Magnetic field modulated electrochemical detection method for horse radish peroxidase

Abstract

The invention discloses a magnetic field modulated electrochemical detection method for horseradish peroxidase, and belongs to the field of electrochemical detection. According to the method, a magnetic field is used for controlling horse radish peroxidase taking magnetic beads as carriers to be in contact with the surface of an electrode of a modified enzyme substrate as required, and ultra-low-amount horse radish peroxidase detection is completed through a time domain detection method and a frequency domain detection method. In time domain detection, an enzyme substrate is fixed on the surface of an electrode, and enzyme is controlled to be far away from and contact with the surface of the electrode through a magnetic field, so that an accurate response signal for shielding noise is obtained. In order to detect a lower amount of enzyme, the magnetic beads are controlled to be in periodic contact with the surface of the electrode by externally adding a periodically changing magnetic field, so that a periodic electrochemical response signal with frequency characteristics is formed. The detection method can be applied to an electrochemical sensing detection system taking horse radish peroxidase or an enzyme-labeled biomarker as a detection object or an electronic mediator, and the detection limit of a traditional horse radish peroxidase electrochemical detection method is improved.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection, specifically relating to a magnetic field-modulated electrochemical detection method for horseradish peroxidase. Background Technology

[0002] Horseradish peroxidase (HRP) is a type of oxidoreductase that catalyzes the oxidation of various substrates through reactions with hydrogen peroxide and related molecules. Due to its catalytic properties, high stability, wide applicability, and ability to amplify weak detection signals, it is widely used in biochemical analysis and bioremediation, particularly in enzyme-linked immunosorbent assays (ELISA). HRP is commonly used as an electrode modifier, biological acceptor, and tag for the detection of proteins, nucleic acids, and H₂O₂; therefore, the quantitative detection of HRP is of great significance. Magnetic microbeads, due to their high specific surface area and enrichment effect caused by superparamagnetic properties, are widely used as carriers for small particles or biomolecules. These properties facilitate collection and transfer via magnetic fields. The method of enriching magnetic beads or surface-modified biomarkers onto the electrode surface using an external magnetic field can significantly improve the sensitivity of such electrochemical biosensors.

[0003] Traditional electrochemical sensors are limited in low-concentration scenarios by background signal interference from unrelated electrochemical processes. This interference weakens the accurate detection of weak electrical signals because they are easily drowned out by noise, resulting in a decreased signal-to-noise ratio. Therefore, there is an urgent need to improve the detection limit and address the detection challenges in these scenarios. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects in the prior art and to provide a magnetic field modulated electrochemical detection method for horseradish peroxidase.

[0005] The specific technical solution adopted in this invention is as follows:

[0006] In a first aspect, the present invention provides a magnetic field-modulated electrochemical detection method for horseradish peroxidase, as detailed below:

[0007] S1: Horseradish peroxidase is coupled to the surface of magnetic beads, and 3,3',5,5'-tetramethylbenzidine is modified on the surface of the electrode.

[0008] S2: By applying an external magnetic field, the magnetic bead is controlled to contact or move away from the electrode surface; when the magnetic bead is away from the electrode surface, horseradish peroxidase does not react with 3,3',5,5'-tetramethylbenzidine; when the magnetic bead contacts the electrode surface, horseradish peroxidase reacts with 3,3',5,5'-tetramethylbenzidine.

[0009] S21: Record the electrochemical response signals generated when the magnetic field is turned on and off, respectively, as the magnetic bead moves away from and contacts the electrode surface; when the magnetic bead contacts the electrode surface, an oxidation signal is generated on the electrode surface; when the magnetic bead moves away from the electrode surface, a reduction signal is generated on the electrode surface, thus obtaining a response signal with noise suppression effect;

[0010] S22: If the difference between the oxidation and reduction signals obtained in S21 is ≥0.3 μA, then the content of the unknown horseradish peroxidase is calculated based on the correlation between the difference and the horseradish peroxidase concentration; otherwise, the frequency domain detection method is used, as detailed below:

[0011] By applying a periodically changing magnetic field to control the periodic contact of magnetic beads with the electrode surface, a periodic electrochemical response signal with frequency characteristics is formed. The frequency characteristics are detected from the periodic electrochemical response signal using a weak signal detection method, and the concentration of the target is calculated based on the correlation between the frequency characteristics and the unknown horseradish peroxidase.

