in the formation and maintenance of biofilms. Therefore, exploring the state of cytochrome c in biofilms is of great significance for studying the function of biofilms and the apoptosis and metabolic activities of bacteria in biofilms.

[0003] The detection of cytochrome c in biofilms generally requires specific sample preparation and detection techniques. Common methods include confocal resonance Raman microscopy combined with electrochemical technology, cryo-electron microscopy, mass spectrometry, proteomics, etc. However, these technologies have problems such as complex sample preparation, slow scanning speed, expensive instruments, complex operation, and low sensitivity. Microneedle technology, which has emerged in recent years, is a newer drug delivery and diagnostic tool. It has gradually entered everyone's field of vision due to its advantages such as fast detection speed and convenient sampling. Common materials for microneedles include metals, silicon, polymers, hydrogels, etc. Among them, hydrogel microneedles are widely used in drug delivery and biosensing due to their good biocompatibility, high swelling rate, easy manufacturing, and degradability. Combining microneedle technology with aptamers and fluorescence detection technology provides a promising method to solve the above problems. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a microneedle for fluorescence detection of cytochrome c in biofilm and a preparation method thereof, wherein MeHA gel is used as a substrate, DNA / MeHA gel is filled into the needle tip, cross-linked by ultraviolet light, and completely dried at low temperature to obtain a fluorescence detection microneedle. The fluorescence detection microneedle prepared by the present invention can penetrate into the biofilm to realize in-situ rapid detection of cytochrome c concentration.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing a microneedle for detecting cytochrome c fluorescence in a biofilm, the steps of the preparation method are:

[0007] 1) dissolving methacrylated hyaluronic acid and photoinitiator in Tris-HCl buffer to prepare MeHA gel;

[0008] 2) adding a DNA fluorescent probe to the MeHA gel to prepare a DNA / MeHA gel;

[0009] 3) Injecting the DNA / MeHA gel into a microneedle mold made of PDMS material and evacuating the mold; then injecting the MeHA gel, leaving it to stand in a dark environment to remove bubbles, irradiating it with ultraviolet light to cure it, drying it in a dark environment in a drying oven, and demolding it after it is completely dried to obtain the microneedle for fluorescence detection of cytochrome c in the biofilm.

[0010] The methacrylated hyaluronic acid is hyaluronic acid, which is prepared by mixing methacrylic anhydride in a water and DMF system in a weight ratio of 1:2 and reacting the mixture under room temperature with continuous stirring for 15-20 hours.

[0011] Preferably, the photoinitiator is photoinitiator Ingacure 2959.

[0012] Preferably, the MeHA gel is obtained by mixing methacrylated hyaluronic acid and photoinitiator Ingacure 2959 in water at a weight ratio of 60:1, shaking for 30-60 minutes to dissolve, and centrifuging at 3500-4500 rpm for 10-20 minutes.

[0013] The DNA / MeHA gel is obtained by adding a DNA fluorescent probe to the MeHA gel and uniformly mixing the mixture.

[0014] Wherein, the ultraviolet light in step 3) is 365nm ultraviolet light.

[0015] The DNA probe is a DNA single strand with complementary pairing added to both ends of the cytochrome c aptamer, and the fluorescent group Cy 5 and the quenching group BNQ-3 are modified at both ends of the DNA strand.

[0016] Wherein, the cytochrome c aptamer is CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC.

[0017] Among them, the complementary paired DNA chains are AAAAAAAAAAAA and TTTTTTTTTTTT.

[0018] Wherein, the DNA fluorescent probe sequence is 5`-AAAAAAAAAAAACCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGCTTTTTTTTTTTT-3`.

[0019] Preferably, the microneedle mold made of PDMS material is a 10x10 needle array, the needle tip height is 300 μm, and the needle body shape is a conical PDMS mold.

[0020] The present invention also provides a microneedle for fluorescence detection of cytochrome c in a biomembrane, which uses MeHA gel as a base filler and DNA / MeHA gel as a needle body filler, fills a microneedle PDMS mold, cross-links with an ultraviolet lamp, and is fully dried in an oven before demoulding.

[0021] Wherein, the sequence of the DNA fluorescent probe loaded by the microneedle for fluorescence detection of cytochrome c in the biofilm is 5`-AAAAAAAAAAAACCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGCTTTTTTTTTTTT-3`.

[0022] The present invention also provides an application of a microneedle for detecting cytochrome c fluorescence in a biofilm, wherein the microneedle is prepared by the preparation method provided by the present invention, and the microneedle for detecting cytochrome c fluorescence in a biofilm is used in the field of biological analysis. Preferably, the microneedle for detecting cytochrome c in a biofilm is used for detecting the microenvironment of a biofilm in marine microbial corrosion.

