An aptamer-based microneedle electrochemical biosensor and a preparation method thereof
By constructing an aptamer-based three-electrode microneedle electrochemical biosensor, the sample limitation problem of traditional cortisol detection methods has been solved, and continuous detection of cortisol with high sensitivity and low detection limit has been achieved, meeting the daily testing needs of the human body.
Patent Information
- Application Number
- CN202311408712.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-27
- Publication Date
- 2026-08-25
- Estimated Expiration
- 2043-10-27
AI Technical Summary
Traditional cortisol detection methods have limitations in sample sourcing and detection, making it difficult to achieve continuous, painless, and sensitive cortisol detection, especially when cortisol concentrations are low.
A three-electrode microneedle electrochemical biosensor based on aptamers was developed. Using polylactic acid microneedles as the substrate material, combined with silver/silver chloride ink and specific modification materials, a working electrode, a reference electrode, and a counter electrode were constructed to achieve highly sensitive detection of cortisol.
It enables continuous and painless detection of cortisol, with a low detection limit, wide detection range, and high sensitivity, making it suitable for daily human testing needs.
Smart Images
Figure CN117368288B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of electrochemical detection technology, and in particular to a microneedle electrochemical biosensor based on aptamers and its preparation method. Background Technology
[0002] Cortisol, a major component of glucocorticoids, is secreted by the adrenal cortex and plays a significant role in carbohydrate metabolism. It is crucial in regulating metabolism, promoting secondary sexual characteristics, and ensuring normal bodily functions. In psychobiological research, cortisol is known as the "stress hormone." It exhibits a significant correlation with psychophysiological states; abnormally elevated cortisol levels can suppress inflammation, inhibit the immune system, and increase blood levels of fats and amino acids. Furthermore, excessively high cortisol levels can contribute to the development of Cushing's disease, characterized by abnormal fat deposition, a weakened immune system, and fragile bones. Therefore, cortisol testing is of great importance in assessing adrenal function, understanding the body's stress response, and diagnosing diseases.
[0003] However, cortisol is a biomarker with low concentrations and a strong circadian rhythm. Detection at a single time point is difficult to reflect the true level of cortisol. Traditional cortisol testing sample sources have certain limitations. For example, saliva and blood samples can only show cortisol levels over a short period; urine sample collection is too cumbersome; hair samples can provide stable cortisol levels over several months, but analyzing hair strands is very costly; sweat samples require excessive sweating during exercise and are affected by many related confounding factors, such as low sweating rate, sample evaporation, sample freshness, and skin contamination.
[0004] A growing body of research indicates a significant correlation between free cortisol levels in tissue fluid and blood. Compared to other peripheral biological fluids such as saliva, sweat, and tears, interstitial fluid (ISF) contains a variety of soluble bioanalytes, including proteins, peptides, metabolites, and nucleic acids closely related to blood. The body's homeostatic feedback loop ensures that the concentration of molecules in ISF is directly correlated with that in circulation. Small and medium-sized molecules, including glucose and ethanol, are found in ISF in proportions similar to those in blood. Cortisol, with its relatively small molecular weight (362.5 g / mol) and lipophilic nature, can diffuse across glandular and capillary epithelial cell membranes. The strong correlation between cortisol levels in ISF and blood facilitates safer online and in-situ biomarker analysis and long-term monitoring, providing stable cortisol levels.
[0005] Since microneedles were first studied in the field of transdermal drug delivery in 1997, they have made significant progress after years of rapid development. Researchers have gradually discovered that by modifying microneedles to create microneedle electrodes, the advantages of microneedles can be leveraged, leading to a surge in research on bioelectrochemical sensors combined with microneedle technology. In the field of electrochemical sensors, microneedle electrodes, made from microneedles, are microelectrodes with a needle height and diameter not exceeding 1 mm. Their small electrode area and short response time significantly enhance the sensitivity of microneedle electrodes, and they do not cause pain when inserted into the skin, improving patient compliance. Microelectrodes have attracted extensive research and applications in the field of electrochemistry, including neurotransmitter detection and the study of the electrochemical properties of bioactive materials. However, the application of microelectrodes for cortisol detection has not yet been reported. Summary of the Invention
[0006] The purpose of this invention is to provide an aptamer-based microneedle electrochemical biosensor and its preparation method. A three-electrode microneedle system is constructed to detect cortisol in tissue fluid, enabling continuous and painless detection, improving patient compliance, and exhibiting low detection limit, wide detection range, and high sensitivity, which can meet the daily needs of human cortisol detection.
