An electrochemical aptamer sensor for detecting adenosine and a preparation method and application thereof
By constructing an electrochemical aptamer sensor using Pt-Cu-MWCNTs catalytic material on a gold electrode, and utilizing the changes in the binding stability of adenosine-adenosine aptamers, rapid, sensitive, and selective detection of adenosine was achieved. This solves the problems of complex operation and high cost of traditional methods and is suitable for on-site detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- EAST CHINA UNIV OF SCI & TECH
- Filing Date
- 2022-10-13
- Publication Date
- 2026-06-26
AI Technical Summary
Existing adenosine detection methods, such as HPLC and enzyme-linked immunosorbent assay (ELISA), are cumbersome and costly, making it difficult to achieve low-cost, sensitive, and selective detection.
Gold electrodes were fabricated using screen printing technology, and combined with trapping aptamers and Pt-Cu-MWCNTs catalytic materials. A double-chain structure was formed through base complementary pairing, and the quantitative detection of adenosine was achieved by utilizing the change in the binding stability of adenosine-adenosine aptamers.
It enables rapid detection of adenosine, with a wide detection range, low detection limit and good stability, suitable for on-site testing, low cost and easy operation.
Smart Images
Figure CN116500105B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of metal nanomaterials, biomolecules and biosensing detection technology, and relates to an electrochemical aptamer sensor for detecting adenosine, a marker of hypoxia. Specifically, it refers to an electrochemical aptamer sensor for detecting adenosine and its preparation method. Background Technology
[0002] Adenosine (AD) is an endogenous purine nucleoside produced during the degradation of purine nucleotides. Primarily composed of 5′-nucleotidases, it is a crucial intermediate in the synthesis of adenosine triphosphate (ATP), adenine, adenosine acid, and vidarabine. It also plays a vital role as a neuromodulator in heart rate regulation, neurotransmission, and respiratory control. Furthermore, adenosine is generated from pre-released adenosine triphosphate in the extracellular space through the action of specific enzymes (collectively called exonucleases). Adenosine is a potential tumor marker and plays an important signaling function in both the peripheral and central nervous systems. In addition, adenosine inhibits synaptic activity, slowing down brain metabolism and thus inducing sleep, which is crucial for successful deep brain stimulation in patients with Parkinson's disease and other brain disorders. Under normal conditions, adenosine concentrations in healthy individuals are maintained at low levels: plasma adenosine concentrations should be around 20 nM, serum adenosine concentrations below 1 mM, and urine adenosine concentrations around 6.7 μM. In hypoxic environments, the body's ATP consumption increases significantly, leading to the breakdown of accumulated adenosine monophosphate (ATP) into adenosine. This results in a marked increase in adenosine concentration within the body, which intensifies with prolonged hypoxia. Under anaerobic conditions, adenosine regulates the body's energy (or oxygen) supply and demand balance and is closely related to ischemia, inflammation, and lung diseases (such as asthma and idiopathic pulmonary fibrosis). Therefore, developing new strategies for highly sensitive and selective adenosine detection is crucial.
[0003] Currently, traditional standard adenosine detection methods are HPLC and enzyme-linked immunosorbent assay (ELISA). HPLC requires preliminary sample separation and standard samples to determine retention time or mass spectrometry for qualitative detection of adenosine; the related kits are lengthy and cumbersome to operate. ELISA, on the other hand, is relatively expensive and demands a high level of skill from the operators. Therefore, developing a low-cost, simple, sensitive, and selective detection method is crucial.
[0004] Electrochemical sensors have attracted increasing attention due to their advantages such as low energy consumption, simple equipment, and ease of miniaturization. They have been widely used in many fields, such as medical diagnostics, environmental monitoring, and food safety monitoring. This experiment, based on an aptamer with high affinity and a high-performance metal composite material, prepared an electrochemical aptamer sensor for detecting adenosine, which is expected to be used for on-site detection. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides an electrochemical aptamer sensor for detecting adenosine and its preparation method. The sensor designed and constructed by this invention enables rapid detection of the hypoxia marker adenosine, exhibiting a wide detection range, a low detection limit, and good stability.
