Preparation of ratio type adriamycin electrochemical aptamer sensor based on single signal molecule

Abstract

The invention relates to the technical field of electrochemical biosensors, in particular to preparation of a ratio type doxorubicin electrochemical aptamer sensor based on a single signal molecule, the sensor realizes double current signal output through the single signal molecule, and calibration-free rapid detection of doxorubicin is realized by utilizing a ratio signal. The method comprises the following specific steps: fixing an adriamycin aptamer modified with methylene blue on the surface of a gold electrode; performing electrochemical scanning in a buffer solution system (such as a 1 * PBS solution) by adopting a square wave voltammetry to obtain two current peak signals; and calculating the ratio of the two current peak signals to determine the doxorubicin concentration. According to the method, the sensitivity and the accuracy of detection are improved, the influence of environmental factors and sensor manufacturing changes on a detection result is reduced, and the calibration-free rapid detection of the concentration of the adriamycin is realized. The sensor can keep signal stability in long-time continuous testing, is suitable for rapid detection of adriamycin and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensors, and in particular to utilizing a signal molecule to generate a dual current signal, and utilizing the ratio value of the dual current signal as an output signal of a sensor to realize the detection and application of the sensor. Background Art

[0002] Doxorubicin is an anti-tumor antibiotic that can inhibit the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA and a wide anti-tumor spectrum. It is effective against a variety of tumors and has a wide range of clinical applications. However, this drug also has strong toxic side effects. Therefore, establishing a sensitive and rapid doxorubicin detection method is of great significance to ensure effective treatment of tumors while minimizing the occurrence of toxic side effects.

[0003] Electrochemical aptamer-based (E-AB) biosensors use aptamers as biorecognition elements to detect target molecules through electrochemical signal conversion mechanisms. Aptamers are single-stranded DNA or RNA sequences that can specifically bind to target molecules (such as proteins, small drug molecules, metal ions, etc.). This binding changes the conformation of the aptamer, which in turn causes changes in the electron transfer rate between the signal molecules modified on the aptamer and the electrode surface, thereby generating a measurable electrical signal. Finally, the electrical signal is detected using electrochemical methods (such as square wave voltammetry, etc.), ultimately achieving the purpose of detecting the target molecule.

[0004] During the sensor preparation process, the differences in the total number of aptamers fixed on the electrode surface by self-assembly make the detection signals of different sensors for the same analyte vary greatly. Therefore, such sensors need to be self-calibrated before each use. This process is not only cumbersome and time-consuming, but also difficult to operate and implement in some complex test systems (such as in vivo tests), which greatly limits the application of such sensors in real-time detection. Based on this, a type of electrochemical aptamer sensor platform that does not require frequent calibration has been developed to address this problem, becoming one of the hot research directions in this direction. Summary of the invention

[0005] The present invention relates to a method for preparing a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule, aiming to provide a method for detecting doxorubicin quickly and with high sensitivity without frequent calibration. Doxorubicin is a broad-spectrum antitumor antibiotic. Although it is widely used in clinical treatment, it also has strong toxic side effects. Therefore, timely and accurate detection of doxorubicin concentration is of great significance for ensuring effective treatment while reducing toxic side effects.

[0006] The innovation of the present invention is to realize dual current signal output through a single signal molecule and realize calibration-free rapid detection of doxorubicin using ratio signals. This method comprises the following steps:

[0007] Modified aptamers: The doxorubicin aptamer modified with methylene blue was fixed to the surface of the gold electrode by self-assembly. The aptamer is a single-stranded DNA or RNA sequence that can specifically bind to doxorubicin. After binding, it will change its conformation, thereby affecting the electrochemical signal.

[0008] Electrochemical scanning: In a buffer system (such as 1×PBS solution), electrochemical scanning is performed using CHI1040C equipment using square wave voltammetry (SWV). The output curve of the sensor shows two current peaks, corresponding to different electrochemical reaction processes.

[0009] Signal ratio calculation: By calculating the ratio of the two current peaks, calibration-free detection of doxorubicin concentration can be achieved. This ratio signal detection method not only improves the sensitivity and accuracy of the detection, but also significantly reduces the impact of environmental factors and sensor manufacturing changes on the detection results.

[0010] Main advantages and technical effects:

[0011] High sensitivity and accuracy: The sensitivity and accuracy of doxorubicin detection are improved through the ratio calculation of dual current signal output.

[0012] Calibration-free detection: No need for frequent self-calibration operations, which simplifies the detection process and is particularly suitable for complex test systems (such as in vivo testing).

[0013] Good stability: The stability test proves that in the buffer system, the sensor can maintain the ratio stability of the current peak signal in long-term continuous testing.

[0014] In summary, the present invention provides a method for preparing a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule, which has the advantages of high sensitivity, no calibration, and simple operation, and is suitable for the rapid detection of doxorubicin.

