Preparation method and application of nanocomposite sensor-based material

Abstract

The application discloses a preparation method and application of a nanocomposite sensor, first, GO-Fe3O4 and EDOT are used to prepare a deposition solution, a GO-Fe3O4 / PEDOT nanocomposite is prepared through an electrochemical method, a glassy carbon electrode is modified as a working electrode, amino-modified DNA is fixed on the GO-Fe3O4 / PEDOT nanocomposite under the action of a catalyst, a biosensor capable of detecting microRNA is prepared, two detection modes of CC method and I-T method are established, the biosensor has good selectivity and high sensitivity, can adapt to the detection precision requirement of the microRNA expression level in serum, and provides important support for developing a detection device for rapidly and on-line detecting the microRNA expression level in serum.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensors, specifically to a method for preparing and applying a sensor based on nanocomposite materials. Background Technology

[0002] Small nucleic acid microRNAs are a broad class of small non-coding RNAs that can inhibit gene expression and protein translation in the human body, thereby affecting metabolism and cell life cycle, thus serving as disease biomarkers. Utilizing this characteristic, a simple and rapid detection of serum microRNA expression levels could effectively predict the occurrence and progression of human diseases. However, existing methods for detecting serum microRNA expression levels involve cumbersome sample pretreatment processes and expensive equipment. Therefore, electrochemical methods for detecting serum microRNA expression levels have become a research hotspot. However, electrochemical biosensors suffer from low detection sensitivity and poor stability, hindering the development of electrochemical detection.

[0003] Electrochemical biosensors are a novel technology for detecting microRNA expression levels in serum, combining electrochemical analysis and biosensing techniques. Compared to traditional detection methods, they offer higher sensitivity, selectivity, and stability, and eliminate the need for cumbersome pretreatment and specialized personnel. Therefore, developing a GO (graphene oxide)-Fe3O4 / PEDOT nanocomposite sensor to enhance the sensitivity and stability of this biosensor is crucial for developing rapid detection devices for microRNA expression levels in serum. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing and applying a sensor based on nanocomposite materials, which enhances the sensitivity and stability of biosensors, establishes a dual-mode detection method of CC and IT, and improves the rapid online detection of microRNA expression levels in serum.

[0005] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0006] A method for preparing a sensor based on GO-Fe3O4 / PEDOT nanocomposite material includes the following steps:

[0007] Ultrapure water was added to the GO-Fe3O4 nanocomposite material, and after ultrasonic dissolution, EDOT (3,4-ethylenedioxythiophene) was added. The ultrasonic dissolution was continued until no oil droplets appeared, and a deposition solution was obtained. The deposition solution was deposited using cyclic voltammetry to obtain the GO-Fe3O4 / PEDOT nanocomposite material.

[0008] The catalyst and aminated DNA were thoroughly mixed to prepare an incubation solution, which was then drop-coated onto the GO-Fe3O4 / PEDOT nanocomposite material. The material was then incubated in a humid environment (humidity greater than 80%). Under the action of the catalyst, the aminated DNA was immobilized on the GO-Fe3O4 / PEDOT nanocomposite material, thus creating a sensor based on the GO-Fe3O4 / PEDOT nanocomposite material.

[0009] Preparation of GO-Fe3O4 nanocomposite materials:

[0010] GO is prepared by adding ultrapure water to GO;

[0011] FeCl3·6H2O and FeSO4·7H2O were mixed and then ultrapure water was added to prepare a mixed solution.

[0012] At an 80°C water bath temperature, the mixed solution was slowly added to the GO suspension. After the addition was complete, the temperature was adjusted to 85°C, and ammonia was quickly added to adjust the pH to 10 (ammonia is used to precipitate Fe). 2+ / Fe 3+ Ions (synthesized magnetite (Fe3O4) particles), are stirred rapidly at a constant temperature, cooled, washed, centrifuged, and dried to obtain GO-Fe3O4 nanocomposite material.

[0013] Furthermore, the mass-to-volume ratio of the GO-Fe3O4 nanocomposite material to the ultrapure water is (1-3) mg:1 mL. EDOT is added after ultrasonic dissolution until no visible solid particles remain.

