In-situ self-amplification nucleic acid-based flexible wound sensor and preparation method thereof

Abstract

The invention belongs to the technical field of electrochemical sensors, and particularly relates to an in-situ self-amplification nucleic acid-based flexible wound sensor and a preparation method thereof. The sensor comprises a flexible substrate, a gold layer and a nucleic acid sensing layer, the nucleic acid sensing layer comprises a tumor necrosis factor-alpha sensor, an interleukin 6 sensor, a transforming growth factor beta1 sensor, a vascular endothelial growth factor sensor, a temperature sensor, a pH sensor, a reference electrode and a counter electrode; the tumor necrosis factor-alpha sensor, the interleukin 6 sensor, the transforming growth factor beta1 sensor and the vascular endothelial growth factor sensor are all modified with rigid DNA tetrahedrons with hairpin DNA H2 and corresponding hairpin DNA H1. The sensor provided by the invention has antifouling and anti-degradation effects; finally, in-situ nucleic acid amplification on the flexible biosensing interface is realized by virtue of a hydrophilic / hydrophobic confinement effect and an auxiliary nucleic acid pre-fixation strategy, so that the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensors, and in particular relates to an in-situ self-amplifying nucleic acid-based flexible wound sensor and a preparation method thereof. Background Art

[0002] Chronic wounds are wounds whose healing process is delayed or stagnant, usually caused by conditions such as diabetes, venous insufficiency or pressure sores. Traditional chronic wound monitoring methods mainly rely on doctors' visual inspection and regular inspections, which is not only subjective, but also prone to missing subtle changes and unable to reflect the actual condition of the wound in a timely manner. Therefore, the development of effective monitoring and management methods is crucial to improving patients' quality of life and reducing medical costs.

[0003] In recent years, with the rapid development of flexible sensor technology, sensor-based intelligent monitoring systems have gradually become a research hotspot. These sensors can monitor physiological parameters such as temperature, humidity, pressure and pH value of wounds in real time, providing objective data support for the wound healing process. Flexible sensors have excellent flexibility and wearability, and can fit skin surfaces of various shapes to meet the needs of different patients. In addition, these sensors are usually small in size and light in weight, and can be connected to mobile devices and cloud platforms to achieve real-time transmission and analysis of data. This combination of technologies not only improves the accuracy of wound monitoring, but also provides possibilities for personalized medicine.

[0004] However, flexible wound sensors still face some problems. For example, the air permeability and softness of some patches cannot meet the needs of wounds; the strain stability of the device is insufficient; the complex microenvironment of the wound contaminates the electrodes and degrades the biorecognition units; and the lack of amplification strategies limits the sensitivity of detecting trace biomarkers.

[0005] In modern medicine, the wound healing process is affected by a variety of biological factors, among which chemical signals play a vital role in regulating wound healing. Existing flexible wound sensors mainly focus on monitoring physical parameters such as temperature, humidity and pressure, but they have obvious deficiencies in detecting chemical signals. This limitation makes it impossible to obtain the changes in chemical molecules at the wound in real time, thus affecting the comprehensive assessment of the wound healing status.

[0006] Detecting chemical molecules in the wound area can not only provide information about the local inflammatory response, but also reveal key physiological changes in the healing process. For example, biomarkers such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) play an important role in the wound healing process. TNF-α and IL-6 are usually associated with inflammatory responses, while TGF-β1 and VEGF play a key role in cell proliferation, migration and angiogenesis.

