An electrochemical sensor based on laser etching and inkjet printing technology, a preparation method thereof and application in AD marker detection

Abstract

The application discloses an electrochemical sensor based on laser etching and inkjet printing technology, a preparation method thereof and application in AD marker detection, and belongs to the technical field of electrochemical analysis, and the preparation process is as follows: vertical graphene is grown on the surface of a substrate by a CVD method, the graphene is etched into a graphene array composed of 2-6 working electrodes and one counter electrode by a laser etching method; an Ag / AgCl reference electrode is inkjet-printed on the substrate, dried, inkjet-printed glue, and after the glue is solidified, a reaction pool is formed in the middle of the substrate, silver conductive wires are printed on the graphene electrodes and the Ag / AgCl reference electrode outside the reaction pool and dried; a chloroauric acid solution is added dropwise into the reaction pool, gold is electrodeposited on the surface of the graphene by a constant voltage method; different AD marker primary antibodies are modified on the working electrodes in the reaction pool and labeled, after incubation, excessive antibodies are washed away by PBS, and the construction of the electrochemical biosensor is completed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of electrochemical analysis, and particularly relates to an electrochemical sensor based on laser etching and inkjet printing technology, a preparation method thereof and application in AD marker detection. BACKGROUND

[0002] Vertical graphene (VG): graphene grown vertically on a ceramic substrate; Electrochemical biosensor based on VG electrode array: Alzheimer's disease marker antibody is modified on the surface of the VG electrode to construct a detection chip for Alzheimer's disease markers.

[0003] Alzheimer's disease is a neurodegenerative disease with a very long duration. At present, the main AD diagnosis methods in clinical practice include cognitive scale score, neuroimaging examination and cerebrospinal fluid molecular marker content detection, etc. The change of the content of the molecular marker in the cerebrospinal fluid can directly reflect the nerve damage of the AD patient, and can find the beginning in the early stage of the disease, thereby greatly advancing the AD diagnosis. However, the acquisition of cerebrospinal fluid is very difficult, and is highly invasive and easy to cause adverse reactions, thereby limiting its application in general population screening. Compared with cerebrospinal fluid, blood is easy to obtain and less harmful, and gradually has the trend of replacing cerebrospinal fluid.

[0004] Scientists have developed a large number of analysis methods, most of which are in the laboratory research stage. The electrochemical method has great potential in the early diagnosis of AD blood markers due to its advantages of high sensitivity, easy miniaturization and easy on-site detection. Most of the current detection methods detect only one marker at a time. However, due to individual differences, a single marker often differs greatly between different individuals, and simultaneous detection of multiple markers can greatly improve the accuracy of AD diagnosis. SUMMARY

[0005] The purpose of the present application is to provide an electrochemical biosensor, a preparation method thereof and application in simultaneous detection of multiple AD markers in order to solve the problems in the above background.

[0006] In order to achieve the above purpose, the application adopts the following solutions:

[0007] A preparation method of an electrochemical sensor based on laser etching and inkjet printing technology, the process is as follows:

[0008] (1) Grow vertical graphene on the surface of the substrate by CVD method, and the height of the vertical graphene is 0.5 μm ~ 5 μm; use laser etching method to etch the graphene into an array of 2 ~ 6 working electrodes and one counter electrode;

[0009] (2) inkjet printing Ag / AgCl reference electrode on the substrate, drying, then inkjet printing glue, after the glue solidification, forming a reaction pool in the middle of the substrate, printing silver wire on the graphene electrode and Ag / AgCl reference electrode outside the reaction pool and drying;

[0010] (3) adding 5mmol / L~15mmol / L chloroauric acid solution into the reaction pool, depositing gold on the surface of graphene by constant voltage method, the deposition time is 100s~1000s, and the potential applied during the electrodeposition is-0.6~-1.8V;

[0011] (4) modifying different AD marker primary antibodies on the working electrode in the reaction pool and labeling, after incubation, washing the excess antibodies with PBS, and completing the construction of the electrochemical biosensor.