[0023] The fluorescent detection microneedle prepared by the present invention can penetrate into the interior of the biofilm to achieve in-situ rapid detection of cytochrome c concentration.

[0024] The present invention adopts fluorescence detection, aptamer and microneedle technology in combination, and the fine tip of the microneedle is used to pierce the interior of the biofilm to achieve rapid collection of biological samples and enrichment in the microneedle. After the DNA probe loaded in the microneedle body binds to the biological target, the fluorescent group and the quenching group are separated, and the signal detection is completed by detecting the change in fluorescence intensity, thereby achieving rapid in situ detection of biological targets in the biofilm. The invention has good biocompatibility and specificity, and has broad application prospects in the fields of fluorescence detection and bioanalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of the synthesis results of methacrylated hyaluronic acid provided in Example 1 of the present invention, wherein A corresponds to the infrared spectrum and B corresponds to the nuclear magnetic resonance hydrogen spectrum.

[0026] Figure 2 The microneedle images provided in Example 2 of the present invention, wherein A corresponds to the optical image and B corresponds to the SEM image.

[0027] Figure 3 This is the specificity test result of the fluorescent microneedle in agarose gel provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the present invention equally.

[0029] A microneedle for fluorescent detection of cytochrome c in biological membranes uses MeHA gel as a microneedle mold filler and a DNA probe is loaded in the microneedle tip.

[0030] The MeHA gel is methacrylated hyaluronic acid mixed with photoinitiator Ingacure 2959, which is fully dissolved in a water system.

[0031] The methacrylated hyaluronic acid is prepared by mixing hyaluronic acid and methacrylic anhydride in a water and DMF system in a weight ratio of 1:2 and reacting the mixture under room temperature with continuous stirring for 15-20 hours.

[0032] The MeHA gel is composed of methacrylated hyaluronic acid and Ingacure 2959 dissolved in water at a weight ratio of 60:1.

[0033] The MeHA gel is prepared by mixing methacrylated hyaluronic acid and Ingacure 2959 in water, shaking for 30-60 minutes to dissolve, and centrifuging at 3500-4500 rpm for 10-20 minutes.

[0034] The DNA / MeHA gel is a mixture of MeHA gel and DNA probe.

[0035] The DNA probe is a cytochrome c aptamer with complementary DNA single strands added to both ends, and the two ends of the DNA strand are modified with a fluorescent group Cy 5 and a quenching group BNQ-3.

[0036] The cytochrome c aptamer is CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC.

[0037] The complementary paired DNA strands are AAAAAAAAAAAA and TTTTTTTTTTTT.

[0038] The invention discloses an application of a microneedle for detecting cytochrome c in a biofilm, and uses the microneedle for detecting cytochrome c in a biofilm for detecting the microenvironment of the biofilm in marine microbial corrosion.

[0039] Example 1

[0040] Preparation of methacrylated hyaluronic acid:

[0041] 1) Dissolve 4 g HA in 200 mL deionized water to prepare a 2% w / v hyaluronic acid solution and stir at room temperature overnight.

[0042] 2) Add 133.3 mL of DMF and 8 mL of MA to the solution, adjust the pH value to between 8 and 9 with 0.5 M NaOH solution, and continue stirring at room temperature for 18 h.

[0043] 3) Separate MeHA using ethanol precipitation method, add 2 times the amount of ethanol to the solution, stir and mix evenly, remove the supernatant, and wash the precipitate with ethanol three times.

[0044] 4) Dissolve the precipitate with ultrapure water and purify it by dialyzing it in a dialysis membrane (MW 8000-14000 kDa) for one week, changing the water every morning and evening.

[0045] 5) The dialyzed MeHA was freeze-dried overnight to obtain a white flocculent, which was stored at -80°C.

[0046] Synthetic methacrylated hyaluronic acid Figure 1 Among them, A is the infrared spectrum and B is the nuclear magnetic resonance hydrogen spectrum.

[0047] Figure 1 A is the infrared spectrum comparison of hyaluronic acid and methacrylated hyaluronic acid. It can be seen from the figure that MeHA has a peak at 1717 cm -1 The vibration absorption peak of -C=O- appeared at 1301cm -1 The enhanced characteristics of -COC- were shown at 949cm -1 The bending vibration peaks of -C=CH- appeared at the positions of HA, while HA had no such bending vibration peaks at these positions. These changes proved the successful modification of methacrylate groups on hyaluronic acid.

[0048] Figure 1B is the H NMR spectrum of methacrylated hyaluronic acid, which proves the successful synthesis of methacrylated hyaluronic acid. According to the ratio of the integrated areas of the three peaks at 1.9, 5.7 and 6.1 ppm, the methylation degree of methacrylated hyaluronic acid is 21.9%.