[0014] Furthermore, the mass-to-volume ratio of the GO-Fe3O4 nanocomposite material to the EDOT is (1-3) mg: 2 μL.

[0015] Furthermore, the cyclic voltammetry (CV) parameters are set as follows: deposition voltage -0.2 to 1.5V, and 10-25 cycles at a scan rate of 0.1V / s, i.e., sweep segments are set to 20-50.

[0016] Furthermore, the catalyst is prepared by adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to phosphate-buffered saline (PBS), respectively. The pH of the phosphate-buffered saline is 7.4.

[0017] Furthermore, the concentration of the catalyst is 0.2M-0.8M. The concentration of the EDC catalyst is 0.8M, and the concentration of the NHS catalyst is 0.2M. The catalyst needs to be prepared and used immediately.

[0018] Furthermore, the concentration of the aminolated DNA is 10. -6 M. Select specific aminolated DNA based on the detection of different microRNAs.

[0019] The amount of amino-modified DNA to be prepared is determined based on the number of incubation electrodes; approximately 20 μL is required per electrode for incubation, resulting in a final concentration of 10. -6 M-aminolated DNA.

[0020] The present invention also provides a sensor based on GO-Fe3O4 / PEDOT nanocomposite material obtained by the above preparation method.

[0021] If the concentration of amino-modified DNA in the laboratory is 10 -5 M can be diluted 5 times with PBS (pH 7.4) to obtain a concentration of 2 × 10⁻⁶. -6 M's DNA, at a concentration of 2 × 10 -6 A 1:1 mixture of DNA from M and a catalyst yields an incubation solution with the target concentration of aminated DNA after the addition of the catalyst. This solution is then drop-coated onto a GO-Fe3O4 / PEDOT nanocomposite material and incubated for 60 minutes in a humid environment. Under the action of the catalyst, the aminated DNA is immobilized on the GO-Fe3O4 / PEDOT nanocomposite material, thus creating a sensor based on the GO-Fe3O4 / PEDOT nanocomposite material.

[0022] This invention also provides an application of a GO-Fe3O4 / PEDOT nanocomposite sensor in dual-mode detection of microRNA. A microRNA-containing sample is drop-coated onto the biosensor and incubated in a humid environment for 30 minutes, during which the microRNA content in the sample is detected.

[0023] Furthermore, the detection methods include the current-time curve method (it) and the time-to-charge method (CC).

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The present invention can realize the synthesis of GO-Fe3O4 / PEDOT nanocomposite material for biosensor substrate, which has good conductivity, large specific surface area and nanoporous morphology. Aminated DNA can be selected to develop a biosensor suitable for detecting the expression level of microRNA in serum.

[0026] (2) The biosensor prepared in this invention uses the CC detection method to detect the expression level of microRNA in serum at different concentrations. The signal inhibition rate of the biosensor is linearly related to the logarithm of the concentration of microRNA expression level in serum, with a linear range of 10. -15 -10 -6 mol / L, detection limit is 5.18 × 10⁻⁶ -15 The expression levels of microRNA in serum at different concentrations were detected using an iterative method. The signal inhibition rate of the biosensor showed a linear relationship with the logarithm of the concentration of microRNA expression levels in serum, within a linear range of 10 mol / L. -15 -10 -6 mol / L, detection limit is 7.36×10 -15 mol / L.

[0027] (3) The dual-mode detection proposed in this invention can achieve rapid detection, on-site detection and mutual verification compared with traditional detection methods.

[0028] (4) The present invention can meet the requirements of the current detection accuracy of microRNA expression level in serum, and provides important support for the development of a detection device for rapid online detection of microRNA expression level in serum. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 CC characterization images of the GO-Fe3O4 / PEDOT nanocomposite sensors prepared in Examples 1-4;

[0031] Figure 2 The images show the FT-IR characterization of the sensors based on GO, GO-Fe3O4 nanocomposite materials, GO-Fe3O4 / PEDOT nanocomposite materials, and GO-Fe3O4 / PEDOT nanocomposite materials in Example 1.