[0006] The technical solution of this invention is as follows:
[0007] An electrochemical aptamer sensor for detecting adenosine utilizes a gold electrode fabricated using screen printing technology. A trapping aptamer (ssDNA 1) is dropped onto the electrode surface, followed by the addition of 6-mercaptohexanol (MCH) to block any unbound active sites. Then, a complex consisting of an adenosine aptamer (ssDNA 2) and a catalytic material (Pt-Cu-MWCNTs) is added. This complex forms a double strand with the ssDNA 1 on the electrode surface through base complementarity. Upon addition of the analyte adenosine, the binding between adenosine and the adenosine aptamer is more stable than that between the adenosine aptamer and the trapping aptamer. The catalytic material (Pt-Cu-MWCNTs) is released from the electrode surface along with the adenosine-adenosine aptamer complex, causing a change in the sensor's catalytic response to hydrogen peroxide, thus achieving the quantitative detection of adenosine.
[0008] This invention also includes an electrochemical aptamer sensor for detecting the hypoxia marker adenosine, the preparation steps of which are as follows:
[0009] (1) Preparation and pretreatment of screen-printed gold electrodes;
[0010] (2) 10 μL of 4 μM ssDNA1 was added to the surface of the treated gold electrode and incubated at 4 °C for 4 h. The electrode was then washed with ultrapure water to remove excess ssDNA1. Then, 5 mM [Fe(CN)6] was added. 3- / [Fe(CN)6] 4- Its cyclic voltammogram and impedance spectrum were tested.
[0011] (3) Add 10 μL of 5 mM 6-mercaptohexanol (MCH) to shield potential non-specific binding sites. After blocking at 37°C for 2 h, rinse the electrode with ultrapure water to remove excess MCH. Then, add 5 mM [Fe(CN)6]... 3- / [Fe(CN)6] 4- Its cyclic voltammogram and impedance spectrum were tested.
[0012] (4) After adding 10 μL of the Pt-Cu-MWCNTs-ssDNA2 complex and incubating at 4 °C for 12 h, the electrode was thoroughly cleaned with ultrapure water. The electrochemical aptamer sensor was then successfully prepared. (The process was repeated in the original text, likely due to an incomplete sentence or missing information.) 3- / [Fe(CN)6] 4- Its cyclic voltammogram and impedance spectrum were tested, and its chronocurrent response to catalyze H2O2 was tested in a PBS buffer solution (pH = 7.4, 10 mM).
[0013] Furthermore, the specific methods for preparing and pretreating the screen-printed gold electrodes are as follows:
[0014] (1) Wipe the cut polyester substrate (PET film) clean with anhydrous ethanol and dry it. Fix it on the screen printing machine. First, print the conductive silver layer and cure it at 120℃ for 40 minutes. Then print the insulating ink layer and cure it at 80℃ for 10 minutes. Fix a plastic mask with a small hole with a diameter of 3mm at an appropriate position on the printed PET board so that the gold particles are fixed at this position. The current intensity during vacuum evaporation is 40mA and the evaporation time is 300s to obtain the screen-printed gold electrode.
[0015] (2) The screen-printed gold electrode (SPGE) prepared above was pretreated by cyclic voltammetry. The electrode was scanned 15 times in 0.5M H2SO4 solution with a voltage range of -0.2V to 1.5V and a scan rate of 0.1V / s. After treatment, the electrode was dried, washed with ultrapure water, and then dried for later use.
[0016] As an improvement, the Pt-Cu-MWCNTs-ssDNA2 composite marker is prepared as follows:
[0017] (1) Synthesis of Pt-Cu-MWCNTs;
[0018] (2) Weigh 0.4 mg Pt-Cu-MWCNTs, add 200 μL of 4 μM activated ssDNA2 solution, and vortex incubate overnight at 8 °C and 1300 rpm.
[0019] (3) After washing three times with PBS buffer to remove unbound ssDNA2, add 50 μL of 5 mM MCH to mask non-specific binding sites, continue incubation for 1 h, then centrifuge and wash four times with PBS buffer (pH = 7.4, 10 mM), disperse the product in 200 μL of PBS buffer (pH = 7.4, 10 mM), and store at 4 °C for later use.
