Method for enhancing electrochemical detection signal of selenocysteine

Abstract

The invention provides a method for enhancing an electrochemical detection signal of selenocysteine, which comprises the following steps: S1, mixing compound protease with selenocysteine with different concentrations in a ratio of 1: 1, and placing in a baking oven at 37 DEG C for 1 hour; s2, carrying out electrochemical detection on the selenocysteine on the surface of the electrode by adopting a square wave voltammetry to obtain an electrochemical signal of the selenocysteine; and S3, analyzing the electrochemical signal, and determining the concentration of selenocysteine. The selenocysteine is treated by the compound protease, the sensitivity of an electrochemical detection signal is remarkably enhanced, the current value generated by 1mu M of selenocysteine is 62nA under the condition that enzyme treatment is not carried out, and the current value of selenocysteine with the same concentration is remarkably increased to 137nA after the compound protease treatment, so that the sensitivity of the electrochemical detection signal is remarkably improved. The obvious current increase proves that the enzyme treatment is effective for improving the detection sensitivity of the selenocysteine, so that the existence of the selenocysteine can be accurately detected under the condition of low concentration.

Description

Technical Field

[0001] The invention relates to the technical field of electrochemical detection of selenocysteine, in particular to a method for enhancing electrochemical detection signals of selenocysteine. Background Art

[0002] Selenium is an essential trace element for the human body and is crucial for maintaining normal physiological functions. When the human body is deficient in selenium, it may lead to decreased immune function and increased risk of cancer and cardiovascular disease. In the body, selenium mainly exists in the form of selenocysteine, which is a component of some enzymes and heme. The physiological function of selenocysteine ​​is closely related to its form of existence in the body. For example, the antioxidant properties of selenium are achieved through glutathione peroxidase containing selenocysteine, which can catalyze the reduction reaction of hydrogen peroxide and lipid peroxides. Selenocysteine ​​is the 21st amino acid in the genetic code, encoded by the UGA codon, and plays an important role in protein synthesis.

[0003] Studies have shown that the selenocysteine ​​molecule, the active center of selenoprotein, has cytotoxic effects on a variety of cancer cell lines, which makes the determination of selenocysteine ​​in the blood important for the prevention and diagnosis of diseases.

[0004] Traditional methods for determining trace amounts of selenium include graphite furnace atomic absorption spectrometry, fluorescence spectrometry, and hydride-atomic fluorescence spectrometry. However, there is currently little research data on the use of electrochemical methods to detect selenocysteine, especially those methods that can enhance the electrochemical detection signal. In the literature "Speciation analysis of selenium using voltammetric techniques", Mara Ochsenkuhn-Petropoulou et al. found that selenocysteine ​​is electrochemically active on a suspended mercury electrode.

[0005] When designing an electrochemical detection system, a series of technical challenges need to be overcome, such as improving the sensitivity, repeatability and solving characterization problems of detection. These challenges increase the design and manufacturing costs of electrochemical detection systems and limit their promotion in practical applications. In order to solve the problems and shortcomings of electrochemical detection technology, researchers have conducted a lot of exploration and research. For example, improving the selectivity and stability of electrochemical sensors by improving electrode materials and electrolytes, or introducing new electrochemical testing methods to expand the application scope of electrochemical detection technology. Nevertheless, these solutions often need to take into account multiple factors such as cost, performance, feasibility, etc., so there are still some difficulties in practical applications. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a method for enhancing the electrochemical detection signal of selenocysteine, using a gold electrode to detect selenium and its related products, scanning by square wave voltammetry, an oxidation peak of selenocysteine ​​appears on the electrode, and further using a composite protease to treat selenocysteine ​​to change its conformation, thereby enhancing its electrochemical activity and improving the sensitivity and accuracy of detection, so as to solve the problems raised in the above background technology.

[0007] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for enhancing the electrochemical detection signal of selenocysteine, comprising the steps of:

[0008] S1. Pretreatment of selenocysteine

[0009] 8 mg / mL composite protease was mixed with different concentrations of selenocysteine ​​in a ratio of 1:1 and placed in a 37°C oven for 1 h;

[0010] S2. Square wave voltammetry detection

[0011] The disposable gold screen-printed electrode is immersed in a 0.03 mol / L borax-NaOH base solution, and the pH value of the base solution is adjusted to 9.5, and selenocysteine ​​solutions of different concentrations are added to the base solution, and then the electrode is deposited at a potential of -0.5 V for 180 seconds to allow selenocysteine ​​to form a thin film on the electrode surface. After the deposition is completed, the selenocysteine ​​on the electrode surface is electrochemically detected by square wave voltammetry to obtain its electrochemical signal;