[0007] Therefore, developing a flexible wound sensor that can simultaneously detect these important chemical signals will greatly promote the development of wound healing monitoring technology and provide more accurate clinical decision support. By monitoring the changes in these biomarkers in real time, doctors can adjust treatment plans in a timely manner, improve healing efficiency and reduce the risk of complications. This research direction not only has important physiological significance, but also provides new opportunities for wound management and personalized medicine. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide an in-situ self-amplifying nucleic acid-based flexible sensor for the incompatibility between flexible sensor devices and human body modulus; low biomarker concentrations that are difficult to accurately detect; complex sensing interface environment, easy degradation of sensing units, and unstable signals under sensor strain. The spinning-based patch used ensures good air permeability and softness to avoid secondary damage to the wound. The DNA tetrahedral structure modified on the electrode surface not only improves the sensing stability under deformation, but also has anti-fouling and anti-degradation effects. Finally, with the help of hydrophilic / hydrophobic confinement effects and auxiliary nucleic acid pre-fixation strategies, in-situ nucleic acid amplification on the flexible biosensor interface is achieved to improve the detection sensitivity.

[0009] In order to solve the above technical problems, the present invention discloses an in-situ self-amplifying nucleic acid-based flexible wound sensor, which comprises a flexible substrate, a gold layer and a nucleic acid sensing layer; the nucleic acid sensing layer comprises a tumor necrosis factor-α sensor, an interleukin 6 sensor, a transforming growth factor β1 sensor, a vascular endothelial growth factor sensor, a temperature sensor, a pH sensor, a reference electrode and a counter electrode; The tumor necrosis factor-α sensor, interleukin 6 sensor, transforming growth factor β1 sensor and vascular endothelial growth factor sensor are all modified with a rigid DNA tetrahedron having a hairpin DNA H2 and a corresponding hairpin DNA H1.

[0010] Specifically, the tumor necrosis factor-α sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.9; The interleukin 6 sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.10; The transforming growth factor β1 sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.11; The vascular endothelial growth factor sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.12.

[0011] The present invention also discloses a method for preparing the above-mentioned in situ self-amplifying nucleic acid-based flexible wound sensor, which specifically comprises the following steps: Step 1: immerse the flexible substrate in a hydrophobic solution, take it out, and wash away the excess hydrophobic solution; Step 2: evaporating a gold layer on the flexible substrate obtained in step 1, and subjecting the substrate to oxygen plasma treatment; Step 3: preparing four inflammatory factor sensors respectively, specifically, reducing the four single strands of the DNA tetrahedron by tri(2-aminoethyl)phosphine chloride, then mixing the four single strands evenly and shaking them to form a rigid DNA tetrahedron with a hairpin DNA H2, cooling and dropping them onto a sensing electrode, incubating and then washing, blocking the binding site with 6-mercapto-1-hexanol, incubating and then washing, and then dropping the hairpin DNA H1 and drying to obtain the inflammatory factor sensor; Step 4: Prepare the temperature sensor, pH sensor, reference electrode and counter electrode in sequence to obtain the in situ self-amplifying nucleic acid-based flexible wound sensor.

[0012] Preferably, the flexible substrate comprises any one of thermoplastic polyurethane elastomer rubber / polyacrylonitrile spun fiber, styrene-butadiene-styrene block copolymer film or flexible polydimethylsiloxane.

[0013] Specifically, in step 1, the hydrophobic solution is a n-hexane solution of octadecyltrichlorosilane; the concentration of the hydrophobic solution is 1 to 5 wt%.

[0014] Specifically, in step 2, the thickness of the gold layer is 80-120 nm; the specific parameters of the oxygen plasma treatment are: treatment at a power of 50-100 W for 30-60 s.

[0015] Specifically, in step three, the molar ratio of the four single chains is 1:1:1:1.

[0016] Specifically, in step 3, the shaking is carried out under the following conditions: shaking at 95° C. for 5 min.

[0017] Specifically, in step 3, the volume ratio of the rigid DNA tetrahedron having the hairpin DNA H2 and the hairpin DNA H1 added dropwise is 1:1.

[0018] Specifically, in step three, the added amount of 6-mercapto-1-hexanol is 1 mM.