[0032] Figure 3 Scanning electron microscope (SEM) images of the GO-Fe3O4 / PEDOT nanocomposite sensors prepared in Examples 1 and 5;

[0033] Figure 4The image shows the CC curves of different concentrations of microRNA detected by the GO-Fe3O4 / PEDOT nanocomposite sensor obtained in Example 1.

[0034] Figure 5 The image shows the it curves of different concentrations of microRNA detected by the GO-Fe3O4 / PEDOT nanocomposite sensor obtained in Example 1.

[0035] Figure 6 The CV scan curve of the GO-Fe3O4 / PEDOT sensor in an iron standard after 50 cycles;

[0036] Figure 7 The interference of the GO-Fe3O4 / PEDOT / DNA biosensor on the detection of different RNAs in PBS 7.4;

[0037] Figure 8 This is a flowchart illustrating the nanocomposite material and detection process of the present invention. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] Example 1

[0044] (1) Preparation of GO-Fe3O4 nanocomposite materials:

[0045] Accurately weigh 40 mg of GO into a weighing bottle and add ultrapure water to prepare 5 mL of graphene oxide suspension. Then weigh 37.2 mg of FeCl3·6H2O and 19.2 mg of FeSO4·7H2O, mix them, and add ultrapure water to prepare 5 mL of mixed aqueous solution. Under an 80°C water bath, slowly add the mixed aqueous solution of FeCl3 and FeSO4 to the graphene oxide suspension. After the addition is complete, adjust the temperature to 85°C and quickly add 30% ammonia to adjust the pH to 10; the ammonia is used to precipitate Fe. 2+ / Fe 3+ Ions were used to synthesize magnetite (Fe3O4) particles. The mixture was stirred rapidly at a constant temperature for 45 min, then cooled to room temperature. The product was washed twice with ultrapure water, centrifuged to remove the supernatant, and then dried in an oven to obtain the GO-Fe3O4 nanocomposite material.

[0046] (2) Preparation of GO-Fe3O4 / PEDOT nanocomposite materials:

[0047] Accurately weigh 10 mg of GO-Fe3O4 nanocomposite material and add it to a weighing bottle. Add 5 mL of ultrapure water with a pipette, stopper the bottle tightly, seal it with sealing film, and place it in an ultrasonic cleaner for ultrasonic dissolution. When there are no visible solid particles, add 10 μL of EDOT, seal the bottle again, and continue ultrasonic dissolution until no oil droplets appear. The sediment is now ready.

[0048] GO-Fe3O4 / PEDOT nanocomposite material was prepared on a glassy carbon electrode by cyclic voltammetry (CV) with the following parameters: deposition voltage of -0.2 to 1.5 V, and a scan rate of 0.1 V / s for 15 cycles (i.e., sweep segments set to 30).

[0049] (3) Catalyst preparation:

[0050] Accurately weigh 0.3834 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.05754 g of NHS (N-hydroxysuccinimide (NHS)) into a 10 mL weighing bottle. Add 2.5 mL of PBS (pH 7.4) to the bottle using a pipette and mix thoroughly to dissolve. This will give you a catalyst with EDC and NHS concentrations of 0.8 M and 0.2 M, respectively. The catalyst should be prepared fresh before use.

[0051] (4) Fabrication of a sensor based on GO-Fe3O4 / PEDOT nanocomposite material:

[0052] The amount of 5'-amino-3-DNA to be prepared is determined based on the number of incubation electrodes. Each electrode requires 20 μL of DNA for incubation, resulting in a final concentration of 10. -6 M-aminolated DNA.

[0053] Using PBS (pH 7.4) to prepare a 10% concentration -5 The amino-coated DNA of M was diluted five times to obtain a concentration of 2 × 10⁻⁶. -6 M-aminolated DNA.