[0007] To achieve the above objectives, the present invention provides a microneedle electrochemical biosensor based on aptamers, which is a three-electrode system. The three-electrode system includes a working electrode, a reference electrode, and a counter electrode. The substrate material of the working electrode, the reference electrode, and the counter electrode is polylactic acid microneedles sputtered with a gold layer.
[0008] The reference electrode is modified with silver / silver chloride ink.
[0009] The modifying materials for the working electrode include 6-mercapto-1-hexanol, 3-aminopropyltriethoxysilane, glutaraldehyde, amino-modified cortisol aptamers, and bovine serum albumin.
[0010] Preferably, the concentrations of the working electrode modification materials are 50 mM / L of 6-mercapto-1-hexanol, 5% of 3-aminopropyltriethoxysilane, 2.5% of glutaraldehyde, 4 μm / L of amino-modified cortisol aptamer, and 10 mg / ml of bovine serum albumin.
[0011] Preferably, the detection limit of the sensor is to detect cortisol concentrations of 1-1000 nM, and the sensitivity of the sensor is 0.71 μA / nM.
[0012] A method for fabricating an aptamer-based microneedle electrochemical biosensor includes the following steps:
[0013] S1 uses a hot pressing method to prepare polylactic acid microneedles. The obtained polylactic acid microneedles are then treated with oxygen plasma, and a gold layer is uniformly sputtered onto them to obtain the substrate materials for the counter electrode, working electrode, and reference electrode.
[0014] Silver / silver chloride ink is sprayed onto the surface of the S2 reference electrode to obtain a silver / silver chloride electrode.
[0015] The S3 working electrode is modified with a five-layer modifier to obtain a sensing electrode for detecting cortisol in tissue fluid.
[0016] S4 assembles the counter electrode, working electrode, and reference electrode into a sensor.
[0017] Preferably, the oxygen plasma treatment in S1 has a power of 50W, a treatment time of 5min, and a sputtered gold layer thickness of 150nm.
[0018] Preferably, the polylactic acid microneedles in S1 are in the shape of a flattened cone or a perfect cone, with a height of 0.6 mm and a base diameter of 0.3 mm.
[0019] The beneficial effects of this invention are:
[0020] Polylactic acid (PLA), with its good biocompatibility and strong mechanical properties, was selected as the matrix material for the microneedle electrode. A cross-linking method was chosen to immobilize the aptamer, with glutaraldehyde, a commonly used chemical cross-linking agent, being employed. Because it contains aldehyde groups, it reacts with the amino groups on the aptamer, ensuring a strong aptamer binding and high immobilization efficiency. By modifying the working electrode with an amino-modified cortisol aptamer, the working electrode is given the ability to detect cortisol in tissue fluid. This allows for the detection of cortisol in tissue fluid. Furthermore, by establishing a three-electrode microneedle system, continuous and painless testing can be achieved, improving patient compliance. The system also features a low detection limit, a wide detection range, and high sensitivity, meeting the needs of daily cortisol testing.
[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0022] Figure 1 This is an optical microscope image of the polylactic acid microneedles, working electrode, counter electrode, and reference electrode of the present invention.
[0023] Figure 2 Test diagrams showing the use of sealing film and pigskin for the working electrode, counter electrode, and reference electrode of this invention;
[0024] Figure 3 The differential pulse voltammetry curves of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in PBS solution are shown.
[0025] Figure 4 This is a standard curve of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in PBS solution;
[0026] Figure 5The following is a differential pulse voltammetry curve of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in simulated tissue fluid:
[0027] Figure 6 This is a standard curve of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in simulated tissue fluid;
[0028] Figure 7 This is a comparison graph showing the current detected by the microneedle electrochemical biosensor of the present invention after incubation in PBS solution containing the same concentration of cortisol for different times.