[0019] Furthermore, the application of the above-mentioned in situ self-amplifying nucleic acid-based flexible wound sensor in the preparation of an instrument for detecting chemical molecules in the wound area is also within the protection scope of the present invention; Specifically, in some embodiments of the present invention, the in situ self-amplifying nucleic acid-based flexible wound sensor is prepared by the above preparation method, which can detect multiple inflammatory factors TNF-α, IL-6, TGF-β1, VEGF, pH and temperature at the wound. The present invention further verifies the advantages of signal amplification, anti-fouling and anti-degradation of the above sensor through comparative experiments, and proves the application prospect of the in situ self-amplifying nucleic acid-based flexible wound sensor provided by the present invention in the preparation of chemical molecular instruments for detecting wound areas.

[0020] Beneficial Effects (1) The flexible spinning fibers of the present invention are uniform in thickness and have good heat dissipation and air permeability; (2) While the spinning substrate is flexible, the nucleic acid cascade amplification reaction is achieved on the flexible spinning substrate with the help of the auxiliary nucleic acid pre-fixation strategy and the confinement effect of the patterned wetting area. Specifically, the hairpin DNA H1 is sealed with 6-mercapto-1-hexanol on the rigid DNA tetrahedron and then dripped onto the electrode surface. When the wound exudate flows through the electrode surface, H1 will redissolve. The patterned hydrophilic and hydrophobic areas will inhibit H1 from flowing out of the sensing area due to their confinement effect, thereby ensuring the amplification reaction.

[0021] The nucleic acid cascade amplification reaction on the flexible spinning substrate reduces the detection line and improves the sensitivity, achieving highly sensitive detection of the target; the specific principle is as follows Figure 1 As shown: When the target reaches the electrode surface, the hairpin of DNA H1 is opened to expose the sticky end and hybridizes with the hairpin H2 on the tetrahedron at the same time, and the target is released. Such a cyclic reaction achieves signal amplification. The strategy of catalytic hairpin self-assembly achieves signal amplification and high-sensitivity detection of trace biomarkers. It has the potential to be applied in the fields of analytical equipment for clinical diagnosis and human health testing; (3) Finally, the rigid DNA tetrahedron structure improves the sensing stability under deformation and solves the disadvantage of insufficient strain stability of the device. At the same time, the anti-fouling and anti-degradation advantages of the rigid DNA tetrahedron also effectively address the problem of complex wound microenvironment contaminating the electrode and degrading the biorecognition unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0023] Figure 1 This is a schematic diagram of the signal amplification principle of the flexible sensor in the present invention; Figure 2 Schematic diagram of the structure of the in situ self-amplifying nucleic acid-based flexible sensor of the present invention, wherein 1 is a tumor necrosis factor-α sensor, 2 is an interleukin 6 sensor, 3 is a transforming growth factor β1 sensor, 4 is a vascular endothelial growth factor sensor, 5 is a pH sensor, 6 is a temperature sensor, 7 is a reference electrode, 8 is a counter electrode, and 9 is a flexible substrate; Figure 3 1 is a scanning electron microscope image of the flexible sensing electrode in Example 1 of the present invention, wherein a is a SEM image of a spun fiber, and b is a SEM image of a spun fiber modified with a gold layer; Figure 4 This is a resistance test diagram of the spun fiber modified with a gold layer under cyclic bending in Example 1 of the present invention; Figure 5 This is a CV test diagram of the spun fiber modified with a gold layer before and after bending in Example 1 of the present invention; Figure 6 The impedance diagram and CV diagram of the nucleic acid-based flexible sensor gradually modified in Example 1 of the present invention, wherein a is the impedance diagram of the nucleic acid-based flexible sensor obtained after gradual modification, and b is the CV diagram of the nucleic acid-based flexible sensor obtained after gradual modification; Figure 7 A statistical diagram of the current response size of the nucleic acid-based flexible sensor with or without an amplification strategy in Example 2 of the present invention; Figure 8 : Characterization diagram of the bending resistance of the nucleic acid-based flexible sensor in Example 3 of the present invention, wherein a is a schematic diagram of the modified single-stranded nucleic acid aptamer chain sensor before and after bending, b is a DPV diagram of the modified single-stranded nucleic acid aptamer chain sensor at different bending times, c is a schematic diagram of the modified rigid DNA tetrahedron sensor before and after bending, and d is a DPV diagram of the modified rigid DNA tetrahedron sensor at different bending times; Fig. 9 This is a characterization diagram of the anti-adsorption and anti-fouling performance of the nucleic acid-based flexible sensor in Example 3 of the present invention, wherein a is the fluorescence intensity obtained by the confocal method for different nucleic acid-based flexible sensors, which is used to reflect the anti-adsorption ability of different nucleic acid-based flexible sensors; b is the protein adsorption rate of different nucleic acid-based flexible sensors measured by the BCA method, which is used to reflect the anti-fouling performance of different nucleic acid-based flexible sensors; Fig.10 This is an integrated test diagram of the nucleic acid-based flexible sensor of the present invention, wherein a is a signal response diagram of the tumor necrosis factor-α sensor to different concentrations of tumor necrosis factor-α, b is a signal response diagram of the vascular endothelial growth factor sensor to different concentrations of vascular endothelial growth factor, c is a signal response diagram of the temperature sensor to temperature changes, d is a signal response diagram of the transforming growth factor β1 sensor to different concentrations of transforming growth factor β1, e is a signal response diagram of the interleukin 6 sensor to different concentrations of interleukin 6, and f is a signal response diagram of the pH sensor to pH changes. DETAILED DESCRIPTION

