A solid electrode for quantitatively detecting selenium monosaccharide and its application

Abstract

The present invention discloses a solid electrode for quantitatively detecting selenium monosaccharide and its application. The selenium monosaccharide is 1β-methylseleno-N-acetyl-D-galactosamine, which is a biomarker of selenium in human urine. The present invention discloses a method for processing and fabricating the electrode: mixing the silver amalgam electrode material in a mass ratio of Hg / Ag = 8 / 2 to 6 / 4, compacting the silver amalgam in the capillary with a copper wire dipped in conductive silver paste, and waiting for it to form an alloy. The present invention also discloses a quantitative analysis method for the monosaccharide molecule. The electrode is activated, dipped in mercury, and enriched. Finally, the test solution containing selenium monosaccharide is detected by square wave voltammetry. The selenium monosaccharide undergoes an oxidation-reduction reaction with mercury under a pulsed voltage to generate an electrical signal, and the peak current of the current response is converted into the concentration of selenium monosaccharide to achieve the quantitative detection of selenium monosaccharide. This method is simple to operate, low in cost, and significantly improves the detection efficiency of selenium monosaccharide.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to a solid electrode for quantitatively detecting selenium monosaccharide and its application. Background Art

[0002] Selenium monosaccharide (1β-selenomethyl-2-N-acetyl-D-galactosamine) has been identified as the main biomarker of selenium metabolites in human urine. Its structure is shown as follows. The quantitative detection of selenium monosaccharide can reflect the selenium metabolism level in the human body. Traditional detection methods include spectroscopy and chromatography, but both of these methods have limitations such as high cost, long detection time, and complex pretreatment, which cannot meet the market demand for rapid detection.

[0003]

[0004] Square wave voltammetry (SWV) is a multifunctional, rapid, highly sensitive, and efficient electroanalytical method. This form of sampling method can increase sensitivity and enable better application analysis. For solid electrodes, applying pulse voltammetry can eliminate the hindrance to electrode reactions caused by adsorption, relatively reduce the consumption of electroactive substances, and also reduce the problem of electrode surface hindrance. Since the current is sampled in both positive and negative pulse directions, the oxidation and reduction reaction peaks of electroactive substances on the electrode surface can be obtained simultaneously in one experiment by subtracting the difference.

[0005] Point-of-Care Testing (POCT) is a new method for on-site immediate analysis, which omits the complex processing procedures during sample transportation and laboratory testing, and can quickly obtain test results. The technical platforms related to POCT are constantly upgraded and have good development prospects. Summary of the Invention

[0006] The purpose of the present invention is to propose a solid electrode for quantitatively detecting selenium monosaccharide and its application in view of the deficiencies of the prior art.

[0007] The purpose of the present invention is achieved through the following technical solutions: A solid electrode for detecting selenium monosaccharide is prepared by the following method:

[0008] (1) Take a silver-mercury alloy capsule weighing 40 - 60 mg, press the cap on the top of the capsule to the bottom until the plastic isolation layer falls off, install the pressed capsule on the chuck of a silver-mercury mixer and start the silver-mercury mixer. After completion, pour out the object in the capsule for later use;

[0009] (2) Prepare the amalgam: successively place liquid mercury and nano silver powder into a capsule, and mix them in a silver amalgamator. The mass ratio of liquid mercury to nano silver powder is 3 - 8:2. After mixing, open the capsule to obtain a paste-like amalgam.

[0010] (3) Take the paste-like amalgam formed in step (2) into a capillary tube, compact it with a copper wire, dip it in conductive silver paste for fixation, and wait for it to form an alloy to obtain a solid amalgam electrode for detecting selenium monosaccharide.

[0011] Furthermore, the parameters set for the silver amalgamator in steps (1) and (2) are: the mixing rotation speed is 3000 - 6000 r / min, and the mixing time is 25 - 35 s.

[0012] A quantitative analysis method for a solid electrode for detecting selenium monosaccharide according to claim 1, comprising the following steps:

[0013] (1) Polish the electrode surface with 2000 - mesh sandpaper.

[0014] (2) Activate the polished electrode.

[0015] (3) Dip the activated electrode in mercury.