[0029] Figure 8 This is a comparison graph showing the current of detecting the same concentration of cortisol on the microneedle electrochemical biosensor modified with different concentrations of cortisol aptamers according to the present invention. Detailed Implementation
[0030] The present invention will be further described below with reference to embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by one of ordinary skill in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.
[0031] Example 1
[0032] An aptamer-based microneedle electrochemical biosensor is a three-electrode system, comprising a working electrode, a reference electrode, and a counter electrode. The substrate material for the working electrode, the reference electrode, and the counter electrode is polylactic acid microneedles sputtered with a gold layer.
[0033] The reference electrode is modified with silver / silver chloride ink.
[0034] The working electrode is modified with 6-mercapto-1-hexanol, 3-aminopropyltriethoxysilane, glutaraldehyde, an amino-modified cortisol aptamer, and bovine serum albumin, at concentrations of 50 mM / L for 6-mercapto-1-hexanol, 5% for 3-aminopropyltriethoxysilane, 2.5% for glutaraldehyde, 4 μm / L for the amino-modified cortisol aptamer, and 10 mg / ml for bovine serum albumin. The electrode itself is not modified.
[0035] The detection limit of the sensor was found to be 1-1000 nM cortisol concentration, and the sensitivity of the sensor was 0.71 μA / nM.
[0036] Example 2
[0037] Preparation method of polylactic acid microneedle substrate
[0038] (1) First, prepare the template solution for making microneedle substrate. Polydimethylsiloxane (PDMS) is selected as the material of microneedle template. The template solution is prepared by mixing PDMS and curing agent in a ratio of 10:1.
[0039] (2) Pour the prepared template solution into the mold, and cure it in a constant temperature and humidity chamber at 60°C for 1 hour. After demolding, the PDMS template is obtained.
[0040] (3) A laser engraving machine is used to engrave a pinhole substrate with a certain array on the surface of the PDMS template. The engraved substrate array is 3×3, so that the polylactic acid microneedle substrate array is 3×3, with a height of 600μm, a bottom diameter of 300μm, a needle spacing of 1000μm, and a needle body shape of conical.
[0041] (4) Polylactic acid (PLA) microneedles were made by hot pressing. PLA solid particles were placed on the above PDMS template at room temperature and then placed in a vacuum drying oven. The heating temperature was adjusted to 200°C to melt the PLA particles. The temperature was maintained for 40 minutes. The melted PLA particles on the PDMS template were pressed with a glass slide and removed after 10 minutes. The microneedles were placed under laboratory temperature conditions and allowed to cool and solidify. The glass slide was removed and the PLA microneedles were taken out with tweezers for use.
[0042] Example 3
[0043] A method for fabricating an aptamer-based microneedle electrochemical biosensor includes the following steps:
[0044] S1 uses a hot-pressing method to prepare polylactic acid (PLA) microneedles, specifically the PLA microneedles obtained in Example 2. The PLA microneedles are flattened cones or perfect cones with a height of 0.6 mm and a base diameter of 0.3 mm. The obtained PLA microneedles are then subjected to oxygen plasma treatment at a power of 50 W for 5 min. A gold layer with a thickness of 150 nm is then uniformly sputtered onto the microneedles, yielding the substrate materials for the counter electrode, working electrode, and reference electrode.
[0045] Silver / silver chloride ink is sprayed onto the surface of the S2 reference electrode to obtain a silver / silver chloride electrode.
[0046] The S3 working electrode was modified with five layers of materials to obtain a sensing electrode for detecting cortisol in tissue fluid. The modifying materials included 6-mercapto-1-hexanol, 3-aminopropyltriethoxysilane, glutaraldehyde, an amino-modified cortisol aptamer, and bovine serum albumin, with concentrations of 50 mM / L for 6-mercapto-1-hexanol, 5% for 3-aminopropyltriethoxysilane, 2.5% for glutaraldehyde, 4 μm / L for the amino-modified cortisol aptamer, and 10 mg / ml for bovine serum albumin.
[0047] S4 assembles the counter electrode, working electrode, and reference electrode into a microneedle electrochemical sensor.
[0048] Example 4
[0049] Based on Example 3, multiple microneedle electrochemical biosensors were prepared. The difference from Example 3 was that in step S3, the concentrations of the amino-modified cortisol aptamers were 1 μm / L, 2 μm / L, 3 μm / L, 4 μm / L, and 5 μm / L, respectively. All other parameters were the same as in Example 3.