[0012] Preferably, the electrode is a planar photolithography electrode or a screen-printed electrode, and the electrode system is a two-electrode system or a three-electrode system.

[0013] Preferably, 3,3',5,5'-tetramethylbenzidine is modified on the electrode surface by drop casting, and the concentration of 3,3',5,5'-tetramethylbenzidine in the drop casting solution is 1-20 mg / mL.

[0014] Preferably, the magnetic bead consists of a magnetic core and a shell surrounding the magnetic core. The magnetic core is made of metal oxide, the shell is made of polystyrene, and the diameter of the magnetic bead is 50 nanometers to 50 micrometers.

[0015] Preferably, the horseradish peroxidase is coupled to the surface of the magnetic beads via antigen-antibody reaction, complementary pairing of nucleic acid chains, or biotinylate.

[0016] Preferably, in step S22, the frequency range of the periodically changing magnetic field is 0.01-100 Hz.

[0017] Preferably, the weak signal detection method is one or more of the following: spectrum analysis, narrowband filtering, phase-locked amplification, sampling integration, and digital averaging.

[0018] Preferably, in step S22, the periodically changing magnetic field is generated by two coils uniformly and tightly wound around a silicon steel core, located on the upper and lower sides of the electrode. By periodically controlling the relay switch, the upper and lower coils are alternately energized to generate an alternating magnetic field.

[0019] Secondly, the present invention provides an immunomarker detection method for horseradish peroxidase based on magnetic field modulation electrochemical detection, as detailed below:

[0020] A first antibody is modified on the surface of magnetic beads, and the target immunomarker is captured using the first antibody to obtain immunomagnetic beads; a second antibody used to capture the target immunomarker is coupled with horseradish peroxidase to obtain a secondary antibody-horseradish peroxidase conjugate; the secondary antibody-horseradish peroxidase conjugate is then coupled to the surface of the immunomagnetic beads; 3,3',5,5'-tetramethylbenzidine is modified on the electrode surface;

[0021] Since the concentration of horseradish peroxidase is directly proportional to the concentration of the target immunomarker, the concentration of the target immunomarker can be obtained by detecting the signal of horseradish peroxidase using the magnetic field-modulated electrochemical detection method of horseradish peroxidase as described in any of the first aspects.

[0022] Preferably, the second antibody is coupled to horseradish peroxidase via biotinylate, glutaraldehyde crosslinking, sodium periodate method, or amide bond; the second antibody-horseradish peroxidase conjugate is coupled to the surface of the immunomagnetic beads by incubation.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] Traditional electrochemical sensors are limited by non-Radidatic signals. In low-target-concentration scenarios, the weak DC time-domain signal is submerged in noise, weakening the accurate detection of the electrical signal. This invention, however, presents a magnetic field-modulated electrochemical detection method for horseradish peroxidase, achieving ultra-low detection of horseradish peroxidase in both the time and frequency domains. In time-domain detection, background noise interference is avoided through a dual-state switching reaction. In frequency-domain detection, the DC signal is converted into a periodic signal of a certain frequency through magnetic field modulation, giving it frequency-domain characteristics. By separating the weak target-concentration signal contained in these periodic signals in the frequency domain, the inherent limitations of traditional magnetic bead electrochemical sensors are overcome, enhancing detection sensitivity. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of a magnetic field-modulated electrochemical detection method for horseradish peroxidase according to the present invention; wherein, (a) is a state diagram when the magnetic bead moves away from and contacts the electrode surface, (b) is a signal diagram generated on the electrode surface when an upward and downward magnetic field is applied, and (c) is a signal diagram generated on the electrode surface when a periodic magnetic field is applied.