[0020] As an improvement, the Pt-Cu-MWCNTs metal composite material is prepared as follows:
[0021] a. Take 50 mg of commercially available acidified multi-walled carbon nanotubes (MWCNTs) and dissolve them in 100 mL of ultrapure water by sonication;
[0022] b. Add 2.9 mL of 0.1 M Cu(NO3)2·3H2O and 4.36 mL of 0.0243 M H2PtCl6·6H2O to the dispersion and stir magnetically for 20 min;
[0023] c. Add 25 mL of freshly prepared 0.79 M sodium borohydride solution dropwise to the reaction mixture and stir at room temperature for several hours;
[0024] d. Centrifuge the precipitate and wash it several times with distilled water to remove unreacted raw materials. The resulting product is then vacuum dried at 60°C.
[0025] As an improvement, the base sequence of ssDNA1 in step (2) is: 5′-CCCAGGTTCTC-(CH2)6-SH-3′.
[0026] As an improvement, the base sequence of ssDNA2 in step (4) is: 5-AGAGAACCTGGGGGAGTATTGCGGAGGAAGGTT-(CH2)6-SH-3′.
[0027] This invention also provides a method for detecting adenosine using an electrochemical aptamer sensor, comprising the following steps: In step (4) of the preparation method of the electrochemical aptamer sensor, a certain concentration of the target substance adenosine is added to the modified electrode, incubated at 37°C for 45 min, washed with ultrapure water, and dried. This electrode is used as the working electrode, Ag / AgCl is used as the reference electrode, and a platinum wire is used as the counter electrode to form a three-electrode system. The electrocatalytic signal of the sensor to H2O2 before and after the addition of adenosine is tested in PBS buffer solution using chronoamperometry to obtain the standard curve of the electrochemical aptamer sensor to adenosine.
[0028] With the above structure, the present invention has the following advantages:
[0029] 1. This invention provides an electrochemical aptamer sensor for detecting adenosine, which has advantages such as wide detection range, low cost, simple operation, and field applicability. It has certain practical application prospects for the detection of adenosine in living organisms and the immediate diagnosis of related physiological diseases. It provides new ideas and methods for the detection of hypoxia markers and related physiological diseases in living organisms.
[0030] 2. The substrate electrode used in this invention is a screen-printed gold electrode, which has a simple preparation process and can be mass-produced. It can also be prepared as a disposable electrode, making it highly applicable in the field of on-site testing.
[0031] 3. This invention uses oligonucleotides (DNA or RNA) selected from a random sequence nucleic acid library using the SELEX system as aptamers to replace traditional antibodies in binding to the analyte. This has significant advantages such as small size, chemical simplicity, easy storage, strong specific binding ability, and ease of further immobilization.
[0032] 4. This invention synthesizes Pt-Cu-MWCNTs metal composite material in a one-pot method and uses it as a marker for an electrochemical aptamer sensor. Pt and Cu nanoparticles can not only immobilize the aptamer, but also amplify the electrochemical signal of H2O2 catalysis after being combined with large surface area and highly conductive multi-walled carbon nanotubes, thereby improving the detection sensitivity to a certain extent. Attached image description:
[0033] Figure 1 This is a schematic diagram of the preparation process of the present invention.
[0034] Figure 2 These are characterization images of the prepared Pt-Cu-MWCNTs, including SEM, TEM, and XRD patterns.
[0035] Figure 3 Cyclic voltammetry and electrochemical impedance spectroscopy of the modified electrochemical aptamer sensor.
[0036] Figure 4 It is the time-current response of the sensor to H2O2 before and after the addition of the target substance adenosine.
[0037] Figure 5 This is the standard curve of the prepared electrochemical aptamer sensor for different concentrations of adenosine. Detailed implementation method:
[0038] The present invention will now be described in further detail with reference to the accompanying drawings.
[0039] Combined with appendix Figure 1 An electrochemical aptamer sensor for detecting adenosine, a marker of hypoxia, includes a gold electrode prepared by screen printing, on which a capture aptamer (ssDNA1) is dropped. Then, MCH is added to block unbound active sites. Finally, a complex consisting of adenosine aptamer (ssDNA2) and Pt-Cu-MWCNTs catalytic material is dropped, which pairs with ssDNA1 on the electrode surface to form a double-stranded structure.
[0040] An electrochemical aptamer sensor for detecting adenosine, a marker of hypoxia, is prepared as follows:
[0041] (1) Preparation and pretreatment of screen-printed gold electrodes;
[0042] (2) 10 μL of 4 μM ssDNA1 was dropped onto the surface of the treated gold electrode and incubated at 4 °C for 4 h. The electrode was then washed with ultrapure water to remove excess ssDNA1.