[0016] (4) Enrich at a potential of 0.0 V in the amperometric i - t curve module of the electrochemical workstation, so as to enrich selenium in the test solution onto the mercury film surface and amplify the detection signal.

[0017] (5) Conduct SWV detection. Utilize the electrochemical reaction between selenium and mercury in selenium monosaccharide under pulsed potential to collect current signals, and determine the concentration of selenium monosaccharide according to the standard curve.

[0018] Furthermore, step (2) is specifically: clean the reference electrode and the auxiliary electrode with ultrapure water. Clip the white electrode clamp on the black reference electrode plug-in tab of the plug-in wire, connect the red electrode to the green reference electrode plug-in tab of the plug-in wire, and clip the green electrode clamp on the copper wire of the working electrode. Place the electrode in a centrifuge tube containing 0.2 M KCl solution, and select the amperometric i - t curve module in the operation window of the electrochemical workstation to activate at -2.2 V for 250 - 350 s.

[0019] Furthermore, the mercury dipping in step (3) is to dip the head of the electrode into mercury sealed with water to pick up mercury, and the mercury dipping time is 30 s - 10 min.

[0020] Furthermore, the parameter settings of the SWV - Square Wave Voltammetry module of the electrochemical workstation in step (4) are:

[0021]

[0022] The enrichment time is 1 min to 15 min.

[0023] Furthermore, step (5) realizes the quantitative detection of selenium monosaccharide through the relationship between the concentration x of selenium monosaccharide and the peak current y detected by square wave voltammetry. Among them, the relationship for calibrating x and y is specifically as follows: The peak current values are detected by SWV at different concentrations of selenium monosaccharide molecules, and the peak current values y at different concentrations x of selenium monosaccharide are obtained, and the x-y relationship is obtained by fitting.

[0024] Furthermore, the relationship between the concentration x of selenium monosaccharide and the peak current value y is y = -5.697x - 1.471.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. The present invention constructs a highly sensitive and rapid POCT detection system for the detection of selenium monosaccharide by making a solid silver amalgam electrode with specific response to selenium monosaccharide and combining square wave voltammetry, realizing the quantitative detection of selenium monosaccharide. Compared with traditional methods, this method is simple to operate, has low cost, and significantly improves the detection efficiency of selenium monosaccharide.

[0027] 2. The selenium monosaccharide detected by the present invention is a selenium metabolism marker in urine. This detection method uses harmonic silver amalgam as the electrode material, and the selenium in selenium monosaccharide and mercury undergo an oxidation-reduction reaction under square wave pulses to generate mercury selenide. When selenium and mercury undergo an oxidation-reduction reaction, electron transfer occurs, which is converted into a current response signal under the SWV method and further into the concentration of selenium monosaccharide, realizing the quantitative detection of selenium monosaccharide molecules. The solid silver amalgam electrode combined with SWV used in the present invention has a faster detection speed and stronger specificity compared with traditional detection methods, realizes the conversion between the current signal intensity and the concentration of selenium monosaccharide, and constructs a specific quantitative detection system for selenium monosaccharide based on this. The detection time is about 11 minutes, and the detection range is 0.1 μM to 10.0 μM.

[0028] 3. Compared with traditional methods, the solid silver amalgam electrode made in the present invention has specificity and high sensitivity for the detection of selenium monosaccharide, and can realize the micro-instant detection of selenium monosaccharide.

[0029] 4. Compared with traditional methods, the quantitative detection of selenium monosaccharide in the present invention does not require pretreatment of the selenium-containing solution and can directly detect, improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the quantitative analysis method for detecting selenium monosaccharide molecules of the present invention;

[0031] Figure 2It is the current response diagram of the present invention under different conditions, where the concentration of selenium monosaccharide is 10 μM. Among them, a is the current response diagram under different silver-mercury ratios, b and c are the current response diagrams under different mixing revolutions and mixing times, d is the current response diagram under different activation times, e is the current response diagram under different mercury dipping times, f is the current response diagram under different enrichment potentials, g is the current response diagram under different enrichment times, h is the current response diagram under different pulse heights, and i is the current response diagram under different square wave frequencies.