[0012] Further, the growth process of the vertical graphene is as follows: placing the substrate into a vapor deposition furnace, taking methane gas as raw material, the flow rate of the methane gas is 3~8 mL / min, the deposition temperature is 750~850℃, and the pressure is ≦10Pa.

[0013] Further, the processing speed of inkjet printing is 140~160 mm / s, and the jet frequency is 100Hz~120Hz.

[0014] Further, the substrate is a silicon wafer, glass or ceramic.

[0015] Further, the working electrode and the counter electrode are both composed of graphene wire and graphene origin, the graphene wire extends from the edge of the substrate to the middle of the substrate, the graphene origin is connected with the graphene wire and located in the reaction pool.

[0016] Further, the size of the substrate is 1.5cm×1.5cm, the size of the reaction pool is 0.8cm×0.8cm, the diameter of the origin is 0.5mm~5mm, and the thickness of the Ag / AgCl reference electrode is 1~3mm.

[0017] Further, the concentration of the chloroauric acid solution is 10mmol / L, the electrodeposition potential is-1.4V, and the added amount is 15~30μL.

[0018] Further, the AD marker primary antibody specifically refers to at least two or more of Aβ40, Aβ42, T-tau, P-tau181, and the added amount of the primary antibody is 2~10μL, and the concentration is 5~15μg / mL.

[0019] The electrochemical sensor based on the laser etching and inkjet printing technology is prepared by the above preparation method.

[0020] Application of the electrochemical sensor based on laser etching and inkjet printing technology in simultaneous detection of multiple AD markers.

[0021] Further, different concentrations of protein mixtures corresponding to the first antibody are added to the electrochemical biosensor reaction cell, the protein concentration gradient is 0.1, 1, 10, 100, 1000 pg / mL, the solvent is PBS with pH=7.4, the change of the differential pulse voltammetry signal of the working electrode before and after incubation of the protein is detected by the electrochemical workstation, the logarithm of the protein concentration and the difference of the DPV signal are plotted, the standard curve corresponding to the marker is obtained, when detecting, the human serum sample is diluted and mixed uniformly with PBS with pH=7.4, then added to the reaction cell, according to the change of the differential pulse voltammetry signal of the working electrode before and after detection by the electrochemical workstation, the corresponding standard curve is brought in to calculate the concentration of the corresponding protein.

[0022] The integrated electrochemical detection sensor constructed by the laser etching and inkjet printing technology can simultaneously detect four AD blood markers Aβ40, Aβ42, T-tau and P-tau181, and the detection limits of the four markers are 0.072, 0.089, 0.071 and 0.051 pg / mL respectively, and the linear range is 0.1-1000 pg / mL, which is better than the ELISA detection method. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The preparation flow chart of the electrochemical biosensor of the application;

[0024] Figure 2 The physical map of the electrochemical biosensor prepared by the application;

[0025] Figure 3 The standard curve of the four markers of the electrochemical biosensor. DETAILED DESCRIPTION

[0026] The technical solutions of the application will be further described in detail in combination with the drawings and examples, but the protection scope of the application is not limited thereto. The concentration of PBS used in the following examples is 10mM, and the pH is 7.4.

[0027] Example 1

[0028] An electrochemical sensor based on laser etching and inkjet printing technology, the preparation process is as follows:

[0029] (1) Preparation of vertical graphene, grow vertical graphene on the surface of ceramic (1.5 cm x 1.5 cm) substrate by CVD method, the process is as follows: put the ceramic substrate into the vapor deposition furnace, use methane gas as raw material, the flow rate of methane gas is 5 mL / min, the deposition temperature is 800℃, the pressure is ≦10 Pa, the growth time is 15 minutes, and the vertical graphene with a height of 0.5 μm is obtained;