[0012] S3. Analyze the electrochemical signal to determine the concentration of selenocysteine.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The present invention significantly enhances the sensitivity of electrochemical detection signals by treating selenocysteine ​​with a composite protease. Without enzyme treatment, the current value generated by 1uM selenocysteine ​​is 62nA, while after the composite protease treatment, the current value of selenocysteine ​​at the same concentration significantly increases to 137nA. This significant increase in current proves the effectiveness of enzyme treatment in improving the sensitivity of selenocysteine ​​detection, so that the present invention can accurately detect the presence of selenocysteine ​​at low concentrations;

[0015] 2. The present invention also establishes a good linear relationship between selenocysteine ​​concentration and electrochemical detection signal, as shown in the following figure Figure 1 and Figure 2As shown, there is an obvious linear correlation between the concentration of selenocysteine ​​and its corresponding current value. This linear relationship not only verifies the accuracy of the detection method, but also provides a reliable mathematical model for quantitative analysis. Through linear regression analysis, the concentration of selenocysteine ​​can be accurately calculated from the current value, which is of great significance for accurately measuring and monitoring the level of selenocysteine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The disclosure of the present invention is described with reference to the accompanying drawings. It should be understood that the drawings are only for illustrative purposes and are not intended to limit the scope of protection of the present invention. In the drawings, the same reference numerals are used to refer to the same components. Among them:

[0017] Figure 1 A schematic diagram of current values ​​corresponding to different concentrations of selenocysteine ​​proposed in one embodiment of the present invention;

[0018] Figure 2 It is a schematic diagram for illustrating the linear relationship between selenocysteine ​​concentration and electrochemical peak current and for predicting the peak current value at a specific concentration, proposed in one embodiment of the present invention;

[0019] Figure 3 This is a schematic diagram of the selenocysteine ​​pretreatment process proposed in one embodiment of the present invention;

[0020] Figure 4 This is a schematic diagram of a process for ensuring accurate detection of selenocysteine ​​by using square wave voltammetry scanning as proposed in one embodiment of the present invention. DETAILED DESCRIPTION

[0021] It is easy to understand that according to the technical solution of the present invention, without changing the essential spirit of the present invention, a person skilled in the art can propose a variety of interchangeable structural modes and implementation modes. Therefore, the following specific implementation modes and drawings are only exemplary descriptions of the technical solution of the present invention, and should not be regarded as the whole of the present invention or as a limitation or restriction to the technical solution of the present invention.

[0022] The present invention is further described in detail below with reference to the accompanying drawings, but is not intended to limit the present invention.

[0023] As an embodiment of the present invention, a method for enhancing the electrochemical detection signal of selenocysteine ​​is proposed, the purpose of which is to use a gold electrode to detect selenium and its related products. Through square wave voltammetry scanning, an oxidation peak of selenocysteine ​​appears on the electrode. By further using a composite protease to treat selenocysteine ​​to change its conformation, its electrochemical activity is enhanced, thereby improving the sensitivity and accuracy of detection.

[0024] In a specific implementation, as an embodiment of the present invention, the steps proposed include:

[0025] S1. Pretreatment of selenocysteine

[0026] 8 mg / mL composite protease was mixed with different concentrations of selenocysteine ​​in a ratio of 1:1 and placed in a 37°C oven for 1 h;

[0027] S2. Square wave voltammetry detection

[0028] A disposable gold screen-printed electrode was immersed in a 0.03 mol / L borax-NaOH base solution, and the pH value of the base solution was adjusted to 9.5. Selenocysteine ​​solutions of different concentrations were added to the base solution. The electrode was then deposited at a potential of -0.5 V for 180 seconds to allow selenocysteine ​​to form a thin film on the electrode surface. After the deposition was completed, the selenocysteine ​​on the electrode surface was electrochemically detected using square wave voltammetry to obtain its electrochemical signal. This process can enhance the electrochemical detection signal of selenocysteine, thereby improving the sensitivity and accuracy of the detection.

[0029] S3. Analyze the electrochemical signal to determine the concentration of selenocysteine.

[0030] It can be understood that the purpose of the composite protease treatment of selenocysteine ​​used in the present invention is that due to its low electrochemical activity, traditional electrochemical detection methods often face the problems of weak signals and low sensitivity when detecting selenocysteine; the electrochemical detection principle adopted is: placing the electrode in an electrolyte solution containing selenocysteine, and using an electrochemical workstation for electrochemical detection. During the detection process, the composite protease can effectively catalyze the redox reaction of selenocysteine, thereby significantly enhancing the electrochemical detection signal.

