Blending device for chemiluminescence detection and application thereof

Abstract

The invention discloses a blending device for chemiluminescence detection and application of the blending device, and relates to the technical field of analysis and detection.The blending device comprises a hollow motor, a chemiluminescence reaction tank support, a mounting support, a chemiluminescence reaction tank, a power source, a motor driver and a speed regulation controller; a chemiluminescence reaction tank bracket is mounted on the hollow motor, and the axis of the chemiluminescence reaction tank bracket and the axis of the hollow motor are not collinear. By arranging the eccentric structure or the inclined structure of the bracket of the chemiluminescence reaction tank, the reaction solution can rotate along with the hollow motor and simultaneously introduces irregular circular motion, so that the turbulent flow in the solution is increased, the uniform mixing effect is improved, the solution can be quickly and uniformly mixed in a reaction system to reach a balanced state, stable chemiluminescence is generated, and the reaction efficiency is improved. The repeatability of an experiment and the relative standard deviation of a parallel experiment are obviously improved, and high-sensitivity detection of cadmium ions is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly to a mixing device for chemiluminescence detection and its application. Background Art

[0002] Chemiluminescence detection is an analytical method with high sensitivity and strong selectivity. Its detection principle is based on the light signal generated in a chemical reaction, with low background noise, enabling rapid detection of ultra-trace substances. This method is easy to operate, usually does not require complex sample pretreatment, the detection equipment has a simple structure and low cost, and is suitable for high-throughput detection. With these advantages, chemiluminescence detection is widely used in environmental monitoring, biomedicine, food safety, drug analysis and other fields.

[0003] The chemiluminescence reaction with luminol (3-aminophthalhydrazide) or its derivative isoluminol (4-aminophthalhydrazide), horseradish peroxidase (HPR) and hydrogen peroxide as reactants is an important chemiluminescence system. General chemiluminescence detectors usually detect chemiluminescence signals from the bottom. When the reactants are added to the chemiluminescence reaction cell, chemiluminescence is immediately generated. Without stirring and mixing, the process of adding reactants will cause uneven concentration of the reactants in the reaction system, with a relatively high local concentration. The process of mixing by relying on concentration gradient diffusion is very slow, resulting in poor repeatability of the detection results. Because the solution addition speed is much greater than the solution diffusion speed, as the solution is added, the local concentration of the solution begins to gradually increase, the reaction rate also gradually increases, and the chemiluminescence intensity gradually increases. When the solution addition is completed, the chemiluminescence reaches the maximum intensity, and then as the solution diffuses and the reactants are consumed, the chemiluminescence intensity begins to gradually decrease. Due to the uncertainty of local flow during each solution addition, the highest local concentration and reaction rate are random, resulting in poor repeatability of the experiment, that is, the relative standard deviation of parallel experimental results is very large.

[0004] To improve the repeatability of detection, one method is to add a chemiluminescence enhancer (such as p-iodophenol or sodium tetraphenylborate) to the reaction system to delay the time when the maximum chemiluminescence intensity value is generated, so that the operator has sufficient time to perform sample addition and mixing operations before detection. However, this method increases the detection time; in addition, the chemiluminescence enhancer delays the time when the maximum chemiluminescence signal is generated by acting on horseradish peroxidase, so this method cannot be used for detection methods based on inhibiting the activity of horseradish peroxidase.

[0005] Another method is to perform chemiluminescence detection by flow injection method. Through the premixing of reactants in the pipeline, a stable chemiluminescence signal is generated when they reach the chemiluminescence detector. However, this method requires the use of reactants to clean and fill the entire pipeline to obtain an effective and stable detection signal. Therefore, this method requires a large amount of reagent and is prone to contamination.

[0006] Generally, chemiluminescence detection is performed by optical detection at the bottom of the chemiluminescence reaction cell and needs to be carried out in a light-tight closed detection chamber. Existing stirring and mixing methods, including magnetic stirring or mechanical oscillation devices placed at the bottom and stirring rods placed above, are not suitable for chemiluminescence detection devices. For this reason, the present invention has developed a chemiluminescence detection mixing device based on a hollow motor and used it for the detection of cadmium ions in solution. Summary of the Invention

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a mixing device for chemiluminescence detection, including a hollow motor, a chemiluminescence reaction cell bracket, a mounting bracket, a chemiluminescence reaction cell, a power supply, a motor driver, and a speed control controller. The mounting bracket is used to be fixedly connected to an external chemiluminescence detector. The hollow motor is installed on the mounting bracket. A chemiluminescence reaction cell bracket is installed on the hollow motor. The chemiluminescence reaction cell bracket is provided with holes, and the chemiluminescence reaction cell is fixed in the holes of the chemiluminescence reaction cell bracket. The axis of the chemiluminescence reaction cell bracket and the axis of the hollow motor are not collinear, and the speed of the hollow motor is adjusted through the motor driver and the speed control controller.