[0030] (2) Use laser etching method to etch graphene into VG array as shown in Figure 1 , that is, form an array of four working electrodes distributed at the four corners of the ceramic substrate and a counter electrode located on the central axis, the working electrode and the counter electrode are both composed of graphene dots and graphene lines connecting the dots, the graphene lines extend from the edge of the ceramic substrate to the center of the ceramic substrate, the graphene dots are connected to the graphene lines and located in the reaction pool;

[0031] (3) Inkjet print Ag / AgCl reference electrode on the ceramic substrate, naturally air dry or dry at 60℃ for 1 hour, the Ag / AgCl reference electrode is located at the other end of the counter electrode on the central axis, inkjet print dam glue, after solidification, a reaction pool with a length of 0.8 cm, a width of 0.8 cm and a height of 3 mm is formed in the middle of the ceramic substrate, print silver wires on the graphene electrode and the reference electrode, naturally air dry or dry at 60℃ for 1 hour, the thickness of the silver wire is 0.5 μm, the silver wire is connected to the reaction pool and located outside the reaction pool, the processing speed of inkjet printing is 150 mm / s, the jet frequency is 100 Hz, the length of the Ag / AgCl reference electrode is 0.6 cm, and the thickness is 2 mm.

[0032] (4) Add 20 μL of 10 mmol / L chloroauric acid solution (20 μL of liquid can cover the reaction pool) to the reaction pool, use graphene as working electrode and counter electrode, Ag / AgCl as reference electrode, apply a potential of -1.4 V, and deposit gold on the surface of graphene in the reaction pool by constant voltage method, the deposition time is 300 s, and a gold layer with a thickness of 1 μm is obtained, which is denoted as VG@Au;

[0033] (5) Construct an electrochemical detection sensor based on VG@Au to detect Aβ40, Aβ42, T-tau, and P-tau181: first, modify the primary antibodies of Aβ40, Aβ42, T-tau, and P-tau181 on the dots of the four working electrodes in the reaction pool and label them as 0, 2, T, and P respectively, the addition amount of the primary antibodies is 3 μL, the concentration is 10 μg / mL, and the reaction is carried out at 37℃ for 1 hour, then wash off the excess antibodies with PBS, and complete the construction of the electrochemical biosensor.

[0034] (6) Construction of standard curves: 20 μL of a mixture of four proteins, Aβ40, Aβ42, T-tau, and P-tau181, was added to the reaction chamber, ensuring that the concentration gradients of the four proteins in the mixture were 0.1, 1, 10, 100, and 1000 pg / mL. PBS (pH 7.4) was used as the solvent. The mixture was incubated at 37°C for 1 hour. Excess protein was then washed away with PBS. The changes in differential pulse voltammetry (DPV) signals before and after protein incubation were detected using an electrochemical workstation. The standard curves for the four protein markers were obtained by plotting the logarithm of the protein concentration against the difference in DPV signals, as shown below. Figure 3 As shown, by Figure 3 It can be seen that the linear equations for this sensor when used for Alzheimer's disease biomarker detection are as follows: the linear equation for Aβ40 is y = 0.548x + 7.232(R). 2 =0.994); The linear equation of Aβ42 is y=0.488x+6.396(R) 2 =0.995); the linear equation of T-tau is y=0.498x+6.586(R) 2 =0.996); The linear equation of P-tau181 is y=0.514x+6.854(R) 2 =0.994), and the detection limits for Aβ40, Aβ42, T-tau, and P-tau181 were calculated to be 0.072 pg / mL, 0.089 pg / mL, 0.071 pg / mL, and 0.051 pg / mL, respectively.

[0035] Application Example 1

[0036] Take 5 μL of human serum sample, add 15 μL of PBS (pH=7.4) for dilution, mix well, and add to the reaction cell of the biosensor prepared in step (5) of Example 1. Based on the changes in differential pulse voltammetry (DPV) signals before and after detection by the four electrodes of the electrochemical workstation, calculate the concentration of the corresponding protein by substituting it into the corresponding standard curve. The specific results are as follows:

[0037]

[0038] As shown in the table above, the two serum samples were not significantly different from the standard ELISA method. Furthermore, ELISA could not detect trace amounts of tau protein, while the sensor in this application could.