[0050] Effect verification
[0051] Observe the surface morphology of the microneedle electrochemical biosensor prepared in Example 3.
[0052] After the polylactic acid microneedles were prepared, Au was sputtered onto them using a magnetron sputtering coating machine, and various modifications were performed. The resulting working electrode, counter electrode, and reference electrode were then observed under an optical microscope to examine the surface morphology of the microneedles and determine their specifications, including the height of the microneedles, the width of the base, and the spacing between the microneedles. Furthermore, the optical microscope allowed for direct determination of whether there were any breaks in the microneedle base tips and the distribution of the needle bodies. Figure 1 These are optical microscope images of the polylactic acid microneedles, working electrode, counter electrode, and reference electrode of this invention, as shown below. Figure 1 As shown in Figures A, B, C, and D, polylactic acid microneedles, working electrode, reference electrode, and counter electrode are respectively. The microneedles are all conical in shape, and the needle bodies are evenly distributed. The needle tips are undamaged and unbroken after completion.
[0053] Mechanical properties of the microneedle electrochemical biosensor prepared in Example 3 were characterized.
[0054] To test whether the microneedle electrochemical biosensor could pierce the skin, piercing performance was tested using sealing film and pigskin. Figure 2 Test diagrams of the sealing film and pigskin used for the working electrode, counter electrode, and reference electrode of this invention are shown below. Figure 2 As shown, each sealing film exhibits a 3×3 array of pores, with each sealing film approximately 120 μm in diameter. This indicates that the microneedle electrode can pierce three layers of sealing film, demonstrating a penetration depth of approximately 400 μm. After inserting a blank microneedle into pigskin and subsequently removing it, staining with Rhodamine B revealed a red mark, confirming the microneedle's ability to penetrate pigskin. The three electrodes in the microneedle electrochemical biosensor were able to pierce three layers of sealing film and pigskin. These results demonstrate that the modification process did not affect the mechanical properties of the microneedles, and that all three electrodes of the fabricated microneedle electrochemical biosensor can penetrate the skin and contact tissue fluid, enabling continuous monitoring of human cortisol levels.
[0055] Electrochemical characterization was performed on the microneedle electrochemical biosensors prepared in Examples 3 and 4.
[0056] High-concentration cortisol solutions were dissolved in phosphate-buffered saline (PBS) solution (0.01 M, pH 7.4) to prepare PBS solutions containing different concentrations of cortisol (0 nM, 1 nM, 100 nM, 200 nM, 400 nM, 600 nM, 800 nM, and 1000 nM).
[0057] Electrochemical characterization was performed using differential pulse voltammetry in an electrochemical workstation. After fixing the detection conditions, the corresponding current magnitude of the microneedle electrochemical biosensor for different concentrations of cortisol was determined by observing the current-time curve. Specifically:
[0058] (1) The microneedle electrochemical sensor prepared in Example 3 was incubated at 37°C for 80 min in 300 μL of cortisol solution containing different concentrations. Then, the time-current curves were obtained by differential pulse voltammetry in PBS solution (0.1 M, pH 7.4) containing 10 mM potassium ferricyanide / potassium ferrocyanide and simulated tissue fluid. The concentration-current curve of cortisol was plotted. The initial potential of differential pulse voltammetry was 0.75 V, the endpoint potential was -0.15 V, the potential increment was 0.005 V, the amplitude was 0.01 ms, and the pulse width was 0.02 ms.
[0059] Figure 3 The differential pulse voltammetry curves of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in PBS solution are shown. Figure 4 This is a standard curve of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in PBS solution; Figure 5 The following is a differential pulse voltammetry curve of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in simulated tissue fluid: Figure 6 This is a standard curve of the microneedle electrochemical biosensor of the present invention detecting different concentrations of cortisol in simulated tissue fluid; for example... Figure 3-6 The microneedle electrochemical biosensor shown has a detection limit of 1 nM, a detection range of 1-1000 nM for cortisol concentration, and a sensitivity of 0.71 μA / nM.