[0026] Figure 2 A schematic diagram showing how an electromagnet controlled by a periodic switch generates a periodically changing magnetic field.

[0027] Figure 3 The graph shows the electrochemical response signals under time-domain detection; where the signals are the electrochemical signals generated when the horseradish peroxidase concentration is (a) 0.1 ng / mL, (b) 5 ng / mL, (c) 10 ng / mL, and (d) 50 ng / mL, respectively.

[0028] Figure 4 The time-domain plots show the periodic electrochemical response signals modulated by the magnetic field under frequency domain detection; where the signals are the electrochemical signals generated when the horseradish peroxidase concentration is (a) 0.1 ng / mL, (b) 1 ng / mL, (c) 10 ng / mL, and (d) 50 ng / mL, respectively.

[0029] Figure 5 The frequency domain diagrams are the periodic electrochemical response signals modulated by the magnetic field under frequency domain detection; where the spectrum diagrams are generated when the horseradish peroxidase concentration is (a) 0.1 ng / mL, (b) 1 ng / mL, (c) 10 ng / mL, and (d) 50 ng / mL, respectively.

[0030] Figure 6 This is a schematic diagram of protein biomarker detection based on the horseradish peroxidase detection method in the embodiments. Detailed Implementation

[0031] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0032] like Figure 1 As shown, this invention provides a magnetic field-modulated electrochemical detection method for horseradish peroxidase, the specific details of which are as follows:

[0033] (1) Horseradish peroxidase is coupled to the surface of magnetic beads.

[0034] In a preferred embodiment of the present invention, in this step, the magnetic bead consists of a magnetic core and a shell surrounding the magnetic core, wherein the magnetic core is made of metal oxide and the shell is made of polystyrene. The diameter of the magnetic bead is 50 nanometers to 50 micrometers.

[0035] In a preferred embodiment of the present invention, in this step, horseradish peroxidase can be coupled to the surface of magnetic beads through antigen-antibody reaction, complementary pairing of nucleic acid chains, biotin-avidin, etc.

[0036] For example, in this embodiment, biotin-functionalized horseradish peroxidase is immobilized on the surface of streptavidin-functionalized magnetic beads via streptavidin-biotin interaction. Specifically, the magnetic bead surface is modified with an antibody, interleukin-6 is used as the detection target, and then horseradish peroxidase is coupled to achieve electrochemical detection of low concentrations of interleukin-6. Aptamers, peptides, etc., can be coupled to the magnetic bead surface; the specific modification material depends on the actual detection requirements to detect other markers such as nucleic acids and viruses, thus enabling applications in the detection and analysis of various immunoassay targets.

[0037] (2) Modify the electrode surface with 3,3',5,5'-tetramethylbenzidine.

[0038] In a preferred embodiment of the present invention, in this step, the electrode is a planar electrode, such as a planar photolithography electrode or a screen printing electrode, and the electrode system is a two-electrode system or a three-electrode system.

[0039] In a preferred embodiment of the present invention, in this step, 3,3',5,5'-tetramethylbenzidine can be modified on the electrode surface by drop casting, wherein the concentration of 3,3',5,5'-tetramethylbenzidine in the drop casting solution is 1 to 20 mg / mL.

[0040] (3) By applying an external magnetic field, the magnetic beads are controlled to contact or move away from the electrode surface. When the magnetic beads are away from the electrode surface, horseradish peroxidase does not react with 3,3',5,5'-tetramethylbenzidine; when the magnetic beads are in contact with the electrode surface, horseradish peroxidase reacts with 3,3',5,5'-tetramethylbenzidine.