[0043] (3) Add 10 μL of 5 mM 6-mercaptohexanol (MCH) to shield possible non-specific binding sites. After sealing at 37°C for 2 h, clean the electrode with ultrapure water to remove excess MCH.
[0044] (4) After adding 10 μL of the Pt-Cu-MWCNTs-ssDNA2 complex and incubating at 4 °C for 12 h, the electrode was thoroughly cleaned with ultrapure water, and the electrochemical aptamer sensor was completed.
[0045] Combined with appendix Figure 2 The preparation and pretreatment methods for the screen-printed gold electrodes are as follows:
[0046] (1) The cut PET board was wiped clean and dried with anhydrous ethanol, and fixed on a screen printing machine. First, a conductive silver layer was printed and cured at 120℃ for 40 min; then an insulating ink layer was printed and cured at 80℃ for 10 min. A plastic mask with small holes of 3 mm in diameter was fixed at an appropriate position on the printed PET board to fix the evaporated gold particles in this position. The current intensity during vacuum evaporation was 40 mA and the evaporation time was 300 s; after evaporation, the electrode was aged at 100℃ for 1 h to obtain the screen-printed gold film electrode.
[0047] (2) The SPGE prepared above was pretreated by cyclic voltammetry (CV). The CV scan was performed in 0.5M H2SO4 solution for 15 cycles, with a voltage range of -0.2V to 1.5V and a scan rate of 0.1V / s. After treatment, the electrode was dried, washed with ultrapure water, and then dried for later use.
[0048] The specific preparation method of the Pt-Cu-MWCNTs metal composite material is as follows:
[0049] a. Take 50 mg of commercially available acidified multi-walled carbon nanotubes (MWCNTs) and dissolve them in 100 mL of ultrapure water by sonication;
[0050] b. Add 2.9 mL of 0.1 M Cu(NO3)2·3H2O and 4.36 mL of 0.0243 M H2PtCl6·6H2O to the dispersion and stir at room temperature for 20 min;
[0051] c. Add 25 mL of freshly prepared 0.79 M sodium borohydride solution dropwise to the reaction mixture and stir at room temperature for several hours;
[0052] d. Centrifuge the precipitate and wash it several times with distilled water to remove unreacted raw materials. The resulting product is then vacuum dried at 60°C.
[0053] The 10 μL Pt-Cu-MWCNTs-ssDNA2 composite marker is prepared as follows:
[0054] a. Weigh 0.4 mg Pt-Cu-MWCNTs, add 200 μL of 4 μM ssDNA2 solution, and vortex incubate overnight at 8 °C and 1300 rpm;
[0055] b. Then wash three times with PBS buffer to remove unbound ssDNA2, add 50 μL of 5 mM MCH to mask non-specific binding sites, and continue incubation for 1 h.
[0056] c. Wash the product four times by centrifugation with PBS buffer (pH=7.4, 10mM), disperse the product in 200μL PBS buffer (pH=7.4, 10mM), and store at 4℃ for later use.
[0057] An electrochemical aptamer sensor for detecting adenosine includes the following steps: (4) A certain concentration of the target substance adenosine is added to the modified electrode, incubated at 37°C for 45 min, washed with ultrapure water, and dried. This electrode is used as the working electrode, Ag / AgCl is used as the reference electrode, and a platinum wire is used as the counter electrode to form a three-electrode system. The electrocatalytic signal of the sensor to H2O2 before and after the addition of adenosine is tested in PBS buffer solution using chronoamperometry to obtain the standard curve of the electrochemical aptamer sensor for adenosine.
[0058] Experimental Example 1
[0059] (1) Synthesis of Pt-Cu-MWCNT-ssDNA2 complex marker
[0060] 50 mg of commercially available acidified multi-walled carbon nanotubes (MWCNTs) were ultrasonically dissolved in 100 mL of ultrapure water. 2.9 mL of 0.1 M Cu(NO3)2·3H2O and 4.36 mL of 0.0243 M H2PtCl6·6H2O were added to the dispersion. After stirring for 20 min, 25 mL of 0.79 M sodium borohydride was added dropwise to the reaction mixture, and the mixture was stirred at room temperature for several hours. The precipitate was filtered and washed several times with distilled water to remove unreacted contents. The resulting product was dried at 60 °C. Weigh 0.4 mg of Pt-Cu-MWCNTs and add 200 μL of 4 μM ssDNA2 solution. Incubate overnight at 8 °C and 1300 rpm using a vortex incubator. Then add 50 μL of 5 mM MCH to mask non-specific binding sites and continue incubation for 1 h. After incubation, wash the product four times with PBS buffer (pH = 7.4, 10 mM) by centrifugation. Disperse the product in 200 μL of PBS buffer and store at 4 °C for later use.