[0024] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0025] The sources of the flexible substrate materials used in the following examples are: thermoplastic polyurethane elastomer rubber (TPU), polyacrylonitrile (PAN, Mw=150000), styrene-butadiene-styrene block copolymer (SEBS) purchased from Sigma-Aldrich, and polydimethylsiloxane (PDMS) purchased from Shanghai Purdue Precision Instrument Technology Co., Ltd.

[0026] The nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.13 in the following examples are as described in Table 1. It should be noted that since the computer-readable carrier of the nucleotide or amino acid sequence table cannot recognize SH-, C6-, and MB-, the sequences in the following examples are based on the modified sequences recorded here: Table 1 Nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.13 in the examples

[0027] Embodiment 1: The in-situ self-amplifying nucleic acid-based flexible sensor of this embodiment uses a TPU / PAN spun fiber layer as a flexible substrate, and includes a TPU / PAN spun fiber layer, a gold layer and a nucleic acid sensing layer in sequence.

[0028] Among them, a sensing area is provided in the nucleic acid sensing layer, and the sensing area specifically includes four inflammatory factor sensors, a temperature (T) sensor and a pH sensor, a reference electrode (RE) and a counter electrode (CE). The four inflammatory factor sensors include a tumor necrosis factor-α (TNF-α) sensor, an interleukin 6 (IL-6) sensor, a transforming growth factor β1 (TGF-β1) sensor and a vascular endothelial growth factor (VEGF) sensor. The specific integrated structure is as follows Figure 2 shown.

[0029] Among them, the TNF-α sensor, IL-6 sensor, TGF-β1 sensor and VEGF sensor are all sensors modified with a rigid DNA tetrahedron (TDNA) having a hairpin DNA H2 and a corresponding hairpin DNA H1 (cDNA). Specifically, the four single strands of the TDNA having a hairpin DNA H2 and the corresponding hairpin DNA H1 are shown in Table 2: Table 2 Four single strands of TDNA and corresponding hairpin DNA H1 of four inflammatory factor sensors