[0041] First, a time-domain detection method is used, as follows:

[0042] The electrochemical response signals generated when the magnetic bead moves away from and contacts the electrode surface were recorded when the magnetic field was turned on and off, respectively. Specifically, when the magnetic bead contacts the electrode surface, an oxidation signal is generated on the electrode surface; when the magnetic bead moves away from the electrode surface, a reduction signal is generated on the electrode surface. The difference between the oxidation and reduction signals is used as the response signal corresponding to the enzyme amount, thus obtaining the baseline noise generated by non-electrochemical processes. Subsequently, the concentration of the target substance (i.e., horseradish peroxidase) can be calculated based on the correlation between the electrochemical response signal and the target substance.

[0043] (4) If the concentration of the object to be detected is low, i.e., the difference between the oxidation signal and the reduction signal in the above time-domain detection method is <0.3 μA, then a frequency-domain detection method is required. That is, the magnetic bead can be periodically contacted with the electrode surface by an externally applied periodically changing magnetic field. The specific frequency-domain detection method is as follows:

[0044] By applying a periodically changing magnetic field to control the periodic contact of magnetic beads with the electrode surface, a periodic electrochemical response signal with frequency characteristics is formed. The frequency characteristics are detected from the periodic electrochemical response signal using a weak signal detection method, and the concentration of the target is calculated based on the correlation between the frequency characteristics and the unknown horseradish peroxidase.

[0045] As a preferred embodiment of the present invention, the weak signal detection method may employ one or more of the following: spectrum analysis, narrowband filtering, phase-locked amplification, sampling integration, and digital averaging, with the aim of detecting frequency characteristics from the time-domain signal of the electrochemical response.

[0046] For example, after obtaining the periodic electrochemical response signal, a fast Fourier transform is performed on the periodic electrochemical response signal to obtain the amplitude-frequency diagram of the signal and perform spectrum analysis. At the frequency point of the modulation frequency, the amplitude peak value that shows a linear relationship with the magnetic bead concentration is obtained. This amplitude peak value is used as the frequency feature, and the content of horseradish peroxidase is calculated based on the correlation between the frequency feature and the detection object.

[0047] In a preferred embodiment of the present invention, the periodically changing magnetic field can be generated by two coils uniformly and tightly wound around a silicon steel core, located on the upper and lower sides of the electrode. By periodically controlling a relay switch, the upper and lower coils are alternately energized, generating an alternating magnetic field. Figure 2 As shown. The device for controlling the magnetic field can consist of an electromagnetic field generating module and a control circuit, responsible for adjusting the period and intensity of the magnetic field. The control circuit includes relays, an adapter power supply, and a main control system. Thermal grease is used to fill the gap between the coil and the copper pipe to promote heat dissipation, and the copper pipe is connected to a cooling pump for circulating cooling.

[0048] It should be noted that in time-domain detection, the position of the magnetic bead relative to the electrode is controlled by energizing the upper or lower coil; in frequency-domain detection, since the frequency characteristics of the periodic electrochemical response signal are generated by the periodic contact of the magnetic bead with the electrode under the drive of a periodically changing magnetic field, the frequency characteristics of the two are actually consistent. As a preferred embodiment of the present invention, the frequency of the periodically changing magnetic field is the same as the frequency of the generated periodic electrochemical response, both of which can be controlled from 0.01 to 100 Hz. In actual use, the period size can be selected according to the analyte.

[0049] The aforementioned periodically changing magnetic field is generated by a device for controlling the magnetic field, specifically an electromagnet controlled by a periodic switch. By controlling the electromagnet to periodically switch on and off, a magnetic force is periodically generated on the magnetic bead, causing it to detach from the electrode surface and then contact the electrode surface.