[0009] Preferably, the axis of the chemiluminescence reaction cell bracket is parallel to the axis of the hollow motor, and a predetermined distance is spaced between the two axes.

[0010] Preferably, the axis of the chemiluminescence reaction cell bracket and the axis of the hollow motor intersect to form an angle, and the angle is greater than 0° and less than 90°.

[0011] The present invention also provides a chemiluminescence detection method for cadmium ions in solution, applying the mixing device for chemiluminescence detection as described above. The method includes the following steps:

[0012] Step 1: Prepare a 1.0×10 -3 mol / L luminol solution, a 1.0×10 -3 mol / L hydrogen peroxide solution, and a 2 mg / L horseradish peroxidase solution;

[0013] Step 2: Use a pipette to transfer 50 μL of 1.0×10 -3 mol / L luminol solution, 50 μL of 1.0×10 -3A hydrogen peroxide solution of [[mol / L]] and 300 μL of a 0.1 mol / L phosphate buffer solution with a pH value of 7.0 are placed in a chemiluminescence reaction cell;

[0014] Step 3: Place the chemiluminescence reaction cell in the hole of the chemiluminescence reaction cell bracket, close the upper cover of the detection chamber of the external chemiluminescence detector, turn on the power, and then use a syringe to inject 100 μL of a 2 mg / L horseradish peroxidase solution into the chemiluminescence reaction cell through the small hole in the upper cover of the detection chamber. Detect the luminescence signal with a chemiluminescence detector, and record the chemiluminescence intensity 20 seconds after adding the horseradish peroxidase solution as I 0 , that is, the blank signal, which is used to calculate the detection limit;

[0015] Step 4: Mix the 2 mg / L horseradish peroxidase solution and the cadmium ion solution in equal volume and incubate for 5 minutes, then take 100 μL of the mixed solution and inject it into the chemiluminescence reaction cell for detecting the chemiluminescence signal I of cadmium ions at different concentrations s .

[0016] The present invention has the following beneficial effects:

[0017] By setting the eccentric structure or inclined structure of the chemiluminescence reaction cell bracket, the reaction solution can be rotated with the hollow motor while introducing an irregular circular motion, increasing the turbulence in the solution, improving the mixing effect. Utilizing the characteristic that the hollow motor shaft is hollow, while not affecting the detection of chemiluminescence signals from the bottom, the reactants can be quickly mixed by the rotation of the motor, enhancing the repeatability of detection.

[0018] By applying this mixing device to the chemiluminescence detection system, the solution can be quickly mixed in the reaction system to reach an equilibrium state, generating stable chemiluminescence, significantly improving the repeatability of the experiment and the relative standard deviation of parallel experiments.

[0019] Based on the inhibitory effect of cadmium ions on the catalytic activity of horseradish peroxidase by this device, a chemiluminescence-based method for detecting cadmium ions is established, realizing the highly sensitive detection of cadmium ions and providing a new method for the analysis of cadmium ions in solution. Description of the Drawings

[0020] Figure 1 is a three-dimensional structural schematic diagram of the mixing device of the present invention;

[0021] Figure 2 is a control schematic diagram of the mixing device of the present invention;

[0022] Figure 3 is a three-dimensional structural schematic diagram of the chemiluminescence reaction cell bracket in the present invention;

[0023] Figure 4It is the graph of the change of the chemiluminescence intensity with time for the detection of cadmium ions in the present invention;

[0024] Figure 5 It is the standard curve for the quantitative detection of cadmium ions in the present invention.

[0025] In the figure: 1, hollow motor; 2, chemiluminescence reaction cell support; 3, mounting bracket; 4, chemiluminescence reaction cell; 5, power supply; 6, motor driver; 7, speed controller. Specific embodiments

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0027] The present invention will be further described in detail below according to the drawings and embodiments.