[0031] Gold has an affinity for selenium, so gold electrodes are often used to detect selenium and its related products. Through square wave voltammetry scanning, an oxidation peak of selenocysteine ​​appears on the electrode. By further treating selenocysteine ​​with a composite protease to change its conformation, its electrochemical activity is enhanced, thereby improving the sensitivity and accuracy of the detection.

[0032] In one embodiment of the present invention, Figure 3 As shown, the pre-treatment steps of selenocysteine ​​in S1 include:

[0033] S1-1. Prepare the composite protease solution to ensure that the concentration of the composite protease solution is 8 mg / mL. If the initial concentration is not consistent, adjust it to the required concentration by dilution or concentration. The operation is as follows:

[0034] S1-11. If the initial concentration is higher than 8 mg / mL, perform the following dilution steps: First, determine the dilution ratio and calculate the required dilution ratio. Suppose the initial concentration is C i , unit mg / mL, then according to the dilution ratio formula Calculate the total volume V required after dilution d , in mL, to ensure that the concentration after dilution is 8 mg / mL; secondly, perform dilution, mixing a certain amount of protease solution with an equal volume of buffer or water to achieve the desired concentration;

[0035] S1-12. If the initial concentration is lower than 8 mg / mL, perform the following concentration steps: First, determine the concentration ratio and calculate the required concentration ratio. Suppose the initial concentration is C i , then according to the concentration ratio formula Calculate the total volume V required after dilution C , unit mL, ensure that the concentration after concentration is 8 mg / mL; secondly, perform concentration, using centrifugation, evaporation or other concentration techniques to concentrate the protease solution to the required concentration.

[0036] S1-2. Prepare selenocysteine ​​solution: Prepare a series of selenocysteine ​​solutions of different concentrations for experimental use. Including: S1-21. Determine the concentration range and set the minimum concentration C min is the minimum selenocysteine ​​concentration required for the experiment, and the maximum concentration C max is the maximum selenocysteine ​​concentration required for the experiment; calculate the mass of selenocysteine ​​required, and for each concentration use the formula m=C×V(2) to calculate the mass of selenocysteine ​​required, where m is the mass of selenocysteine ​​required, C is the required concentration, and V is the required volume of the solution. S1-22. Weigh the selenocysteine ​​powder. According to the calculated mass value of the required selenocysteine, accurately weigh the selenocysteine ​​powder. S1-23. Under sterile conditions, use a sterile Eppendorf tube to dissolve the weighed selenocysteine ​​powder in an appropriate amount of solvent, and use a magnetic stirrer or vortex mixer to assist in dissolution. S1-24. Use a pipette to transfer the solution to a sterile Eppendorf tube, and further dilute to the precise concentration as needed. S1-25. Verify the actual concentration of the solution to ensure that it meets the experimental requirements, and place the mixed Eppendorf tube in a constant temperature oven that has been set and maintained at 37°C for a 1-hour enzyme-catalyzed reaction. After the reaction is completed, remove the Eppendorf tube from the oven, allow the mixture to cool to room temperature, and prepare for electrochemical detection.

[0037] In one embodiment of the present invention, Figure 4 As shown, in S2, the steps of using square wave voltammetry for detection include:

[0038] S2-1. Preparation: Electrode preparation: Use disposable gold screen-printed electrode (Screen-Printed Electrode, SPE). Prepare the base solution: 0.03 mol / L borax-NaOH solution, adjust the pH to 9.5, and add different concentrations of selenocysteine ​​to the base solution. S2-2. Immerse the electrode in the selenocysteine ​​solution and apply a deposition potential (-0.5 V) for 180 seconds to allow selenocysteine ​​to deposit on the electrode surface.

[0039] S2-3. Square wave voltammetry detection. It is understood that square wave voltammetry is an electrochemical technique that enhances the signal by superimposing a small square wave on the deposition potential. In specific implementation, a square wave voltammetry scanning method is required to ensure accurate detection of selenocysteine. In this scanning method, the basic parameters of the square wave voltammetry that need to be set are: the initial potential, that is, the deposition potential is set to E in = -0.5V, the termination potential (a potential more negative than the initial potential) is set to E fi =-1.0V, square wave amplitude (the potential difference between the peak and valley of the square wave, the frequency of the square wave, affects the resolution and sensitivity of the signal) ΔE is set to [0.01V, 0.1V], frequency f (the frequency of the square wave, affects the resolution and sensitivity of the signal) is set to [50, 100], step potential (the potential increment of each scan) ΔE st Set to [0.001V,0.01V].

[0040] S2-4. Data acquisition: Use an electrochemical workstation to record the current response and the peak current value at each concentration.

