A magnetic enrichment luminescence sensing device for opaque samples

Abstract

The present invention discloses a magnetic enrichment luminescent sensing device for opaque samples, belonging to the field of bio-optical sensing. The transparent substrate in the device serves as a working electrode; the bottom of the reaction pool is sealed and packaged with the transparent substrate; the lower surface of the transparent substrate is arranged in contact with the annular electromagnetic coil; the photomultiplier tube detector is arranged at the center of the annular electromagnetic coil and is arranged in contact with the lower surface of the transparent substrate; the photomultiplier tube detector and the annular electromagnetic coil form a longitudinal magnetic field, and the functional magnetic beads in the reaction pool are captured to the upper surface of the transparent substrate, and a light signal is obtained through an oxidation-reduction reaction; the top cover of the reaction pool is provided with a reference electrode jack and a counter electrode jack; the reference electrode is inserted into the electrolyte in the reaction pool through the reference electrode jack; the counter electrode is inserted into the electrolyte in the reaction pool through the counter electrode jack. The present invention can achieve high sensitivity and accuracy detection of opaque samples.

Description

Technical Field

[0001] The invention relates to the field of bio-optical sensing, and in particular to a magnetic enrichment luminescent sensing device for opaque samples. Background Art

[0002] In the field of biochemical analysis and medical diagnosis, chemiluminescence technology is widely used to detect target molecules and is the mainstream technology for immunoassays. Among them, functional magnetic beads are used as solid phase extraction materials, playing a key role in sample separation. When used, the target to be detected interacts specifically with the functionalized molecules on the surface of the functional magnetic beads, thereby achieving capture. Then, the functional magnetic beads capture the target to be detected to the substrate interface to achieve signal detection. However, the traditional magnetic enrichment chemiluminescence technology has the following limitations: the magnetic field strength and direction generated by the permanent magnet are fixed, and cannot be flexibly adjusted according to different samples and detection conditions, and it is difficult to adapt to complex and diverse detection needs. The detector and the permanent magnet are located on both sides of the interface, making it difficult to directly apply this technology to the detection of opaque samples (such as blood), limiting its scope of application. Uneven magnetic field distribution may lead to uneven distribution of magnetic beads at the substrate interface, affecting the accuracy and repeatability of the detection. For opaque samples, additional pretreatment steps (such as centrifugation, etc.) are often required to make them transparent, which increases the complexity of the workflow and reduces the detection efficiency.

[0003] Therefore, there is an urgent need to provide a magnetic enrichment chemiluminescent sensing device that can be directly used for opaque samples to overcome the important problems of traditional methods for rapid and easy detection. Summary of the invention

[0004] The purpose of the present invention is to provide a magnetic enrichment luminescence sensing device for opaque samples, which breaks through the limitation that the traditional method can only be used to detect transparent samples. The present invention adopts the unique design of the annular electromagnetic coil, which can flexibly adjust the magnetic field strength to ensure the effective capture of the target. The magnetic field can also be dynamically adjusted to adapt to the changes in the detection; reduce nonspecific adsorption and improve the specificity of the detection; have a good synergistic effect with the detector, and enhance the effect of the detection.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] A magnetic enrichment luminescence sensing device for opaque samples, the device comprising: a transparent substrate, a reaction cell, a reference electrode, a counter electrode, a ring electromagnetic coil and a photomultiplier tube detector;

[0007] The transparent substrate serves as a working electrode; the transparent substrate contacts an electrolyte;

[0008] The reaction pool is used to contain electrolyte and functionalized magnetic beads;

[0009] The bottom of the reaction pool is sealed and packaged using the transparent substrate;

[0010] The lower surface of the transparent substrate is arranged in contact with the annular electromagnetic coil;

[0011] The photomultiplier tube detector is arranged at the center of the annular electromagnetic coil and is arranged in contact with the lower surface of the transparent substrate; the annular electromagnetic coil has a built-in photomultiplier tube detector, and after power is turned on, the photomultiplier tube detector and the annular electromagnetic coil form a longitudinal magnetic field, which pulls the functional magnetic beads in the reaction pool to the upper surface of the transparent substrate and obtains a light signal through a chemical reaction;

[0012] The top cover of the reaction cell is provided with a reference electrode jack and a counter electrode jack; the reference electrode is inserted into the reaction cell through the reference electrode jack; and the counter electrode is inserted into the reaction cell through the counter electrode jack.

[0013] Optionally, the transparent substrate is ITO glass or FTO glass.

[0014] Optionally, the reference electrode is an Ag / AgCl electrode, a saturated calomel electrode, a mercury oxide electrode or a mercurous sulfate electrode.

[0015] Optionally, the counter electrode is a platinum electrode or a graphite rod electrode.

[0016] Optionally, the reference electrode socket and the counter electrode socket are both located directly above the transparent substrate.

[0017] Optionally, the bottom of the reaction pool is sealed and packaged with the upper surface of the transparent substrate via an O-ring.