[0032] Figure 3 It is the fitting curve diagram of the concentration x of selenium monosaccharide and the peak current y; Specific Embodiments

[0033] The following combines examples to further describe in detail the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0034] Example 1: Fabrication of a Selenium Monosaccharide-Specific Solid Silver Amalgam Electrode

[0035] (1) Take a silver amalgam capsule, press the lid on the top of the capsule to the bottom until the plastic isolation layer falls off, install the pressed capsule on the chuck of the silver amalgam mixer and start the silver amalgam mixer. After completion, pour out the object in the capsule for later use.

[0036] (2) Mix the silver amalgam; use a pipette to take 3 or 4 drops of liquid mercury into the capsule, calculate and weigh the nano silver powder (60 - 120 nm) according to the mass ratio of Hg / Ag = 7 / 3, place it in the capsule, and mix it in the silver amalgam mixer. After completion, open the capsule to obtain a paste-like silver amalgam, pour it into a glass capillary with an inner diameter of 1.0 mm for about 1 cm, and cut the capillary from the flat end into a length of 3 cm.

[0037] (3) Assemble the electrode. Cut the copper wire into a length approximately equal to the length of the centrifuge tube. Dip the copper wire in conductive silver glue and compact the paste. If it is not firm, it can be fixed with AB glue and left for 1 day until a stable alloy is formed to obtain a solid silver amalgam electrode for detecting selenium monosaccharide.

[0038] Moreover, the silver amalgam electrode and the electrolytic cell used in subsequent experiments can be recycled and uniformly processed. Wash the reference electrode and the counter electrode with ultrapure water. After washing, put the reference electrode back into the saturated KCl solution, air-dry the auxiliary electrode, and store it at room temperature; after washing the electrolytic cell with ultrapure water, ultrasonically clean it for 30 seconds, put it into a constant temperature incubator for drying, and take it out after drying is completed.

[0039] Example 2

[0040] The preparation method of the selenium monosaccharide (1β-methylseleno-2-N-acetyl-D-galactosamine) solution is as follows:

[0041] Dissolve 30 mg of the powder sample standard of selenium monosaccharide in 10 mL of ultrapure water to prepare a 1 mM stock solution, and ultrasonicate until completely dissolved. Dilute it proportionally to prepare Se standard solutions with different concentrations.

[0042] The electrode prepared according to the steps of Example 1 was used as the working electrode, where the mass ratios of silver to mercury were 2 / 8, 2.5 / 7.5, 3 / 7, 3.5 / 6.5, 4 / 6 respectively, the mixing rotation speed was 4000 r / min, and the mixing time was 30 s. The rest was the same as in Example 1. A platinum wire was used as the auxiliary electrode, and a silver-silver chloride electrode was used as the reference electrode to form a three-electrode system. The system was activated in a 5 mL centrifuge tube containing 4 mL of 0.2 M KCl solution for 3 min, dipped in mercury for 30 s, 4 mL of 0.1 M HCl solution was added to the electrolytic cell, and then 40 μL of 1 mM selenium monosaccharide was added to form. Enrich for 1 min at 0.0 V, and the square wave frequency f for SWV detection was 90 s －1 , and the pulse height a was 0.025 V. The experimental results are as Figure 2 (a). The electrode prepared with a silver-mercury mass ratio of 3 / 7 had a more obvious current response compared to other experimental groups.

[0043] Example 3

[0044] The difference between Example 3 and Example 2 was that the silver-mercury mass ratio for making the electrode was 3 / 7, and the combinations of mixing rotation speed (r / min) and time (s) were 4000, 25 s; 4000, 30 s; 4000, 35 s; 3000, 30 s; 3000, 35 s; 3000, 40 s; 3000, 45 s; 3000, 50 s. The rest was the same as in Example 2, and the experimental results Figure 2 (b) showed that: when other conditions were the same, the current response was significantly greater than that of other experimental groups when the mixing parameters were 4000 r / min and 30 s. Similarly, the silver-mercury ratio for making the electrode was 3 / 7, and the combinations of mixing rotation speed (r / min) and time (s) were 5000, 3 s; 5000, 4 s; 5000, 5 s; 6000, 3 s; 6000, 4 s; 6000, 5 s. The rest was the same as in Example 2, and the experimental results Figure 2 (c) showed that: the current response values and stabilities of each experimental group were relatively poor. By comparing with Figure 2 (b), it could be determined that the optimal mixing parameters for the electrode were 4000 r / min and 30 s.