[0049] Example 2

[0050] Preparation of microneedles for cytochrome c detection in biofilms:

[0051] 1) MeHA (30 mg mL -1 )、Photoinitiator (Ingacure 2959, 0.5mgmL -1 ) was dissolved in Tris-HCl buffer to prepare MeHA gel. DNA fluorescent probe was added to MeHA gel to prepare DNA / MeHA gel.

[0052] 2) Take 100 μL DNA / MeHA gel and inject it into the mold, evacuate for 3 minutes, repeat twice. Then, inject 200 μL MeHA gel, let it stand in the dark for 30 minutes to remove bubbles, cure it under 365nm UV light for 3 minutes, and dry it in a dark oven at 35°C for 8 hours. De-mold it after it is completely dried.

[0053] The image of the prepared microneedle for detecting cytochrome c in biofilm is shown in Figure 2 , where A is the optical image and B is the SEM image.

[0054] Figure 2 Figures A and B demonstrate the successful preparation of the microneedle. The overall size of the microneedle is 1×1cm, and it is transparent or milky white. The 10x10 needle array is neatly arranged, the needle tip height is 300μm, the needle body shape is conical, the needle body radius is 150μm, the needle spacing is 100μm, the needle body distribution is regular, and the needle tip morphology is good.

[0055] Example 3

[0056] Microneedle biocompatibility test:

[0057] Take 100 μL MeHA gel and DNA / MeHA gel, solidify them with 365 nm UV lamp for 3 min, and sterilize them under ultraviolet irradiation in a clean bench for 30 min. Mix the solidified MeHA gel and DNA / MeHA gel with 1 mL of Pseudomonas aeruginosa bacterial solution, and use 1 mL of bacterial solution as the control group, and culture them for 3 h. Then, apply gradient dilutions on the plate and observe the results of the plate application.

[0058] The bacterial concentration of the pure bacterial solution was lower than that of the other two groups, about 7.6×10 8 CFU mL -1The concentrations of the other two groups of bacterial suspensions were almost the same, both within 1×10 9 CFU mL -1 This is enough to prove that MeHA gel has no obvious toxic effect on bacteria, and DNA probe added to MeHA gel has almost no harm to bacterial growth. The bacterial concentration of pure bacterial solution is lower than that of the two groups of bacterial solutions added with gel. The reason is speculated to be that the gel has dried to a certain extent during the UV sterilization process, resulting in the gel needing to absorb water to reach equilibrium after being added to the bacterial solution, resulting in part of the bacterial solution being absorbed, causing the bacterial solution concentration to increase.

[0059] Example 4

[0060] Cytochrome c concentration test in microneedle biofilm:

[0061] The Eh 40 steel was immersed in the bacterial solution of Pseudomonas aeruginosa for 3 days to form a biofilm. The Eh 40 steel was taken out and immersed in solutions of different concentrations of Cyt c for 30 minutes, and gently rinsed with PBS buffer to achieve a balance of Cyt c concentration inside and outside the biofilm. Then the metal sheet was taken out from the Cyt c solution and rinsed with PBS to remove the Cyt c solution on the surface of the metal sheet. DNA / MeHAMNs were inserted into the biofilm for 20 minutes and then taken out. The fluorescence imaging results of the fluorescent microneedle tip were characterized by LSM.

[0062] Cytochrome c concentration is 50-300 μg mL -1 As the concentration of cytochrome c increases, the fluorescence intensity of the microneedle tip gradually increases. This proves that the fluorescent microneedle can detect the target concentration in the biofilm and has an obvious fluorescence response.

[0063] Example 5

[0064] Microneedle-specific tests:

[0065] Prepare agarose gel containing L-cysteine, ascorbic acid, glucose (10 mM), prepare agarose gel containing L-cysteine, ascorbic acid, glucose (10 mM), prepare agarose gel containing L-cysteine, ascorbic acid, glucose (10 mM), prepare Cyt c (100 μg mL -1 The microneedle was inserted into the agarose gel for 20 minutes and then taken out. The fluorescence imaging results of the fluorescent microneedle tip were characterized by laser confocal fluorescence microscopy.

[0066] Figure 3 The specificity test results of the fluorescent microneedle in agarose gel provided in Example 5 are shown in Figure 5. To avoid the presentation of the results being affected by unclear images, Figure 3The results can be described in words as follows: the colors of the four pictures in the first row and the first picture in the second row are consistently darker, and the colors of the last two pictures in the second row are consistently brighter. In other words, there was no obvious fluorescence response when the fluorescent microneedles acted on blank, L-cysteine, glucose, ascorbic acid, and a mixture of the above three agarose gels, and obvious fluorescence responses appeared when they acted on agarose gels containing only cytochrome c and containing L-cysteine, glucose, ascorbic acid, and cytochrome c. That is, the response of the fluorescent microneedles to L-cysteine, ascorbic acid, and glucose was weak, almost the same as that of the control group. The presence of non-targets had no effect on the detection of the fluorescent microneedles. This shows that the fluorescent microneedles have good specificity and can specifically identify and detect cytochrome c.