[0050] The working principle of this invention is as follows: Traditional electrochemical sensors are limited by non-Radidatic signals, especially in low target concentration scenarios, where weak DC time-domain signals are submerged in noise, weakening the accurate detection of electrical signals. This invention, however, creates a magnetic field-modulated electrochemical detection method for horseradish peroxidase, achieving ultra-low detection of horseradish peroxidase in both the time and frequency domains. In time-domain detection, a dual-state switching reaction avoids background noise interference. When the magnetic field force is upward, the peroxidase is carried away from the electrode surface, and the enzyme catalytic reaction is in a closed state. The signal generated on the electrode surface at this time is considered an interference signal generated by the non-Radidatic process. The magnetic bead remains at the gas-liquid interface of the electrochemical reaction system. Due to surface tension, the magnetic bead does not detach from the droplet but is fixed to the top of the droplet by the magnetic field. When the magnetic field force is downward, the enzyme catalytic reaction is activated, and an oxidation signal is generated on the electrode surface. By subtracting the background signal from the closed state from the current response in the activated state, the true signal related to the enzyme concentration can be obtained, thus shielding against background noise interference. In the presence of low-target-object backgrounds, the true signal is submerged in background noise and difficult to detect. Therefore, this invention introduces a method to convert time-domain detection to frequency-domain detection. This method uses magnetic field modulation to convert the time-domain DC signal into a periodic signal of a certain frequency. Under this frequency magnetic field modulation, horseradish peroxidase, carried by magnetic beads, is periodically moved away from and brought into contact with the electrode surface, generating periodically changing oxidation and reduction signals, thus giving it frequency-domain characteristics. By separating the weak target-object signal contained in these periodic signals in the frequency domain, the inherent limitations of traditional magnetic bead electrochemical sensors are overcome, enhancing detection sensitivity.

[0051] For example, by modifying the surface of magnetic beads with interleukin-6 antibodies to capture interleukin-6 in the analyte, and then conjugating a secondary antibody-horseradish peroxidase conjugate as a label and signal tag, extremely low levels of interleukin-6 can be detected using a magnetic field modulation method. This detection method can be applied to detect various immunomarkers that use horseradish peroxidase as a signal amplification molecule, such as proteins, nucleic acids, and viruses.

[0052] In addition, based on the above-mentioned electrochemical detection method for horseradish peroxidase, the present invention also provides a method for detecting immunomarkers, as follows:

[0053] A first antibody was modified onto the surface of magnetic beads, and the target immunomarker was captured using the first antibody to obtain immunomagnetic beads. A second antibody, which can also capture the target immunomarker, was conjugated with horseradish peroxidase to obtain a secondary antibody-horseradish peroxidase conjugate. The obtained secondary antibody-horseradish peroxidase conjugate was then conjugated onto the surface of the immunomagnetic beads. 3,3',5,5'-tetramethylbenzidine was modified onto the electrode surface.

[0054] Since the concentration of horseradish peroxidase is directly proportional to the concentration of the target immune marker, the concentration of the target immune marker can be calculated by detecting horseradish peroxidase, as follows:

[0055] An external magnetic field is used to control whether the magnetic beads are in contact with or away from the electrode surface. When the magnetic beads are away from the electrode surface, horseradish peroxidase does not react with 3,3',5,5'-tetramethylbenzidine. When the magnetic beads are in contact with the electrode surface, horseradish peroxidase reacts with 3,3',5,5'-tetramethylbenzidine.

[0056] The electrochemical response signals generated when the magnetic field is turned on and off, and when the magnetic bead moves away from and contacts the electrode surface, are recorded respectively. When the magnetic bead contacts the electrode surface, an oxidation signal is generated on the electrode surface; when the magnetic bead moves away from the electrode surface, a reduction signal is generated on the electrode surface, thus obtaining a response signal with noise suppression effect.

[0057] If the difference between the obtained oxidation and reduction signals is ≥0.3 μA, the concentration of the target immunomarker is calculated based on the correlation between this difference and the horseradish peroxidase concentration; otherwise, a frequency domain detection method is used, as detailed below:

[0058] By applying a periodically changing magnetic field to control the periodic contact of magnetic beads with the electrode surface, a periodic electrochemical response signal with frequency characteristics is formed. The frequency characteristics are detected from the periodic electrochemical response signal using a weak signal detection method, and the concentration of the target immunomarker is calculated based on the correlation between the frequency characteristics and the target immunomarker.