[0015] Beneficial effects of the present invention:

[0016] 1. Based on the characteristics of aptamers, this method has high specificity;

[0017] 2. The ratio signal of the dual current peak signal can be used to avoid calibration operations, making the detection of doxorubicin more convenient and timely, which is conducive to promoting the clinical application of doxorubicin electrochemical aptamer biosensor;

[0018] 3. The output of dual current signals achieved by adjusting the test parameters of the square wave voltammetry method can greatly reduce the production cost of the sensor. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 (A) is a schematic diagram of the E-AB detection principle of the present invention, and (B) is a square wave voltammetry measurement curve diagram;

[0021] Figure 2 The double peaks generated during the test of the doxorubicin electrochemical aptamer sensor of the present invention vary with the target concentration;

[0022] Figure 3 This is a stability curve of the doxorubicin electrochemical aptamer sensor of the present invention in PBS solution;

[0023] Figure 4 It is a curve diagram showing the relationship between the current ratio signal and the target concentration of the aptamer sensor of the present invention in a PBS solution;

[0024] Figure 5 This is a peak diagram of the doxorubicin sensor in PBS solution under different test conditions according to an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments, and those skilled in the art may also adopt other alternatives to implement some known technologies; and the accompanying drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0026] It should be noted that the references to "one embodiment", "an embodiment", "an exemplary embodiment", "some embodiments" and the like in the specification indicate that the embodiments described may include specific features, structures or characteristics, but not every embodiment may include the specific features, structures or characteristics. In addition, when a specific feature, structure or characteristic is described in conjunction with an embodiment, it should be within the knowledge of a person skilled in the art to implement such feature, structure or characteristic in conjunction with other embodiments (whether or not explicitly described).

[0027] In general, a term can be understood, at least in part, from its use in context. For example, depending, at least in part, on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in the singular sense, or can be used to describe a combination of features, structures, or characteristics in the plural sense. Additionally, the term "based on" can be understood as not necessarily intended to convey an exclusive set of factors, but can instead, depending, at least in part, on the context, allow for the presence of other factors that are not necessarily explicitly described.

[0028] Example 1

[0029] Electrode cleaning:

[0030] 1.1. Using CHI1040C equipment, place the electrode in the electrolytic cell, select Ag / AgCl as the reference electrode and platinum wire as the counter electrode.

[0031] 1.2. Electrochemically clean the electrode in 0.5M NaOH and 0.5M H2SO4 to remove surface impurities and oxides. After cleaning, rinse the electrode with deionized water and dry it for later use.

[0032] Aptamer modification:

[0033] 2.1. Take 2 μL of the thiol-modified doxorubicin aptamer, centrifuge, add 2 μL of 20 mM tris(2-carboxyethyl)phosphine hydrochloride solution (Tcep), and react at room temperature for 1 hour.

[0034] 2.2. Dilute the aptamer solution to a concentration of 1 μM using PBS solution.

[0035] Sensor preparation:

[0036] 3.1. Rinse the cleaned gold electrode with deionized water, soak it in the aptamer solution, and self-assemble at room temperature for 2 hours to make the aptamer firmly attached to the electrode surface.

[0037] 3.2. The electrode was then transferred into 1 mL of 20 mM 6-mercapto-1-hexanol (MCH) solution and placed in a 4°C refrigerator overnight to further enhance the stability and sensitivity of the aptamer.

[0038] Electrochemical testing:

[0039] 4.1. Take out the prepared electrode from the refrigerator and connect it to the CHI1040C instrument.

[0040] 4.2. In 10 mL of PBS solution, square wave voltammetry (SWV) was used to scan different concentrations of doxorubicin solution at multiple frequencies and the induced current was recorded.

[0041] 4.3. Scan the two current peak signals output by the electrode, calculate their ratio, and draw a curve showing the relationship between the ratio signal size and the doxorubicin concentration.

[0042] Data processing and analysis:

[0043] 5.1. In the buffer system, the current peak signal at different doxorubicin concentrations was measured by square wave voltammetry.

[0044] 5.2. Calculate the ratio signal of the two current peaks and perform data processing to reduce the impact of environmental factors and sensor manufacturing changes on the detection results.

[0045] 5.3. According to the change of ratio signal, a standard curve of doxorubicin concentration is established, and finally the purpose of calibration-free and rapid detection of doxorubicin concentration is achieved.

[0046] The present invention covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present invention. In order to make the public have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, but those skilled in the art can fully understand the present invention without the description of these details. In addition, in order to avoid unnecessary confusion about the essence of the present invention, well-known methods, processes, procedures, components and circuits are not described in detail.

[0047] 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.

Claims

1. Preparation of a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule, characterized in that: A dual current signal output is achieved through a single signal molecule, and a ratio signal is used to achieve calibration-free rapid detection of doxorubicin, which includes the following steps: Step 1, fixing the doxorubicin aptamer modified with methylene blue onto the surface of the gold electrode by a self-assembly method; Step 2: Scanning by square wave voltammetry in a buffer system (1×PBS solution) to detect two current peak signals output by the sensor; Step 3: By calculating the ratio of the two current peak signals, calibration-free detection of doxorubicin concentration is achieved.

2. The preparation of a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule according to claim 1, characterized in that: The aptamer was immobilized on the surface of the gold electrode by a self-assembly method.

3. The preparation of a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule according to claim 1, characterized in that: The square wave voltammetry (SWV) method scans the induced current at different doxorubicin concentrations at multiple frequencies and calculates the ratio of two current peaks.

4. The preparation of a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule according to claim 1, characterized in that: By calculating the ratio of two current peak signals, the influence of environmental factors and sensor manufacturing changes on the detection results is reduced, thereby realizing a calibration-free rapid detection method for doxorubicin.

5. The preparation of a ratiometric doxorubicin electrochemical aptamer sensor based on a single signal molecule according to claim 1, characterized in that: The stability test proves that in a buffer system (1×PBS solution), the sensor can maintain the ratio stability of the current peak signal during long-term continuous testing.

Images