[0030] The preparation method of the in situ self-amplifying nucleic acid-based flexible sensor of this embodiment comprises the following specific steps: (1) Weigh 0.5 g of TPU and PAN respectively, dissolve them in 6.134 g of dimethylformamide and let them stand for 3 days before electrospinning. The specific spinning parameters are: volume flow rate 1.25 mL·h -1 , the distance from the tip to the collection was 26 cm, and the voltage was 20 kV; the TPU / PAN fibers were collected on tin foil, and the spun fiber membrane was obtained after 4 h; (2) Soaking the spun fiber membrane in a 4 wt% hexane solution of octadecyltrichlorosilane for 10 min, then taking it out and washing it with alcohol three times to remove excess hydrophobic solution; (3) A mask was covered on the spun film and 100 nm thick nano-gold was evaporated to obtain a conductive substrate for the sensor. Then, the mask was covered to expose the sensing area and the sensing area was plasma cleaned (CPC-B, CIFInternational Group Co., Ltd) at a power of 100 W for 30 s. (4) Four inflammatory factor sensors were prepared respectively. The specific steps were as follows: four linear single strands of TDNA were mixed with tris(2-aminoethyl)phosphine chloride at a concentration 1000 times its own, and the reaction was carried out at room temperature in the dark for 2-3 h to reduce the disulfide bonds, so that the aptamer could be fixed on the gold layer through the Au-S bond. Then, the four single strands were mixed at a molar ratio of 1:1:1:1 and shaken at 95 °C for 5 min, and then gradually cooled at room temperature. During the cooling process, the four single strands self-paired and folded, and the hairpin structure spontaneously formed. Through mutual pairing, the four DNA single strands were self-assembled into a TDNA with hairpin DNA H2. 10 µL of the above TDNA was taken out and dropped onto the sensing electrode, incubated at 4 °C overnight, and then washed with PBS. 1 mM 6-mercapto-1-hexanol (MCH) was added to block the binding site, incubated for 1-2 h, and then washed with PBS. Finally, 10 µL of hairpin DNA H1 was added and dried at room temperature for 1-2 h before electrochemical testing. (5) The reference electrode is prepared in the patterning process in step 3, specifically a circular arc-shaped gold nanolayer with a thickness of 100 nm; (6) To prepare the shared reference electrode, 100 nm thick silver was sputtered onto the designed reference electrode area. The CHI 760E electrochemical workstation was used to conduct the electrochemical test at 50 mV s in a chlorinated solution consisting of 0.01 M HCl and 0.1 M KCl. -1 Ag / AgCl electrode was prepared by CV 3 cycles from −1.0 to −0.2 V at a scan rate of 0.1 M PBS; (7) To prepare the pH electrode, the PANI layer was electropolymerized, and a 0.1 M aniline / 0.1 M HCl solution was dropped onto the hydrophilic sensing area on the Au / nanofiber membrane. The electrode was then subjected to a CV from −0.2 to 1 V at 100 mV s using a CHI 760E electrochemical workstation. -1 Loop 25 times; (8) The temperature electrode is prepared by patterned evaporation of a 100 nm thick gold nanolayer under a designed serpentine mask.

[0031] In order to prove the successful preparation of the spun fiber membrane, the morphology of the spun fiber membrane prepared in step (1) was characterized. The prepared spun fiber membrane was attached to the SEM sample stage, and the two ends of the film were connected to the sample stage with conductive glue. Gold was sprayed on the electrodes, and then the spun fiber was observed under a scanning electron microscope. The results are shown in Figure 2. Figure 3 As shown in Figure 2, scanning electron microscopy showed that the spun fiber membrane had uniform thickness and gold nanoparticles were uniformly attached to the spinning surface.

[0032] The spun fiber membrane prepared in step (1) was gold-deposited to verify that the flexible sensing electrode still had good electrical and electrochemical properties under repeated bending. The handle and head of the prepared flexible sensing electrode were connected with conductive silver glue and wires and connected to the system digital source meter. The resistance test was performed when the electrode was bent repeatedly for different times. The results are shown in Figure 2. Figure 4 As shown, the resistance of the spun fiber membrane remains stable under repeated bending, demonstrating the good electrical properties of the flexible sensing electrode.

[0033] Subsequently, the above-mentioned spun fiber membrane with gold vapor deposition was used as the working electrode, a 1 cm × 1 cm platinum sheet was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The three electrodes were then immersed in an electrolyte solution (3 mmol / L K4Fe(CN)6 +0.5 mol / L Na2SO4 aqueous solution) for electrochemical testing. The results are shown in Figure 2. Figure 5 As shown, the peak current and peak spacing in the CV graph of the flexible sensing electrode remain basically unchanged before and after being bent 1000 times, proving the good chemical properties of the flexible sensing electrode.