[0060] (2) The microneedle electrochemical sensor prepared in Example 3 was incubated in 300 μL of a 400 nM cortisol solution at 37 °C for different times: 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, and 120 min. Then, the time-current curve was measured by differential pulse voltammetry in PBS solution (0.1 M, pH 7.4) containing 10 mM potassium ferricyanide / potassium ferrocyanide. The initial potential of the differential pulse voltammetry was 0.75 V, the endpoint potential was -0.15 V, the potential increment was 0.005 V, the amplitude was 0.01 ms, and the pulse width was 0.02 ms. Figure 7 This is a comparison graph showing the current detected by the microneedle electrochemical biosensor of the present invention after incubation in PBS solution containing the same concentration of cortisol for different times. Figure 7 It can be seen that the detection of cortisol is most sensitive when the constant temperature incubation time is 80 min.
[0061] (3) The microneedle electrochemical sensor prepared in Example 4 was incubated in 300 μL of a 400 nM cortisol solution at 37 °C for 80 min. Then, the time-current curve was measured by differential pulse voltammetry in a PBS solution (0.1 M, pH 7.4) containing 10 mM potassium ferricyanide / potassium ferrocyanide. The initial potential of the differential pulse voltammetry was 0.75 V, the endpoint potential was -0.15 V, the potential increment was 0.005 V, the amplitude was 0.01 ms, and the pulse width was 0.02 ms. Figure 8 This is a comparison of the currents detected by the same concentration of cortisol on the microneedle electrochemical biosensor modified with different concentrations of cortisol aptamers according to the present invention. Figure 8 It can be seen that the detection of cortisol is most sensitive when the concentration of cortisol modified on the microneedle electrochemical biosensor is 4 μm / L.
[0062] Therefore, the present invention employs the above-mentioned aptamer-based microneedle electrochemical biosensor, which can be used for the detection of cortisol in tissue fluid, and can achieve continuous painless detection, improving patient compliance. It also has a low detection limit, a wide detection range, and high sensitivity, which can meet the daily needs of human cortisol detection.
[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A microneedle electrochemical biosensor based on aptamers, characterized in that: It is a three-electrode system, which includes a working electrode, a reference electrode, and a counter electrode. The substrate material of the working electrode, the reference electrode, and the counter electrode is polylactic acid microneedles sputtered with a gold layer. The reference electrode is modified with silver / silver chloride ink. The modifying materials for the working electrode include 6-mercapto-1-hexanol, 3-aminopropyltriethoxysilane, glutaraldehyde, amino-modified cortisol aptamers, and bovine serum albumin.
2. The aptamer-based microneedle electrochemical biosensor according to claim 1, characterized in that: The concentrations of the working electrode modification materials were 50 mM / L of 6-mercapto-1-hexanol, 5% of 3-aminopropyltriethoxysilane, 2.5% of glutaraldehyde, 4 μm / L of amino-modified cortisol aptamer, and 10 mg / ml of bovine serum albumin.
3. The aptamer-based microneedle electrochemical biosensor according to claim 1, characterized in that: The sensor has a detection limit of 1-1000 nM for cortisol concentration and a sensitivity of 0.71 μA / nM.
4. A method for preparing an aptamer-based microneedle electrochemical biosensor as described in any one of claims 1-3, characterized in that, Includes the following steps: S1 uses a hot pressing method to prepare polylactic acid microneedles. The obtained polylactic acid microneedles are then treated with oxygen plasma, and a gold layer is uniformly sputtered onto them to obtain the substrate materials for the counter electrode, working electrode, and reference electrode. Silver / silver chloride ink is sprayed onto the surface of the S2 reference electrode to obtain a silver / silver chloride electrode. The S3 working electrode is modified with a five-layer modifier to obtain a sensing electrode for detecting cortisol in tissue fluid. S4 assembles the counter electrode, working electrode, and reference electrode into a sensor.
5. The method for fabricating an aptamer-based microneedle electrochemical biosensor according to claim 4, characterized in that: The oxygen plasma treatment in S1 has a power of 50W, a treatment time of 5min, and a sputtered gold layer thickness of 150nm.
6. The method for fabricating an aptamer-based microneedle electrochemical biosensor according to claim 4, characterized in that: The polylactic acid microneedles in S1 are flattened cones or perfect cones, with a height of 0.6 mm and a base diameter of 0.3 mm.