[0061] Characterization diagram of the prepared product is shown below Figure 2 MWCNTs appeared in the XRD patterns.
[0062] The absorption of Pt(111), Cu(110), Cu(200), and Cu(220) crystal planes indicates the successful loading of Pt and Cu particles onto MWCNTs. SEM and TEM images also show the good loading of Pt and Cu particles onto carbon nanotubes, indicating the successful synthesis of the material.
[0063] (2) Preparation of SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 modified electrode
[0064] 10 μL of 4 μM ssDNA1 was added to the surface of the treated gold electrode and incubated at 4 °C for 4 h. The electrode was then washed with ultrapure water to remove excess ssDNA1. Next, 10 μL of 6-mercaptohexanol (MCH) was added to block potential non-specific binding sites, and the electrode was blocked at 37 °C for 2 h. The electrode was then washed with ultrapure water to remove excess MCH. Finally, 10 μL of the Pt-Cu-MWCNT-ssDNA2 complex was added, and the electrode was incubated at 4 °C for 12 h. The electrode was then thoroughly washed with ultrapure water to obtain the SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 modified electrode.
[0065] (3) Determination of sensor modification steps
[0066] Electrochemical tests were performed on the SPGE, SPGE / ssDNA1, SPGE / ssDNA1 / MCH, SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2, and the SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 / AD modified electrodes in Experimental Example (2) using cyclic voltammetry and electrochemical impedance spectroscopy. The test solution was 5 mM [Fe(CN)6]. 3- / [Fe(CN)6] 4- Solution, test results as follows Figure 3 As shown, after assembling ssDNA1 on SPGE, the redox peak current (curve b) decreased compared to bare SPGE (curve a) due to the formation of the aptamer layer. Similarly, due to the severe inhibition of electron transfer by MCH, the peak current continued to decrease as MCH (curve c) and the labeled material (Pt-Cu-MWCNTs-ssDNA2) were coated on the electrode surface. However, after adding the target adenosine, the redox peak current increased (curve e), which was due to the release of the adenosine-ssDNA2 complex from the electrode surface.
[0067] Impedance can be represented by the diameter of the semicircle in the high-frequency EIS spectrum. A larger diameter results in a higher impedance and correspondingly lower conductivity. Compared to curve a, the impedance values of the SPGE / ssDNA1 modified electrode (curve b) and the SPGE / ssDNA1 / MCH modified electrode (curve c) are increased, indicating successful bonding of ssDNA1 and MCH to the electrode surface. The impedance value of the SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 modified electrode (curve d) is slightly increased, indicating successful bonding of the labeled material (Pt-Cu-MWCNTs-ssDNA2). Upon addition of AD, adenosine forms a complex with ssDNA2, which is released from the electrode surface, resulting in a decrease in the impedance value of the modified electrode (curve e). The electrochemical impedance spectroscopy results are consistent with the cyclic voltammetry results, indicating that the sensor with the modified electrode has been successfully fabricated.
[0068] (4) Determination of the target substance adenosine
[0069] In (2), a certain concentration of the target substance adenosine was added dropwise to the modified electrode. After incubation at 37°C for 45 min, it was washed with ultrapure water and dried. A three-electrode system was constructed using this electrode as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire as the counter electrode. The catalytic electrochemical test of the sensor before and after the addition of adenosine was performed in PBS buffer solution using a chronoamperometry. The test results of the It response are as follows: Figure 4 As shown.
[0070] The results showed that after adding 100 nM adenosine, the prepared sensor showed a significant decrease in its It catalytic response to H2O2. This indicates that after the addition of the target analyte adenosine, the catalytic material on the electrode surface was released from the electrode to stabilize the binding between the adenosine-adenosine aptamer and the adenosine aptamer-capture aptamer, thus reducing the catalytic response of the sensor to H2O2. The experimental results were consistent with the expected results, and the quantitative detection of adenosine was achieved.