[0034] In order to prove the successful modification of nucleic acid-based flexible sensing electrodes, this example further uses a gold-deposited spinning fiber membrane as a working electrode, a 1 cm × 1 cm platinum sheet as a counter electrode, and Ag / AgCl as a reference electrode. The electrodes before and after modification were immersed in an electrolyte solution (3 mmol / L K4Fe(CN)6 + 0.5 mol / L Na2SO4 aqueous solution) for electrochemical testing. The electrodes used for testing were divided into gold-deposited (Blank) spinning-based electrodes, TDNA-modified (TDNA) gold spinning-based electrodes, TDNA and MCH-modified (TDNA+MCH) gold spinning-based electrodes, and TDNA, MCH and hairpin DNA H1-modified (TDNA+MCH+cDNA) gold spinning-based electrodes. The results are shown in Figure 2. Figure 6 As shown, the impedance, peak current and peak spacing of the electrode gradually increased after each step of modification, proving the success of the step-by-step modification of the electrode.

[0035] For integrated sensor monitoring, a wearable biosensing system was placed on the wound and wirelessly transmitted the multiplexed sensing data to a user interface, enabling comprehensive recording of TNF-α, IL-6, TGF-β1, VEGF, pH, and temperature measurements from day 1 to day 4. Fig.10 This is an integrated test diagram of the in-situ self-amplifying nucleic acid-based flexible sensor of this embodiment, as shown in FIG. Fig.10 As shown, the integrated sensor realizes integrated sensing of TNF-α, IL-6, TGF-β1, VEGF, temperature and pH.

[0036] Embodiment 2: The difference between this embodiment and embodiment 1 is that the in-situ self-amplifying nucleic acid-based flexible sensor constructed in this embodiment uses commercially available SEBS film as a flexible substrate, and other structures and preparation methods are the same as those in embodiment 1.

[0037] A nucleic acid-based flexible sensor was prepared by the same method as steps (2) to (8) of Example 1. In order to further demonstrate the signal amplification effect of the in situ self-amplifying nucleic acid-based flexible sensor in Example 2, the current response of the in situ self-amplifying nucleic acid-based flexible sensor prepared in this example and the nucleic acid-based flexible sensor without amplification strategy to different IL-6 concentrations was used to verify whether the amplification strategy was successful.

[0038] Specifically, the electrode modified with the common aptamer is used as a control group without amplification strategy. Specifically, the common aptamer is also a DNA tetrahedral structure, and its four single strands are SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The specific construction method refers to Example 1.

[0039] A 1 cm × 1 cm platinum sheet was used as the counter electrode and Ag / AgCl was used as the reference electrode. The three electrodes were immersed in an electrolyte solution (0.1 M PBS) for electrochemical testing. The results are shown in Figure 7 As shown, when exposed to different IL-6 concentrations, the unfolded H1 modified with MB signal molecules exposes the sticky end and opens the hairpin located on the top of the tetrahedral DNA H2, and methylene blue (MB) is decorated on the biosensing electrode according to the concentration relationship of IL-6. Since the affinity between H1-H2 is higher than the affinity between H1-target, the protein will be released from the intermediate and cyclically participate in the cascade reaction of H1 and H2, thereby achieving the recycling of the target. For different concentrations of IL-6, the flexible sensing electrodes with the amplification strategy all showed a larger current response, proving the successful implementation of the amplification strategy.

[0040] Embodiment 3: The difference between this embodiment and embodiment 1 is that the in situ self-amplifying nucleic acid-based flexible sensor constructed in this embodiment uses flexible PDMS as a flexible substrate, and other structures and preparation methods are the same as those in embodiment 1.