[0045] Example 4

[0046] The difference between Example 4 and Example 3 was that the mixing rotation speed was 4000 r / min, the mixing time was 30 s, and the activation times were 1 min, 3 min, 5 min, 10 min, and 15 min respectively. The rest was the same as in Example 3, and the results Figure 2(d) shows that when other conditions are the same, as the activation time increases, the peak value of the current response first increases and then decreases to gradually stabilize. When the electrode activation time is 5 min, the current response is significantly improved and relatively stable.

[0047] Example 5

[0048] The difference between Example 5 and Example 4 is that the activation time is 5 min, and the mercury dipping times are 30 s, 1 min, 3 min, 5 min, and 10 min respectively. The rest is the same as in Example 4, and the experimental results Figure 2 (e) shows that when other conditions are the same, as the mercury dipping time increases, the peak value of the current response first increases significantly and then gradually stabilizes. When the electrode mercury dipping time is 1 min, the electrode response reaches the peak value.

[0049] Example 6

[0050] The difference between Example 6 and Example 5 is that the mercury dipping time is 1 min, and the enrichment potentials are -0.4 V, -0.2 V, -0.1 V, 0.0 V, and 0.1 V respectively. The rest is the same as in Example 5, and the experimental results Figure 2 (f) shows that when other conditions are the same, as the enrichment potential changes from negative to positive, the peak value of the current response first gradually increases and then decreases. When the enrichment potential is 0.0 V, the detection effect of the electrode is the best.

[0051] Example 7

[0052] The difference between Example 7 and Example 6 is that the enrichment potential is 0.0 V, and the enrichment times are 1 min, 3 min, 5 min, 10 min, and 15 min respectively. The rest is the same as in Example 6, and the experimental results Figure 2 (g) shows that when other conditions are the same, as the enrichment time increases, the peak value of the current response shows a trend of first increasing and then gradually stabilizing. When the enrichment time is 5 min, the current response is significantly enhanced and more stable than that of other experimental groups.

[0053] Example 8

[0054] The difference between Example 8 and Example 7 is that the enrichment time is 5 min, and the pulse heights a are 0.001 V, 0.0025 V, 0.005 V, 0.01 V, 0.025 V, and 0.05 V respectively. The rest is the same as in Example 7, and the experimental results Figure 2 (h) shows that when other conditions are the same, as the pulse height a increases, the peak value of the current response increases linearly.

[0055] Example 9

[0056] The difference between Example 9 and Example 8 is that the pulse height a is 0.025 V, and the square wave frequencies f are 10 s -1 , 30 s-1 , 50 s -1 , 70 s -1 , 90 s -1 , the rest is the same as in Example 8, and the experimental results Figure 2 (i) show that: when other conditions are the same, as f 1 / 2 / gradually increases, the peak value of the current response increases linearly.

[0057] The optimal conditions for each step can be summarized as follows: the ratio of silver to mercury is 3 / 7, the mixing rotation speed is 4000 r / min, the mixing time is 30 s, the activation time is 5 min, the mercury dipping time is 1 min, the enrichment time is 5 min, and the square wave frequency f is 90 －1 , and the pulse height a is 0.025 V.

[0058] Example 10: Quantitative detection method for selenomono - saccharide (1β - methylseleno - N - acetyl - D - galactosamine)

[0059] Using the electrode fabricated in Example 1 as the working electrode, a platinum wire as the auxiliary electrode, and a silver - silver chloride electrode as the reference electrode to form a three - electrode system. The system is placed in a 5 mL centrifuge tube containing 4 mL of 0.2 M KCl solution. Select the amperometric i~t curve module in the operation window of the electrochemical workstation and activate it at - 2.2 V for 300 s (the sensitivity is set to 10 -3 ). Dip the working electrode in mercury for 1 min. In 0.1 M HCl buffer solution, add different volumes of 1 mM selenomono - saccharide to form test solutions with different concentrations. Select the amperometric i~t curve module in the operation window of the electrochemical workstation, and the system is enriched at 0.0 V for 300 s (sensitivity 10 -5 ). Finally, detect the current response by SWV method, where the analyte is selenomono - saccharide, and the concentrations are 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 2.0 μM, 4.0 μM, 6.0 μM, 8.0 μM, 10.0 μM respectively.