[0071] (5) Determination of the standard curve
[0072] Prepare adenosine standard solutions with concentrations of 10 nM, 20 nM, 50 nM, 100 nM, 500 nM, and 1 μM, and store them at 4°C for later use.
[0073] Using an SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 modified electrode as the working electrode, the working electrode was first incubated with adenosine standard solutions of different concentrations at 37°C for 45 minutes to form an SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 / AD modified electrode. Then, this working electrode was combined with a platinum wire electrode (counter electrode) and Ag / AgCl (reference electrode) to construct a three-electrode system. PBS solution was used as the detection solution to construct an electrochemical aptamer sensor.
[0074] The current signal was obtained using a chronoamperometry method. The principle is as follows: AD was incubated on the surface of the SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 modified electrode. The stronger specific binding of AD to ssDNA2 caused the Pt-Cu-MWCNTs-ssDNA2 / AD complex to be released from the electrode surface, resulting in a decrease in the catalytic current signal of the sensor for H2O2. Moreover, the intensity of the decrease in current signal is proportional to the adenosine concentration within a certain range.
[0075] At 37℃, the constructed sensor was used to detect different concentrations of adenosine to obtain current signals. The current signal from the SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 modified electrode was used as the blank value, and the current signal from the SPGE / ssDNA1 / MCH / Pt-Cu-MWCNTs-ssDNA2 / AD modified electrode was used as the detected value. The difference (ΔI) between the blank value and the detected value was calculated. A standard curve was obtained by plotting the logarithm of adenosine concentration on the x-axis and the current difference on the y-axis (see...). Figure 5 ).
[0076] The standard curve shows that within the adenosine concentration range of 10 nM to 1 μM, the difference in current signal detected by the prepared sensor is directly proportional to the logarithm of the adenosine concentration, and its linear regression equation is ΔI = 19.07lgC. AD-9.25(R 2 =0.9994). The blank value was measured multiple times, and the relative standard deviation of the blank value was calculated. According to the formula: detection limit = 3 × standard deviation of blank / slope of standard curve, the detection limit of the electrochemical immunosensor was calculated to be 0.4 nM (S / N = 3).
[0077] (6) Determination of actual samples
[0078] The suitability of the prepared electrochemical adapter sensor was verified by measuring the adenosine concentration in healthy human serum samples using a standard addition method. Previous reports indicated that the adenosine concentration in normal human blood ranges from 50 nM to 100 nM. Experimentally, after adding serum samples, the ΔI was measured to be 26.25 μA (N = 3), and substituting this into a linear regression equation yielded an adenosine concentration of approximately 72.6 nM.
[0079] Furthermore, to evaluate the accuracy of the prepared sensor in detecting adenosine in real samples, we used a spiked recovery method to spike different concentrations of adenosine (20 nM, 100 nM) into healthy human serum samples diluted 10-fold. The constructed electrochemical aptamer sensor was then incubated with these samples, thoroughly washed, and tested using a chronoamperometry method. The results are shown in Table 1. The recoveries for different concentrations of samples ranged from 98.4% to 99.85%, with relative standard deviations all below 3%. These results indicate that the constructed sandwich-type adenosine electrochemical aptamer sensor is feasible for determining adenosine in serum samples.
[0080] Table 1. Analysis of the prepared adenosine electrochemical aptamer sensor in actual samples.
[0081]
[0082] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.
Claims
1. An electrochemical aptamer sensor for detecting adenosine, characterized in that, The method involves fabricating a gold electrode using screen printing technology, on which a capture aptamer ssDNA 1 is attached. Unbound active sites are then blocked with 6-mercaptohexanol. Next, a complex of adenosine aptamer ssDNA 2 and catalytic material Pt-Cu-MWCNTs is added dropwise. This forms a stable double-stranded structure on the electrode surface through base complementarity with ssDNA 1. Upon addition of the analyte adenosine, the catalytic material on the electrode surface is released from the electrode because the binding between adenosine and the adenosine aptamer is more stable than that between the adenosine aptamer and the capture aptamer. This causes a change in the catalytic response of the prepared sensor to hydrogen peroxide, thus achieving the quantitative detection of adenosine. The Pt-Cu-MWCNTs metal composite material is prepared by the following method: a. Take 50 mg of commercially available acidified multi-walled carbon nanotubes (MWCNTs) and dissolve them in 100 mL of ultrapure water by sonication; b. Add 2.9 mL of 0.1 M Cu(NO3)2·3H2O and 4.36 mL of 0.0243 M H2PtCl6·6H2O to the dispersion, and stir magnetically for 20 min; c. Add 25 mL of freshly prepared 0.79 M sodium borohydride solution dropwise to the reaction mixture and stir at room temperature for several hours; d. Centrifuge the precipitate and wash it several times with distilled water to remove unreacted raw materials. The resulting product is then vacuum dried at 60 °C.