[0041] Specifically, the preparation method of the flexible PDMS substrate of this embodiment comprises the following steps: The PDMS monomer and the cross-linking agent are uniformly mixed at a mass ratio of 10:1, and then centrifuged at 5000-10000 rpm for 5-20 minutes to remove bubbles. Then, the mixture is spin-coated on a silicon wafer substrate at 300-1000 rpm for 30-180 seconds in a spin coater to form a flexible PDMS coating on the silicon wafer substrate, which is then cured to obtain a flexible PDMS coating.

[0042] The nucleic acid-based flexible sensor was prepared by the same method as steps (2) to (8) of Example 1, and the in-situ self-amplifying nucleic acid-based flexible sensor prepared in Example 3 was applied to the experimental test of TDNA for increasing the strain stability and antifouling property of the electrode. The specific process is as follows: the flexible sensor in Example 3 is divided into two groups: single-stranded nucleic acid aptamer chain Apt / Au and TDNA / Au, wherein the single-stranded nucleic acid aptamer chain in Apt / Au is selected from SEQ ID NO.13, and the tetrahedral structure does not need to be synthesized, and it can be obtained by directly dropping it on the electrode surface to form an Au-S bond with gold.

[0043] The above Apt / Au and TDNA / Au were used as working electrodes, a 1 cm × 1 cm platinum sheet was used as the counter electrode, and Ag / AgCl was used as the reference electrode. The three electrodes were immersed in an electrolyte solution (0.1 M PBS) for electrochemical testing. The results are shown in Figure 8 As shown, the current response of the flexible sensing electrode without TDNA modification changes greatly at different bending times, while the current of the flexible sensing electrode modified with TDNA remains basically unchanged at different bending times, proving that TDNA has better bending stability than ordinary single-stranded nucleic acid aptamer chains.

[0044] Then the flexible sensors were divided into three groups: Au, Apt / Au, and TDNA / Au. First, the fluorescence intensity of the three different samples was measured by confocal microscopy to characterize the anti-adsorption of the rigid DNA tetrahedron. The specific contents are as follows: 10 μL of bovine serum albumin (BSA) (1 mg mL -1 ), then the electrode was placed in a 37 ℃ incubator for 1 hour and then taken out and rinsed with deionized water three times. The excitation light was 488 nm and the emission light was 520~530 nm during the confocal experiment. Fig. 9 As shown in a in the figure. Due to its stable three-dimensional structure and large steric hindrance, TDNA is not easy to bind or adsorb to the surface, so the TDNA / Au group has the lowest fluorescence intensity, which proves the good anti-adsorption property of TDNA.

[0045] The protein adsorption rate was then evaluated by the BCA method. In the first step, BSA (20 mg) was dissolved in 0.8 mL of NaCl solution (0.9 wt%) at 37 °C with gentle shaking to form a uniform standard protein solution. The protein solution was then added to the NaCl solution to obtain a concentration gradient solution (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg mL -1In the second step, 20 μL of standard protein solution and 200 μL of BCA-A: BCA-B (50:1 v / v) solution were added to the wells of the 96-well plate and incubated at 37 °C for 4 h. The analysis was performed using a UV-visible spectrophotometer at a wavelength of 562 nm, and a standard curve was drawn with protein content as the horizontal axis and absorbance as the vertical axis. In the third step, the sensor sample (surface area of ​​about 0.05 mm 2 ) were immersed in triplicate in 100 μL of 0.5 mg mL -1 The samples were incubated in BSA solution at 37 °C for 4 hours. Then 20 μL of each well was poured into another 96-well plate and the absorbance was measured according to the second step. Finally, the protein adsorption on each sample was calculated by comparing the results with the standard curve. The results are shown in Fig. 9 As shown in b, antifouling performance generally refers to the ability of a material to reduce or prevent the attachment of pollutants (such as proteins, cells, particles in sewage, etc.) in a specific environment. The rigidity and fixed three-dimensional structure of TDNA provide a certain steric hindrance, which makes it more difficult for proteins to form a stable bond with its surface. In addition, the negative charge of DNA molecules (provided by the phosphate backbone) also plays a certain repulsive role, further reducing the adsorption of proteins. It can effectively prevent proteins or other biological molecules from attaching to its surface through physical and chemical means, thereby improving the antifouling ability of the material. The TDNA / Au group has the lowest protein adsorption rate, proving that TDNA has good antifouling performance.