[0054] The concentration is 2×10 -6 The 5'-amino-DNA of M is mixed with a catalyst (EDC+NHS mixed catalyst, EDC concentration 0.8M, NHS concentration 0.2M) at a volume ratio of 1:1 to obtain the target concentration of amino-DNA incubation solution after adding the catalyst. This solution is then drop-coated onto GO-Fe3O4 / PEDOT nanocomposite material and incubated in a humid environment for 60 min. Under the action of the catalyst, the amino-DNA is immobilized on the GO-Fe3O4 / PEDOT nanocomposite material, thus fabricating a sensor based on GO-Fe3O4 / PEDOT nanocomposite material.

[0055] Examples 2-4

[0056] Same as Example 1, except that the number of CV deposition layers is changed from 15 to 10, 20 and 25 respectively.

[0057] The CC characterization images of the sensors based on GO-Fe3O4 / PEDOT nanocomposite materials prepared in Examples 1-4 are shown below. Figure 1 As shown, it can be seen that 15 is the optimal number of sedimentary layers for CV deposition.

[0058] Figure 1The figure shows a comparison of the concentration (CC) of sensors with different deposition cycles on an iron standard (A) and corresponding linear fitting (B), where a represents 10 cycles, b represents 15 cycles, c represents 20 cycles, and d represents 25 cycles. The results are shown in the figure; the electrode with 15 deposition cycles exhibits better sensing performance and the largest electroactive surface area. Therefore, all subsequent GO-Fe3O4 / PEDOT sensors prepared in this experiment used the CV deposition method with 15 cycles.

[0059] Figure 2 The images show the FT-IR characterization of the GO, GO-Fe3O4 nanocomposite, GO-Fe3O4 / PEDOT nanocomposite, and GO-Fe3O4 / PEDOT-based sensor from Example 1. In Figure a, a represents the infrared spectrum of GO; b represents the infrared spectrum of GO-Fe3O4; c represents the infrared spectrum of GO-Fe3O4 / PEDOT; and d represents the infrared spectrum of DNA / GO-Fe3O4 / PEDOT. It can be seen that Fe3O4 and GO form a nanocomposite, GO-Fe3O4 is successfully doped into the GO-Fe3O4 / PEDOT nanocomposite, and aminated DNA is successfully immobilized on the surface of the GO-Fe3O4 / PEDOT nanocomposite.

[0060] Example 5

[0061] Same as Example 1, except that the GO-Fe3O4 nanocomposite material is replaced with graphene oxide.

[0062] Figure 3 The images show scanning electron microscope (SEM) images of the GO-Fe3O4 / PEDOT nanocomposite sensors prepared in Examples 1 and 5. (A) is a 20,000x magnified SEM image of the GO / PEDOT nanocomposite; (B) is a 50,000x magnified SEM image of the GO / PEDOT nanocomposite; (C) are 20,000x magnified SEM images of the GO-Fe3O4 / PEDOT nanocomposite; and (D) are 50,000x magnified SEM images of the GO-Fe3O4 / PEDOT nanocomposite. It can be seen that the GO-Fe3O4 nanocomposite possesses a porous microstructure, providing a large surface area, making it an optimal choice as a modifier material for sensors.

[0063] Example 6

[0064] Prepare 10 solutions of water treated with DEPC (diethyl pyrocarbonate) respectively. -6 10 -7 10 -8 10 -9 10 -10 10 -11 10 -12 10 -1310 -14 10 -15 MicroRNA samples of mol / L were drop-coated onto the GO-Fe3O4 / PEDOT nanocomposite sensor prepared in Example 1, and incubated in a humid environment for 30 min. The CC detection method was used for the experiment, and the results are as follows: Figure 4 As shown in Figure A, the sensor quantitatively detects microRNA. 24 CC comparison; Figure B shows the corresponding linear fit; microRNA 24 Concentration from 10 -15 Up to 10 -6 mol L -1 (a→j) shows that there is a good linear relationship between the concentration of microRNA and the response signal, and the linear equation is y=-0.01462x+0.404(R 2 =0.993), the linear range is 10. -15 -10 -6 mol / L, detection limit is 5.18 × 10⁻⁶ -15 mol / L.