[0028] The present application provides a mixing device for chemiluminescence detection, as Figures 1 to 3 shown, including a hollow motor 1, a chemiluminescence reaction cell support 2, a mounting bracket 3, a chemiluminescence reaction cell 4, a power supply 5, a motor driver 6 and a speed controller 7. The mounting bracket 3 is used to be connected and fixed to an external chemiluminescence detector. The hollow motor 1 is installed on the mounting bracket 3. The central part of the hollow motor 1 is a central hole with a certain diameter that completely passes through the motor central axis, which does not affect the detection of chemiluminescence signals from the bottom. The chemiluminescence reaction cell support 2 is installed on the hollow motor 1. The chemiluminescence reaction cell support 2 is provided with hole positions, and the chemiluminescence reaction cell 4 is fixed in the hole positions of the chemiluminescence reaction cell support 2. The axis of the chemiluminescence reaction cell support 2 and the axis of the hollow motor 1 are not collinear. The rotation speed of the hollow motor 1 is adjusted by the motor driver 6 and the speed controller 7.

[0029] The motor driver 6 is used to provide a driving signal for the hollow motor 1 to realize the rotation of the hollow motor 1. At the same time, the speed controller 7 controls the rotation speed of the hollow motor 1 by applying an external voltage of 0 - 5V or a PWM signal. The rotation speed of the hollow motor 1 is adjusted by the motor driver 6 and the speed controller 7 to achieve the best mixing effect. The hollow motor 1 drives the chemiluminescence reaction cell support 2 and the chemiluminescence reaction cell 4 to rotate, so that the reactants in the chemiluminescence reaction cell 4 are quickly mixed.

[0030] The hollow motor 1, the power supply 5, the motor driver 6, the speed controller 7 and the external chemiluminescence detector are all prior arts and will not be elaborated here.

[0031] To enhance the mixing effect, two schemes of eccentricity or inclination are adopted for the chemiluminescence reaction cell bracket 2. In the eccentric structure, the axis of the chemiluminescence reaction cell bracket 2 is parallel to the axis of the hollow motor 1, and a predetermined distance is spaced between the two axes. At the same time, it is necessary to ensure that the placed chemiluminescence reaction cell 4 does not collide with the inner wall of the hollow motor 1. In the inclined structure, the axis of the chemiluminescence reaction cell bracket 2 intersects with the axis of the hollow motor 1 to form an angle, and the angle is greater than 0° and less than 90°. At the same time, it is ensured that the placed chemiluminescence reaction cell 4 does not collide with the inner wall of the hollow motor 1. These two structures can introduce irregular circular motion while the reaction solution rotates with the hollow motor 1, increasing the turbulence in the solution and improving the mixing effect.

[0032] The chemiluminescence reaction cell bracket 2 is processed by 3D printing technology according to the diameter of the chemiluminescence reaction cell 4 (the diameter should be smaller than the diameter of the central hole of the hollow motor 1).

[0033] Based on the inhibitory effect of cadmium ions on the catalytic activity of horseradish peroxidase, different concentrations of cadmium ions result in different catalytic abilities of horseradish peroxidase, thereby generating chemiluminescence of different intensities. This application provides a chemiluminescence detection method for cadmium ions in solution, using the mixing device for chemiluminescence detection as described above, including the following steps:

[0034] Step 1: Prepare a 1.0×10 -3 mol / L luminol solution, a 1.0×10 -3 mol / L hydrogen peroxide solution, and a 2mg / L horseradish peroxidase (HRP) solution, specifically including the following steps:

[0035] Step 1.1: Prepare the luminol solution: Accurately weigh 17.72mg of luminol, dissolve it with a 1.0×10 -2 mol / L sodium hydroxide solution and make the volume up to 100mL to obtain a 1.0×10 -3 mol / L luminol solution, and store it in the dark.

[0036] Step 1.2: Prepare the hydrogen peroxide solution: Gradually dilute the 30% hydrogen peroxide solution with deionized water to 1.0×10 -3 mol / L;

[0037] Step 1.3: Prepare the horseradish peroxidase (HRP) solution: Accurately weigh 1mg of horseradish peroxidase freeze-dried powder, dissolve it with a 0.1mol / L phosphate buffer solution with a pH value of 7.0 and dilute it to 2mg / L.

[0038] Step 2: Use a pipette to transfer 50μL of the 1.0×10 -3 mol / L luminol solution, 50μL of the 1.0×10-3 A hydrogen peroxide solution of [[mol / L]] and 300 μL of a 0.1 [[mol / L]] phosphate buffer solution with a pH value of 7.0 are placed in the chemiluminescence reaction cell 4.