[0041] S2-5, data processing: Process the collected concentration and peak current data, and observe the relationship between them by drawing a scatter plot of concentration versus peak current. Specific implementation method: Collect the peak current data of selenocysteine ​​at different concentrations obtained by square wave voltammetry; secondly, use chart software or data analysis tools to draw a scatter plot with the concentration of selenocysteine ​​as the horizontal coordinate and the peak current as the vertical coordinate; finally, observe the distribution of data points on the scatter plot to determine whether there is a linear relationship between them, and determine the best fit line through linear regression analysis, so as to derive the linear equation between peak current and concentration. It should be noted that this process not only intuitively shows the relationship between concentration and peak current, but also provides a mathematical model for quantitative analysis.

[0042] Based on the above technical concept, in order to verify that there is an obvious linear correlation between the concentration of selenocysteine ​​and its corresponding current value, this relationship can be accurately deduced from the current value by linear regression analysis. Figure 1 , Figure 2 As shown, the present invention uses the following selenocysteine ​​concentration (C) and the corresponding peak current (I peak )Data table example:

[0043]

[0044]

[0045] The specific example steps are: Use chart software (such as Excel, Origin, Python's matplotlib library, etc.) to draw a scatter plot of selenocysteine ​​concentration versus peak current. Perform linear regression analysis to determine the linear relationship between peak current and concentration. The linear regression model can be expressed as:

[0046]

[0047] In the formula, I peak is the peak current value measured in electrochemical detection, N is the number of data points, ∑C is the sum of all concentration values, is the intercept, which indicates the expected value of the peak current when the concentration C is 0, ∑I peak is the sum of all peak current values, It is the amount by which the peak current value changes when the concentration changes by one unit, and is used to reflect the strength of the linear relationship between concentration and peak current.

[0048] The correlation coefficient r was set to evaluate the model fit:

[0049]

[0050] It should be noted that in linear regression analysis, r can be used to evaluate the fit of the model. The correlation coefficient r takes the value of [-1,1]. If the absolute value of r is closer to 1, the linear relationship between concentration and peak current value is stronger. If r is close to 0, it means that there is no obvious linear relationship between concentration and peak current value.

[0051] When performing calculations according to the above example, it is known that N = 6 (number of data points), ∑C = 15,

[0052] ∑I peak =1124.61, intercept After optimization, when the linear relationship between peak current and concentration is required to present the best fitting straight line, the adjusted linear regression model is: I peak=30.25C+111.81(5), 30.25 means that the peak current increases by 30.25nA on average for every 1uM increase in selenocysteine ​​concentration, and 111.81 means that when the selenocysteine ​​concentration is 0uM, the expected value of the peak current is 111.81nA. At this time, It shows that there is a very strong linear relationship between the data and the model, which verifies the effectiveness of the model. Through this model, the peak current can be predicted according to the concentration of selenocysteine, or conversely, the concentration of selenocysteine ​​can be estimated according to the peak current.

[0053] Thus, it is found that the present invention significantly enhances the sensitivity of electrochemical detection signals by treating selenocysteine ​​with a composite protease. Without enzyme treatment, the current value generated by 1uM selenocysteine ​​is 62nA, while after composite protease treatment, the current value of selenocysteine ​​at the same concentration significantly increases to 137nA. This significant increase in current proves the effectiveness of enzyme treatment in improving the sensitivity of selenocysteine ​​detection, so that the present invention can accurately detect the presence of selenocysteine ​​even at low concentrations.

[0054] The technical scope of the present invention is not limited to the contents in the above description. Those skilled in the art can make various deformations and modifications to the above embodiments without departing from the technical idea of ​​the present invention, and these deformations and modifications should all fall within the protection scope of the present invention.

Claims

1. A method for enhancing the electrochemical detection signal of selenocysteine, characterized in that: Includes steps: S1. Pretreatment of selenocysteine 8 mg / mL composite protease was mixed with different concentrations of selenocysteine ​​in a ratio of 1:1 and placed in a 37°C oven for 1 h; S2. Square wave voltammetry detection The disposable gold screen-printed electrode is immersed in a 0.03 mol / L borax-NaOH base solution, and the pH value of the base solution is adjusted to 9.5, and selenocysteine ​​solutions of different concentrations are added to the base solution, and then the electrode is deposited at a potential of -0.5 V for 180 seconds to allow selenocysteine ​​to form a thin film on the electrode surface. After the deposition is completed, the selenocysteine ​​on the electrode surface is electrochemically detected by square wave voltammetry to obtain its electrochemical signal; S3. Analyze the electrochemical signal to determine the concentration of selenocysteine.