[0065] Example 7

[0066] Prepare 10 solutions of water treated with DEPC respectively -6 10 -7 10 -8 10 -9 10 -10 10 -11 10 -12 10 -13 10 -14 10 - 15 MicroRNA samples of mol / L were drop-coated onto the GO-Fe3O4 / PEDOT nanocomposite sensor prepared in Example 1. After incubation in a humid environment for 30 min, the samples were dissolved in 5 mL of NaNO2 solution prepared with PBS (pH 8.0). The iterative detection method was used, with the detection voltage set to 0.75 V. The results are as follows: Figure 5 As shown in Figure A, the sensor quantitatively detects microRNA. 24 The comparison of it; Figure B shows the corresponding linear fit; microRNA 24 Concentration from 10 -15 Up to 10 -6 mol L -1 (a→l). It can be seen that there is a good linear relationship between the concentration of microRNA and the response signal, with the linear equation being y = -0.0495x + 0.382(R). 2=0.994), linear range is 10 -15 -10 -6 mol / L, detection limit is 7.36×10 -15 mol / L.

[0067] Example 8

[0068] The GO-Fe3O4 / PEDOT sensor prepared in Example 1 was subjected to 50 CV scans (scan rate 0.1 V / s) in an iron standard solution. Figure 6 Observing the scanning results, the CV conversion signal remained basically unchanged after 50 cycles, with the Fe redox peaks around +0.25V and +0.05V, respectively. This indicates that the sensor has good stability, and the deposited material on the electrode surface will not fall off during the experiment.

[0069] Example 9

[0070] To evaluate the specificity of the GO-Fe3O4 / PEDOT / DNA biosensor (Example 1), the sensor's resistance to other possible interferences, including microRNA, was investigated under the same conditions. 21 M1 (RNA with a single base mismatch, curve b), M2 (RNA with a double base mismatch) 24 Curve c), M3 (triple-base mismatched RNA) 24 (Curve d), microRNA 21 (Curve e) Five RNAs. GO-Fe3O4 / PEDOT / DNA were then followed by 10... -6 mol L -1 Different RNAs were incubated for 30 min, washed three times with PBS 7.4 buffer, and then subjected to CV and CC scans directly in PBS 7.4 solution. Finally, they were compared with microRNAs. 24 (Curve f) Incubate and detect. Figure 7 The comparative results show that this biosensor is similar to RNA. 24 The CV and CC values ​​changed most significantly after incubation, while smaller changes were observed after reacting with the other four RNAs. Therefore, the GO-Fe3O4 / PEDOT / DNA sensor is effective for microRNA detection. 24 The detection has good selectivity.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of a GO-Fe3O4 / PEDOT nanocomposite sensor in dual-mode detection of microRNA, characterized in that, The preparation method of the sensor based on GO-Fe3O4 / PEDOT nanocomposite material includes the following steps: Ultrapure water was added to the GO-Fe3O4 nanocomposite material, and the mixture was dissolved by ultrasonication. Then, EDOT was added, and the mixture was dissolved by ultrasonication again to obtain a deposition solution. The deposition solution was deposited using cyclic voltammetry to obtain the GO-Fe3O4 / PEDOT nanocomposite material. The catalyst and amino-modified DNA were mixed to prepare an incubation solution, which was then drop-coated onto GO-Fe3O4 / PEDOT nanocomposite material for incubation treatment, thus obtaining a sensor based on GO-Fe3O4 / PEDOT nanocomposite material; the catalyst was a mixed catalyst prepared by adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to phosphate buffer. The mass-to-volume ratio of the GO-Fe3O4 nanocomposite material to the EDOT is (1-3) mg: 2 μL; The detection methods include the current-time curve method and the time-electrode method.

2. The application according to claim 1, characterized in that, The mass-to-volume ratio of the GO-Fe3O4 nanocomposite material to the ultrapure water is (1-3) mg: 1 mL.

3. The application according to claim 1, characterized in that, The cyclic voltammetry parameters are set as follows: -0.2 to 1.5V, with a scan rate of 0.1V / s for 10-25 cycles.

4. The application according to claim 1, characterized in that, The concentration of the catalyst is 0.2M-0.8M.

5. The application according to claim 1, characterized in that, The concentration of the amino-modified DNA was 10. -6 M.

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