[0039] Step 3: Place the chemiluminescence reaction cell 4 into the hole position of the chemiluminescence reaction cell bracket 2, close the upper cover of the detection chamber of the external chemiluminescence detector, turn on the power supply 5, and then use a syringe to inject 100 μL of a 2 [[mg / L]] horseradish peroxidase solution into the chemiluminescence reaction cell 4 through the small hole in the upper cover of the detection chamber. Detect the luminescence signal with a chemiluminescence detector (for the detection result reference Figure 4 ), and record the chemiluminescence intensity 20 seconds after adding the horseradish peroxidase solution as I 0 , that is, the blank signal, which is used to calculate the detection limit.

[0040] Step 4: Mix the 2 [[mg / L]] horseradish peroxidase solution and the cadmium ion solution in equal volume and incubate for 5 minutes. Then take 100 μL of the above mixed solution and inject it into the chemiluminescence reaction cell 4 to detect the chemiluminescence signal I of cadmium ions at different concentrations s .

[0041] This application also provides the data for detecting cadmium ion solutions at different concentrations (refer to Table 1), which is used to detect the chemiluminescence signal I of cadmium ions at different concentrations s , to establish a standard curve or detect actual samples.

[0042] Table 1 Experimental data

[0043]

[0044] The results show that when the concentration (c) of Cd 2+ is in the range of 5 - 35 μg / L, the chemiluminescence intensity (I s ) is inversely proportional to the concentration of Cd 2+ (refer to Figure 5 ), and the calibration equation is I s = -26.814c + 1486.6 (R 2 = 0.9996), and the detection limit is 2.6 μg / L (the amount of the analyte corresponding to 3 times the standard deviation of the reagent blank signal).

[0045] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A mixing device for chemiluminescence detection, characterized in that: The invention comprises a hollow motor (1), a chemiluminescent reaction pool support (2), a mounting support (3), a chemiluminescent reaction pool (4), a power supply (5), a motor driver (6) and a speed controller (7); the mounting support (3) is used for connecting and fixing with an external chemiluminescent detector; the hollow motor (1) is mounted on the mounting support (3); a chemiluminescent reaction pool support (2) is mounted on the hollow motor (1); the chemiluminescent reaction pool support (2) is provided with a hole; the chemiluminescent reaction pool (4) is fixed in the hole of the chemiluminescent reaction pool support (2); the axis of the chemiluminescent reaction pool support (2) and the axis of the hollow motor (1) are not collinear; the rotation speed of the hollow motor (1) is adjusted by the motor driver (6) and the speed controller (7).

2. A mixing device for chemiluminescence detection according to claim 1, characterized in that: The axis of the chemiluminescent reaction pool support (2) is parallel to the axis of the hollow motor (1), and a predetermined distance is spaced between the two axes.

3. A mixing device for chemiluminescence detection according to claim 1, characterized in that: The axis of the chemiluminescent reaction pool support (2) and the axis of the hollow motor (1) intersect with each other to form an angle, which is greater than 0° and less than 90°.

4. A chemiluminescent detection method for cadmium ions in a solution, characterized in that: Using the mixing device for chemiluminescence detection as described in any one of claims 2 and 3, the method comprises the following steps: Step 1: Prepare 1.0×10 -3 mol / L luminol solution, 1.0×10 -3 mol / L hydrogen peroxide solution and 2mg / L horseradish peroxidase solution; Step 2: Use a pipette to transfer 50 μL of 1.0×10 -3 mol / L luminol solution, 50 μL of 1.0×10 -3 mol / L hydrogen peroxide solution and 300 μL of 0.1 mol / L phosphate buffer solution with a pH value of 7.0 are placed in a chemiluminescent reaction cell (4); Step 3: Place the chemiluminescent reaction pool (4) in the hole of the chemiluminescent reaction pool bracket (2), close the detection chamber cover of the external chemiluminescent detector, turn on the power supply (5), and then use a syringe to inject 100 μL of 2 mg / L horseradish peroxidase solution into the chemiluminescent reaction pool (4) through the small hole in the detection chamber cover, and use the chemiluminescent detector to detect the luminescent signal. The chemiluminescent intensity 20 seconds after adding the horseradish peroxidase solution is recorded as I0, i.e., the blank signal, which is used to calculate the detection limit; Step 4: Mix equal volumes of 2 mg / L horseradish peroxidase solution and cadmium ion solution and incubate for 5 minutes. Then, inject 100 μL of the mixed solution into the chemiluminescent reaction pool (4) to detect the chemiluminescent signals of different concentrations of cadmium ions. s .

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