[0059] The following specific embodiments illustrate the specific implementation methods and technical effects of the present invention.

[0060] Example 1

[0061] like Figure 2 As shown, the horseradish peroxidase sensor in this example uses periodically switched electromagnets to generate a periodically changing magnetic field. Electromagnets are placed at appropriate positions above and below the working electrode in the planar electrode. When the upper electromagnet is energized and the lower electromagnet is de-energized, the magnetic force on the magnetic bead is upward, carrying the horseradish peroxidase carried by the bead away from the electrode surface, stopping the electrochemical reaction. When the upper electromagnet is de-energized and the lower electromagnet is energized, the magnetic force on the magnetic bead is downward, carrying the horseradish peroxidase to the electrode surface, starting the electrochemical reaction. For time-domain detection, controlling the energization of the upper or lower electromagnets allows for the on / off control of the electrochemical reaction, outputting a corresponding electrochemical signal. In frequency-domain detection, periodically switching the upper and lower electromagnets on and off generates a periodic electrochemical response signal.

[0062] Electrochemical detection experiments were conducted based on the horseradish peroxidase electrochemical sensor of this embodiment to verify the technical effect of the invention. The experiment used chronoamperometry, where horseradish peroxidase was coupled to the surface of magnetic beads via streptavidin-biotin interaction. Different enzyme concentration gradients were set: for time-domain detection, the concentration gradients were 50 ng / mL, 10 ng / mL, 5 ng / mL, and 0.1 ng / mL; for frequency-domain detection, the concentration gradients were 50 pg / mL, 10 pg / mL, 1 pg / mL, and 0.1 pg / mL. A three-electrode planar electrode system was used: a gold electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a carbon electrode as the counter electrode. A constant potential of -0.4 V was applied to the electrodes. 10 μL of a 10 mg / mL solution of 3,3',5,5'-tetramethylbenzidine was dropped onto the surface of the working electrode and dried in a vacuum oven for 2 hours. The magnetic field modulation period was set to 40 s. 200 μL of HAc-NaAc buffer containing 30 mM hydrogen peroxide and horseradish peroxidase-coupled magnetic beads was added to the electrode surface, with a magnetic bead concentration of 0.1 mg / mL. The sampling rate was 0.1 s, and each test was repeated three times. The final time-domain and frequency-domain detection results are as follows: Figure 3 , Figure 4 and Figure 5 As shown. Figure 3 The signals are reduction and oxidation signals generated by different enzyme concentrations under time-domain testing. As the enzyme concentration increases, the signal recorded under the oxidation state gradually increases, while the response under the reduction state remains almost unchanged. Figure 4 and Figure 5 The time-domain signal plots and spectra of enzyme concentrations of 50 pg / mL, 10 pg / mL, 1 pg / mL, and 0.1 pg / mL are obtained using a frequency domain detection method. Figure 4 It can be seen that the amplitude of the modulated chronoamperometric response is significantly correlated with the concentration of horseradish peroxidase. Figure 5 The results showed that the intensity of the main peak in the spectrum increased with increasing enzyme concentration.