[0060] The results are as Figure 3 , it can be seen that as the concentration of selenomono - saccharide increases, the current increases accordingly, indicating that the peak current is proportional to the concentration of selenomono - saccharide. The fitting curve relationship between the concentration x of selenomono - saccharide and the peak current y detected by square - wave voltammetry is y = - 5.697x - 1.471, and the standard deviation R 2 = 0.999, the detection limit is 0.0186 μM, and the quantification limit is 0.0559 μM; where the unit of y is μA and the unit of x is μM.

[0061] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A solid electrode for detecting selenium monosaccharide, characterized in that, it is prepared by the following method: (1) Take a silver amalgam capsule, press the cap on the top of the capsule to the bottom until the plastic isolation layer falls off, install the pressed capsule on the chuck of the silver amalgam mixer and start the silver amalgam mixer. After completion, pour out the object in the capsule for later use; (2) Blend silver amalgam; sequentially place liquid mercury and nano silver powder into the capsule and blend them in the silver amalgam mixer. The mass ratio of liquid mercury to nano silver powder is 3-8:

2. After blending, open the capsule to obtain a paste-like silver amalgam; (3) Take the paste-like silver amalgam obtained in step (2) into a capillary tube. After compacting the paste-like silver amalgam, fix it with a copper wire dipped in conductive silver paste and wait for it to form an alloy to obtain a solid silver amalgam electrode for detecting selenium monosaccharide; the selenium monosaccharide is 1β-methylseleno-2-N-acetyl-D-galactosamine; The parameters set for the silver amalgam mixer in step (1) and step (2) are that the blending rotation speed is 3000-6000 r / min and the blending time is 25-35 s.

2. A quantitative analysis method based on the solid electrode for detecting selenium monosaccharide described in claim 1, characterized in that, it includes the following steps: (1) Polish the electrode surface with 2000-mesh sandpaper; (2) Activate the polished electrode; (3) Dip the activated electrode in mercury; (4) Enrich at a potential of 0.0 V in the amperometric i-t curve module of the electrochemical workstation, so as to enrich selenium in the test solution on the mercury film surface and expand the detection signal; (5) Perform SWV detection, utilize the electrochemical reaction of selenium and mercury in selenium monosaccharide under the pulsed potential, collect the current signal, and determine the concentration of selenium monosaccharide according to the standard curve.

3. The quantitative analysis method of the solid electrode for detecting selenium monosaccharide described in claim 2, characterized in that, the specific step (2) is: Wash the reference electrode and the auxiliary electrode with ultrapure water, clamp the white electrode clip on the black plug of the reference electrode connection wire, connect the red electrode to the green plug of the reference electrode connection wire, and clamp the green electrode clip on the copper wire of the working electrode; Place the electrode in a centrifuge tube containing 0.2 M KCl solution, and select the amperometric i-t curve module in the operation window of the electrochemical workstation to activate at -2.2 V for 250 s to 350 s.

4. The quantitative analysis method of the solid electrode for detecting selenium monosaccharide described in claim 2, characterized in that, the mercury dipping in step (3) is to dip the head of the electrode into mercury with water liquid seal to pick up mercury, and the mercury dipping time is 30 s to 10 min.

5. The quantitative analysis method of the solid electrode for detecting selenium monosaccharide described in claim 2, characterized in that, the parameter settings of the SWV - Square Wave Voltammetry module of the electrochemical workstation in step (4) are: The enrichment time is 1 min to 15 min.

6. The quantitative analysis method of the solid electrode for detecting selenium monosaccharide described in claim 2, characterized in that, Step (5) realizes the quantitative detection of selenium monosaccharide through the relationship between the concentration x of selenium monosaccharide and the peak current y detected by square wave voltammetry; among them, the relationship for calibrating x and y is specifically: the peak current values are detected by SWV at different concentrations of selenium monosaccharide molecules, the peak current value y at different concentrations x of selenium monosaccharide is obtained, and the relationship of x-y is obtained by fitting.

7. The quantitative analysis method of the solid electrode for detecting selenium monosaccharide as described in claim 6, characterized in that The relationship between x and y is y ＝ -5.697x - 1.471.

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