[0046] The present invention provides an in-situ self-amplifying nucleic acid-based flexible wound sensor and a method for preparing the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. An in situ self-amplifying nucleic acid-based flexible wound sensor, characterized in that: The in-situ self-amplifying nucleic acid-based flexible wound sensor comprises a flexible substrate, a gold layer and a nucleic acid sensing layer; the nucleic acid sensing layer comprises a tumor necrosis factor-α sensor, an interleukin 6 sensor, a transforming growth factor β1 sensor, a vascular endothelial growth factor sensor, a temperature sensor, a pH sensor, a reference electrode and a counter electrode; The tumor necrosis factor-α sensor, interleukin 6 sensor, transforming growth factor β1 sensor and vascular endothelial growth factor sensor are all modified with a rigid DNA tetrahedron having a hairpin DNA H2 and a corresponding hairpin DNA H1.

2. The in situ self-amplifying nucleic acid-based flexible wound sensor according to claim 1, characterized in that: The tumor necrosis factor-α sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.9; The interleukin 6 sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.10; The transforming growth factor β1 sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the nucleotide sequence corresponding to the hairpin DNA H1 is shown as SEQ ID NO.11; The vascular endothelial growth factor sensor has a rigid DNA tetrahedron with a hairpin DNA H2, and its four single-stranded nucleotide sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively, and the corresponding nucleotide sequence of the hairpin DNA H1 is shown as SEQ ID NO.

12.

3. The method for preparing the in situ self-amplifying nucleic acid-based flexible wound sensor according to any one of claims 1 or 2, characterized in that: The specific steps are: Step 1: immerse the flexible substrate in a hydrophobic solution, take it out, and wash away the excess hydrophobic solution; Step 2: evaporating a gold layer on the flexible substrate obtained in step 1, and subjecting the substrate to oxygen plasma treatment; Step 3: preparing four inflammatory factor sensors respectively, specifically, reducing the four single strands of the DNA tetrahedron by tri(2-aminoethyl)phosphine chloride, then mixing the four single strands evenly and shaking them to form a rigid DNA tetrahedron with a hairpin DNA H2, cooling and dropping them onto a sensing electrode, incubating and then washing, blocking the binding site with 6-mercapto-1-hexanol, incubating and then washing, and then dropping the hairpin DNA H1 and drying to obtain the inflammatory factor sensor; Step 4: Prepare the temperature sensor, pH sensor, reference electrode and counter electrode in sequence to obtain the in situ self-amplifying nucleic acid-based flexible wound sensor.

4. The preparation method according to claim 3, characterized in that: In step 1, the hydrophobic solution is a n-hexane solution of octadecyltrichlorosilane; the concentration of the hydrophobic solution is 1 to 5 wt%.

5. The preparation method according to claim 3, characterized in that: In step 2, the thickness of the gold layer is 80-120 nm; the specific parameters of the oxygen plasma treatment are: 50-100 W power for 30-60 s.

6. The preparation method according to claim 3, characterized in that: In step three, the molar ratio of the four single chains is 1:1:1:

1.

7. The preparation method according to claim 3, characterized in that: In step 3, the shaking is carried out under the following conditions: shaking at 95° C. for 5 min.

8. The preparation method according to claim 3, characterized in that: In step 3, the volume ratio of the rigid DNA tetrahedron having the hairpin DNA H2 and the hairpin DNA H1 added dropwise is 1:

1.

9. The preparation method according to claim 3, characterized in that: In step 3, the amount of 6-mercapto-1-hexanol added is 1 mM.

10. Use of the in situ self-amplifying nucleic acid-based flexible wound sensor according to claim 1 or 2 in the preparation of an instrument for detecting chemical molecules in a wound area.

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