[0063] Traditional horseradish peroxidase electrochemical sensors evaluate enzyme activity by recording the electrochemical signals generated after different amounts of enzyme oxidize 3,3',5,5'-tetramethylbenzidine substrate solutions. The time-domain detection method described in this invention records the signals for both the on and off states of the electrochemical reaction, using the difference between these signals as the response to the enzyme concentration, thus shielding against noise from non-electrochemical processes. However, when the enzyme concentration is low, such as... Figure 3At concentrations as low as 0.1 ng / mL, the response and noise levels are very close, making them difficult to distinguish. Therefore, the magnetic field-modulated electrochemical detection method for horseradish peroxidase proposed in this invention adjusts the magnetic field to move magnetic microbeads away from or into contact with the electrode surface at a specific frequency, generating periodic electrochemical signals. These time-domain electrochemical signals are then converted to the frequency domain, and combined with weak signal detection methods to improve detection sensitivity and achieve detection of ultra-low concentration target analytes. Figure 5 As shown, for Figure 4 The experimental data were subjected to Fast Fourier Transform (FFT) to obtain the amplitude-frequency spectrum of the signal, and spectral analysis was performed. A clear amplitude peak was observed at the modulation frequency. The amplitude of the modulated chronoampere response was significantly correlated with the enzyme concentration; therefore, the enzyme concentration could be calculated based on the amplitude peak. Although the square wave signal was weak and masked by noise at the lowest concentration of 0.1 pg / mL, the amplitude spectrum still showed a clear peak at 0.025 Hz after signal processing.

[0064] Therefore, this invention uses magnetic field modulation of enzyme-loaded magnetic beads to control electrochemical reactions, obtaining accurate signals with noise shielding. At the same time, the above-mentioned spectral analysis method improves the detection limit of the magnetic bead electrochemical sensor, which has significant advantages for the efficient detection of ultra-low concentrations of horseradish peroxidase.

[0065] Example 2

[0066] like Figure 6 As shown, in this embodiment, the interleukin-6 electrochemical sensor utilizes a magnetic field modulation method to detect horseradish peroxidase, thereby detecting low concentrations of interleukin-6 protein. Interleukin-6 primary antibody is coupled to magnetic beads, which are then used to capture interleukin-6 in the sample. After capture, a secondary antibody-horseradish peroxidase conjugate is coupled as a label and signal tag to prepare immunomagnetic beads. The amount of interleukin-6 is directly proportional to the amount of coupled horseradish peroxidase. The amount of enzyme coupled to the magnetic beads is detected using a magnetic field modulation method, thus obtaining the amount of interleukin-6. Using the same method as in Example 1, a buffer solution containing immunomagnetic beads is dropped onto the electrode surface. An upper electromagnet and a lower electromagnet are placed at appropriate positions above and below the working electrode of the planar electrode. When the upper electromagnet is energized and the lower electromagnet is de-energized, the magnetic force on the magnetic beads is upward, causing the horseradish peroxidase-labeled immunomagnetic beads to be carried away from the electrode surface, stopping the electrochemical reaction. The collected periodic electrochemical signals were subjected to fast Fourier transform to obtain the amplitude spectrum, and the peak value at 0.025 Hz was taken as the response value corresponding to the interleukin-6 concentration.