2. A method for enhancing the electrochemical detection signal of selenocysteine ​​according to claim 1, characterized in that: In S1, the pretreatment steps of selenocysteine ​​include: Prepare the composite protease solution to ensure that the concentration of the composite protease solution is 8 mg / mL. If the initial concentration is not consistent, adjust it to the required concentration by dilution or concentration. The operation is as follows: If the initial concentration is higher than 8 mg / mL, perform the following dilution steps: First, determine the dilution ratio and calculate the required dilution ratio. Assume that the initial concentration is C i , then according to the dilution ratio formula Calculate the total volume V required after dilution d , ensuring that the concentration after dilution is 8 mg / mL; secondly, perform dilution, mixing a certain amount of protease solution with an equal volume of buffer or water to achieve the desired concentration; If the initial concentration is lower than 8 mg / mL, perform the following concentration steps: First, determine the concentration ratio and calculate the required concentration ratio. Set the initial concentration as C i , then according to the concentration ratio formula Calculate the total volume V required after dilution C , ensuring that the concentration after concentration is 8 mg / mL; secondly, performing concentration to concentrate the protease solution to the required concentration.

3. A method for enhancing the electrochemical detection signal of selenocysteine ​​according to claim 1, characterized in that: In S1, a selenocysteine ​​solution is prepared. The specific steps of preparing selenocysteine ​​solutions of different concentrations include: First, determine the concentration range and set the minimum concentration C min is the minimum selenocysteine ​​concentration required for the experiment, and the maximum concentration C max is the highest selenocysteine ​​concentration required for the experiment; calculate the mass of selenocysteine ​​required, and for each concentration use the formula m=C×V to calculate the mass of selenocysteine ​​required, where m is the mass of selenocysteine ​​required, C is the required concentration, and V is the required volume of the solution; Secondly, weighing the selenocysteine ​​powder, and accurately weighing the selenocysteine ​​powder according to the calculated mass value of the required selenocysteine; Again, under sterile conditions, using a sterile Eppendorf tube, dissolve the weighed selenocysteine ​​powder in an appropriate amount of solvent, and use a magnetic stirrer or vortex mixer to assist in dissolution; From this time, use a pipette to transfer the solution into a sterile Eppendorf tube and further dilute to the exact concentration as needed; Finally, verify the actual concentration of the solution to ensure that it meets the experimental requirements, and place the mixed Eppendorf tube in a constant temperature oven set and maintained at 37°C for a 1-hour enzyme-catalyzed reaction; after the reaction is completed, remove the Eppendorf tube from the oven and allow the mixture to cool to room temperature.

4. The method for enhancing the electrochemical detection signal of selenocysteine ​​according to claim 1, characterized in that: In S2, square wave voltammetry scanning was used to ensure accurate detection of selenocysteine, where In this scanning mode, the basic parameters of square wave voltammetry that need to be set are: the initial potential is set to E in =-0.5V, the termination potential is set to E fi =-1.0V, square wave amplitude ΔE is set to [0.01V, 0.1V], frequency f is set to [50, 100], step potential ΔE st Set to [0.001V,0.01V].

5. A method for enhancing the electrochemical detection signal of selenocysteine ​​according to claim 1 or 4, characterized in that: The specific steps of the square wave voltammetry scan used include: Data acquisition, using an electrochemical workstation to record the current response and record the peak current value at each concentration; and Data processing, the operations are as follows: Use chart software or data analysis tools to draw a scatter plot with the concentration of selenocysteine ​​as the horizontal axis and the peak current value as the vertical axis; observe the distribution of data points on the scatter plot to determine whether there is a linear relationship, and determine the best fit line by constructing a linear regression model to analyze and obtain the linear equation between the peak current value and the concentration.

6. A method for enhancing the electrochemical detection signal of selenocysteine ​​according to claim 5, characterized in that: The constructed linear regression model is: In the formula, I peak is the peak current value measured in electrochemical detection, N is the number of data points, ∑C is the sum of all concentration values, is the intercept, which indicates the expected value of the peak current when the concentration C is 0, ΣI peak is the sum of all peak current values, It is the amount by which the peak current value changes when the concentration changes by one unit, and is used to reflect the strength of the linear relationship between concentration and peak current.

7. A method for enhancing the electrochemical detection signal of selenocysteine ​​according to claim 6, characterized in that: After the linear regression model is built, it is also necessary to set the correlation coefficient r to evaluate the model fit. Among them, the correlation coefficient r takes values ​​of [-1,1]. If the absolute value of r is closer to 1, it means that the linear relationship between the concentration and the peak current value is stronger. If r is close to 0, it means that there is no obvious linear relationship between the concentration and the peak current value.

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