2. The method for preparing an electrochemical aptamer sensor for detecting adenosine according to claim 1, characterized in that, Includes the following steps: (1) Preparation and pretreatment of screen-printed gold electrodes; (2) The capture aptamer ssDNA1 was dropped onto the surface of the treated gold electrode and incubated at 4 °C for 4 h. The electrode was then washed with ultrapure water to remove excess ssDNA1. (3) Add 10 μL of 5 mM 6-mercaptohexanol (MCH) to the electrode surface obtained in step (2) to shield possible non-specific binding sites. After sealing at 37 °C for 2 h, clean the electrode with ultrapure water to remove excess MCH. (4) 10 μL of Pt-Cu-MWCNTs-ssDNA2 complex was dropped onto the electrode surface obtained in step (3). After incubation at 4 °C for 12 h, the electrode was thoroughly cleaned with ultrapure water. The electrochemical aptamer sensor was then prepared.
3. The method for preparing an electrochemical aptamer sensor for detecting adenosine according to claim 2, characterized in that, The specific methods for preparing and pretreating the screen-printed gold electrodes are as follows: (1) Wipe the cut PET board clean with anhydrous ethanol and dry it. Fix it on the screen printing machine. First, print the conductive silver layer and cure it at 120 °C for 40 min. Then print the insulating ink layer and cure it at 80 °C for 10 min. Fix a plastic mask with a small hole with a diameter of 3 mm at an appropriate position on the printed PET board so that the gold particles are fixed at this position. The current intensity during vacuum evaporation is 40 mA and the evaporation time is 300 s to obtain the screen-printed gold electrode. (2) The SPGE prepared above was pretreated by cyclic voltammetry. The CV scan was performed in 0.5 M H2SO4 solution for 15 cycles, with a voltage range of -0.2 V to 1.5 V and a scan rate of 0.1 V / s. After treatment, the electrode was dried, washed with ultrapure water, and then dried for later use.
4. The method for preparing an electrochemical aptamer sensor for detecting adenosine according to claim 2, characterized in that, The specific preparation method of the Pt-Cu-MWCNTs-ssDNA2 complex marker is as follows: (1) Synthesis of Pt-Cu-MWCNTs; (2) Weigh 0.4 mg Pt-Cu-MWCNTs, add 200 μL of 4 μM activated ssDNA2 solution, and vortex incubate overnight at 8 ℃ and 1300 rpm; (3) After washing three times with PBS buffer to remove unbound ssDNA2, add 50 μL, 5 mM MCH to mask non-specific binding sites, continue incubation for 1 h, then wash four times with PBS buffer pH = 7.4, 10 mM by centrifugation, disperse the product in 200 μL PBS buffer pH = 7.4, 10 mM, and store at 4 ℃ for later use.
5. The method for preparing an electrochemical aptamer sensor for detecting adenosine according to claim 2, characterized in that, The ssDNA1 sequence is 5´-CCCAGGTTCTC-(CH2)6-SH-3´, and the ssDNA2 sequence is 5´-AGAGAACCTGGGGGAGTATTGCGGAGGAAGGTT-(CH2)6-SH-3´.
6. The application of an electrochemical aptamer sensor for detecting adenosine according to claim 1, or an electrochemical aptamer sensor prepared by the preparation method of any one of claims 2-5 for detecting adenosine, for non-diagnostic purposes, characterized in that, Includes the following steps: A certain concentration of the target compound adenosine was added dropwise to the modified electrode in the electrochemical aptamer sensor. After incubation at 37 °C for 45 min, the electrode was washed with ultrapure water and dried. This electrode was used as the working electrode, and Ag / AgCl was used as the reference electrode and a platinum wire counter electrode to form a three-electrode system. The sensor's response to H2O2 before and after the addition of adenosine was measured using a chronoamperometry in PBS buffer solution. The quantitative detection of adenosine is achieved by measuring the magnitude of changes in the catalytic electrical signal.