[0039] It should be pointed out finally that the above embodiments are only used for illustrating the technical solutions of the embodiments of the present application but not for limiting the same, and those skilled in the art should understand that any equivalent replacement or obvious modification to the embodiments of the present application without changing the performance or purpose, which is not against the spirit of the present application, should be covered in the scope of the present application claimed.

Claims

1. A method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology, characterized in that, The process is as follows: (1) Vertical graphene is grown on the substrate surface by CVD method. The height of the vertical graphene is 0.5μm ~ 5μm. The graphene is etched into an array of 2~6 working electrodes and one counter electrode by laser etching method. (2) Inkjet print Ag / AgCl reference electrode on substrate, dry it, inkjet print adhesive, after adhesive solidifies to form a reaction cell in the middle of substrate, print silver wires on graphene electrode and Ag / AgCl reference electrode outside reaction cell and dry them. (3) Add 5 mmol / L to 15 mmol / L chloroauric acid solution to the reaction cell, and electrodeposit gold on the graphene surface by constant voltage method for 100 s to 1000 s. The potential applied during electrodeposition is -0.6 to -1.8 V. (4) Modify and label the working electrode in the reaction cell with primary antibodies of different AD markers. After incubation, wash away excess antibody with PBS to complete the construction of electrochemical biosensors.

2. The method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology according to claim 1, characterized in that, The growth process of the vertical graphene is as follows: the substrate is placed in a vapor deposition furnace, methane gas is used as raw material, the methane gas flow rate is 3~8 mL / min, the deposition temperature is 750~850℃, and the pressure is ≦10Pa.

3. The method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology according to claim 1, characterized in that, The inkjet printing processing speed is 140~160 mm / s; the jetting frequency is 100Hz~120Hz.

4. The method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology according to claim 1, characterized in that, Both the working electrode and the counter electrode are composed of graphene lines and graphene origins. The graphene lines extend from the edge of the substrate to the center of the substrate, and the graphene origins and graphene lines are connected, with the graphene origins located within the reaction cell.

5. The method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology according to claim 4, characterized in that, The substrate is 1.5cm × 1.5cm in size, the reaction cell is 0.8cm × 0.8cm in size, the diameter of the origin is 0.5mm to 5mm, and the thickness of the Ag / AgCl reference electrode is 1 to 3mm.

6. The method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology according to claim 1, characterized in that, The chloroauric acid solution has a concentration of 10 mmol / L, an electrodeposition potential of -1.4 V, and an addition amount of 15~30 μL.

7. The method for fabricating an electrochemical sensor based on laser etching and inkjet printing technology according to claim 5, characterized in that, The primary antibody for AD markers is at least two of Aβ40, Aβ42, T-tau, and P-tau181, with an addition amount of 2-10 μL and a concentration of 5-15 μg / mL.

8. An electrochemical sensor based on laser etching and inkjet printing technology prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the electrochemical sensor based on laser etching and inkjet printing technology according to claim 8 for non-diagnostic purposes in the simultaneous detection of multiple AD markers.

10. The application according to claim 9, characterized in that, First, add a mixture of proteins with different concentration gradients corresponding to the primary antibody to the electrochemical biosensor reaction cell. The protein concentration gradients are 0.1, 1, 10, 100, and 1000 pg / mL, and the solvent is PBS at pH 7.

4. Use an electrochemical workstation to detect the changes in differential pulse voltammetry (DPV) signals before and after protein incubation at the working electrode. Plot the difference between the logarithm of the protein concentration and the DPV signal to obtain the standard curve for the corresponding protein biomarker. During detection, dilute and mix the human serum sample with PBS at pH 7.4, add it to the reaction cell, and calculate the concentration of the corresponding protein biomarker by substituting the changes in differential pulse voltammetry signals before and after detection at the working electrode of the electrochemical workstation into the standard curve of the corresponding protein biomarker.

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