[0067] This invention utilizes a magnetic field to control horseradish peroxidase, carried by magnetic beads, to contact the modified enzyme substrate on an electrode surface as needed. Ultra-low levels of horseradish peroxidase are detected using both time-domain and frequency-domain detection methods. In time-domain detection, the enzyme substrate is immobilized on the electrode surface, and the magnetic field controls the enzyme's movement away from and contact with the electrode surface, obtaining a precise response signal shielded from noise. To detect even lower amounts of enzyme, a periodically varying external magnetic field controls the magnetic beads to periodically contact the electrode surface, thereby forming a periodic electrochemical response signal with frequency characteristics. The frequency characteristics are detected from the periodic electrochemical response signal using a weak signal detection method, achieving electrochemical detection of trace amounts of peroxidase or trace analytes on the electrode surface. This detection method can be applied to electrochemical sensing systems using horseradish peroxidase or enzyme-labeled biomarkers as the detection target or electron mediator, improving the detection limit of traditional horseradish peroxidase electrochemical detection methods.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A magnetic field-modulated electrochemical detection method for horseradish peroxidase, characterized in that, Specifically as follows: S1: Horseradish peroxidase is coupled to the surface of magnetic beads, and 3,3',5,5'-tetramethylbenzidine is modified on the surface of the electrode. S2: By applying an external magnetic field, the magnetic bead is controlled to contact or move away from the electrode surface; when the magnetic bead is away from the electrode surface, horseradish peroxidase does not react with 3,3',5,5'-tetramethylbenzidine; when the magnetic bead contacts the electrode surface, horseradish peroxidase reacts with 3,3',5,5'-tetramethylbenzidine. S21: Record the electrochemical response signals generated when the magnetic field is turned on and off, respectively, as the magnetic bead moves away from and contacts the electrode surface; when the magnetic bead contacts the electrode surface, an oxidation signal is generated on the electrode surface; when the magnetic bead moves away from the electrode surface, a reduction signal is generated on the electrode surface, thus obtaining a response signal with noise suppression effect; S22: If the difference between the oxidation and reduction signals obtained in S21 is ≥0.3 μA, then the content of the unknown horseradish peroxidase is calculated based on the correlation between the difference and the horseradish peroxidase concentration; otherwise, the frequency domain detection method is used, as detailed below: By applying a periodically changing magnetic field to control the periodic contact of magnetic beads with the electrode surface, a periodic electrochemical response signal with frequency characteristics is formed. The frequency characteristics are detected from the periodic electrochemical response signal using a weak signal detection method, and the concentration of the target is calculated based on the correlation between the frequency characteristics and the unknown horseradish peroxidase.

2. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, The electrode is a planar photolithography electrode or a screen-printed electrode, and the electrode system is a two-electrode system or a three-electrode system.

3. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, 3,3',5,5'-tetramethylbenzidine was modified on the electrode surface by drop casting, and the concentration of 3,3',5,5'-tetramethylbenzidine in the drop casting solution was 1-20 mg / mL.

4. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, The magnetic bead consists of a magnetic core and a shell surrounding the magnetic core. The magnetic core is made of metal oxide, and the shell is made of polystyrene. The diameter of the magnetic bead is 50 nanometers to 50 micrometers.

5. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, The horseradish peroxidase is coupled to the surface of the magnetic beads via antigen-antibody reaction, complementary pairing of nucleic acid chains, or biotin-avidin.

6. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, In S22, the frequency range of the periodically changing magnetic field is 0.01-100 Hz.

7. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, The weak signal detection method is one or more of the following: spectrum analysis, narrowband filtering, phase-locked amplification, sampling integration, and digital averaging.

8. The magnetic field-modulated electrochemical detection method for horseradish peroxidase according to claim 1, characterized in that, In S22, the periodically changing magnetic field is generated by two coils uniformly and tightly wound around a silicon steel core, located on the upper and lower sides of the electrode. By periodically controlling the relay switch, the upper and lower coils are alternately energized to generate an alternating magnetic field.

9. An immunomarker detection method for horseradish peroxidase based on magnetic field modulation electrochemical detection, characterized in that, Specifically as follows: A first antibody is modified on the surface of magnetic beads, and the target immunomarker is captured using the first antibody to obtain immunomagnetic beads; a second antibody used to capture the target immunomarker is coupled with horseradish peroxidase to obtain a secondary antibody-horseradish peroxidase conjugate; the secondary antibody-horseradish peroxidase conjugate is then coupled to the surface of the immunomagnetic beads; 3,3',5,5'-tetramethylbenzidine is modified on the electrode surface; Since the concentration of horseradish peroxidase is directly proportional to the concentration of the target immune marker, the concentration of the target immune marker can be obtained by detecting the signal of horseradish peroxidase using the magnetic field-modulated electrochemical detection method for horseradish peroxidase as described in any one of claims 1 to 8.

10. The method for detecting immunomarkers according to claim 9, characterized in that, The second antibody is coupled to horseradish peroxidase via biotinylate, glutaraldehyde crosslinking, sodium periodate method, or amide bond; the second antibody-horseradish peroxidase conjugate is coupled to the surface of the immunomagnetic beads by incubation.

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