[0067] The detection microneedle prepared by the present invention can detect the concentration of cytochrome c in the biofilm and has an obvious fluorescence response; moreover, the microneedle has good specificity and can specifically identify and detect cytochrome c.

[0068] The present invention adopts fluorescence detection, aptamer and microneedle technology in combination, and the fine tip of the microneedle is used to pierce the interior of the biofilm to achieve rapid collection of biological samples and enrichment in the microneedle. After the DNA probe loaded in the microneedle body binds to the biological target, the fluorescent group and the quenching group are separated, and the signal detection is completed by detecting the change in fluorescence intensity, thereby achieving rapid in situ detection of biological targets in the biofilm. The invention has good rapid swelling ability, biocompatibility and specificity, and has broad application prospects in the fields of fluorescence detection and bioanalysis.

[0069] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a microneedle for fluorescence detection of cytochrome c in a biomembrane, characterized in that: The steps of the preparation method are: 1) dissolving methacrylated hyaluronic acid and photoinitiator in Tris-HCl buffer to prepare MeHA gel; 2) adding a DNA fluorescent probe to the MeHA gel to prepare a DNA / MeHA gel; 3) Injecting the DNA / MeHA gel into a microneedle mold made of PDMS material and evacuating the mold; then injecting the MeHA gel, leaving it to stand in a dark environment to remove bubbles, irradiating it with ultraviolet light to cure it, drying it in a dark environment in a drying oven, and demolding it after it is completely dried to obtain the microneedle for fluorescence detection of cytochrome c in the biofilm.

2. The method for preparing a microneedle for fluorescent detection of cytochrome c in a biofilm according to claim 1, characterized in that: The methacrylated hyaluronic acid is hyaluronic acid, which is prepared by mixing methacrylic anhydride in a water and DMF system in a weight ratio of 1:2 and reacting under room temperature with continuous stirring for 15-20 hours.

3. The method for preparing a microneedle for detecting cytochrome c fluorescence in a biofilm according to claim 1, characterized in that: The MeHA gel is obtained by mixing methacrylated hyaluronic acid and photoinitiator Ingacure 2959 in water at a weight ratio of 60:1, shaking for 30-60 minutes to dissolve, and centrifuging at 3500-4500 rpm for 10-20 minutes.

4. The method for preparing a microneedle for fluorescent detection of cytochrome c in a biofilm according to claim 1, characterized in that: The DNA / MeHA gel is obtained by adding a DNA fluorescent probe to the MeHA gel and uniformly mixing the mixture.

5. The method for preparing a microneedle for fluorescent detection of cytochrome c in a biofilm according to claim 1, characterized in that: In step 3), the ultraviolet light is 365 nm ultraviolet light.

6. The method for preparing microneedles for fluorescence detection of cytochrome c in biofilm according to claim 1, characterized in that: The DNA probe is a cytochrome c aptamer with complementary DNA single strands added to both ends, and the two ends of the DNA strand are modified with a fluorescent group Cy 5 and a quenching group BNQ-3.

7. The method for preparing microneedles for detecting cytochrome c fluorescence in biofilm according to claim 6, characterized in that: The sequence of the DNA fluorescent probe is 5`-AA AAAAAAAAAACCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGCTTTTTTTTTTTT-3`.

8. A microneedle for fluorescence detection of cytochrome c in biofilm, characterized in that: The microneedle for detecting cytochrome c fluorescence in the biofilm uses MeHA gel as a base filler, DNA / MeHA gel as a needle body filler, a DNA probe is loaded in the microneedle tip, a microneedle PDMS mold is filled, cross-linked by ultraviolet light, and demolded after being fully dried in an oven.

9. The microneedle for detecting cytochrome c fluorescence in biofilm according to claim 8, characterized in that: The sequence of the DNA fluorescent probe loaded by the microneedle for fluorescent detection of cytochrome c in the biofilm is 5`-AAAAAAAAAAAACCGTGTCTGGGGCCGACCGGCGCATT GGGTACGTTGTTGCTTTTTTTTTTTT-3`.

10. An application of a microneedle for fluorescence detection of cytochrome c in a biofilm, characterized in that: The intra-biofilm cytochrome c fluorescence detection microneedle is prepared by the method described in any one of claims 1 to 7, and the intra-biofilm cytochrome c detection microneedle is used in the field of biological analysis.

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