[0018] Optionally, the transparent substrate is square; the reaction pool is cylindrical; and the reaction pool is tangent to four sides of the transparent substrate.

[0019] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0020] The present invention provides a magnetic enrichment luminescence sensing device for opaque samples, which uses a photomultiplier tube detector (PMT) and a ring electromagnetic coil to form a longitudinal magnetic field, thereby realizing the capture of functional magnetic beads in a reaction pool. When in use, the functionalized magnetic beads can be directly adsorbed to the surface of a transparent substrate, and signal detection is formed at the bottom, effectively avoiding the key technical problem that the top light detection technology in conventional chemiluminescence technology is interfered by opaque samples, and can be directly used for environmental molecule detection such as blood and turbid liquid, greatly improving the detection efficiency. The present invention solves the limitation that the current technology can only be used for transparent sample detection. The device uses a transparent substrate as a working electrode, and the bottom of the reaction pool is sealed and packaged by a transparent substrate. The lower surface of the transparent substrate is connected to the ring electromagnetic coil, and the photomultiplier tube detector is located on the same side and in contact with the lower surface of the transparent substrate. The ring electromagnetic coil has a built-in photomultiplier tube detector. After power is turned on, a longitudinal magnetic field is formed around the reaction pool, which can effectively pull the functional magnetic beads in the reaction pool to the upper surface of the transparent substrate. The chemical reaction is used to generate a light signal, thereby realizing the detection of opaque samples. At the same time, the top cover of the reaction cell is provided with a reference electrode jack and a counter electrode jack, and the reference electrode and the counter electrode are respectively inserted into the electrolyte of the reaction cell through the jacks. The present invention combines the efficient target enrichment ability of functional magnetic beads with the high sensitivity and specificity of luminescence detection, and can quickly and accurately detect target molecules in opaque samples, and has the characteristics of rapidity, simplicity, high sensitivity, and strong specificity. The present invention can have broad application prospects in the fields of biomedical diagnosis, environmental monitoring, and food safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 A schematic diagram of a process flow of a magnetic enrichment luminescence sensing device for opaque samples provided in Example 1 of the present invention;

[0023] Figure 2 It is the electrochemiluminescence intensity-voltage curve;

[0024] Figure 3 It is a schematic diagram of the linear curve for the detection of Abeta protein;

[0025] Figure 4 Schematic diagram of the linear detection curve of phosphorylated tau (p-tau) protein. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The purpose of the present invention is to provide a magnetic enrichment luminescence sensing device for opaque samples, which can achieve high-sensitivity and accurate detection of opaque samples.

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] like Figure 1 As shown, the present invention provides a magnetic enrichment luminescence sensing device for opaque samples, the device comprising: a transparent substrate 1, a reaction cell 2, a reference electrode 7, a counter electrode 8, a ring electromagnetic coil 3 and a photomultiplier tube detector 4;

[0030] The transparent substrate 1 is used as a working electrode; the transparent substrate 1 is conductive glass; the transparent substrate 1 contacts the electrolyte;

[0031] The reaction pool 2 is used to contain electrolyte and functionalized magnetic beads;

[0032] The bottom of the reaction pool 2 is sealed and packaged with the transparent substrate 1;

[0033] The lower surface of the transparent substrate 1 is arranged in contact with the annular electromagnetic coil 3; the magnetic particles in the electrolyte or the analyte labeled with the magnetic particles can be enriched on the surface of the transparent substrate 1 to the greatest extent through the action of the annular electromagnetic coil 3, which has a great improvement on the electrochemical luminescence. When the annular electromagnetic coil 3 is not working, the analyte cannot be adsorbed on the surface of the working electrode, thereby realizing the in-situ renewal of the electrode surface and improving the electrochemical luminescence efficiency.

[0034] The photomultiplier tube detector 4 is arranged at the center of the annular electromagnetic coil 3 and is arranged in contact with the lower surface of the transparent substrate 1 to achieve a distance contact between the photomultiplier tube detector 4 and the transparent substrate 1. The annular electromagnetic coil 3 and the detector 4 are located on the same side of the reaction pool 2.

[0035] The photomultiplier tube detector 4 is arranged at the center of the annular electromagnetic coil 3 and is in contact with the lower surface of the transparent substrate 1; the annular electromagnetic coil 3 has a built-in photomultiplier tube detector 4. After power is turned on, the photomultiplier tube detector 4 and the annular electromagnetic coil 3 form a longitudinal magnetic field, which pulls the functional magnetic beads in the reaction pool 2 to the upper surface of the transparent substrate 1 and obtains a light signal through a chemical reaction.

[0036] The top cover of the reaction cell 2 is provided with a reference electrode jack 5 and a counter electrode jack 6; the reference electrode 7 extends into the electrolyte in the reaction cell 2 through the reference electrode jack 5; the counter electrode 8 extends into the electrolyte in the reaction cell 2 through the counter electrode jack 6.

[0037] The transparent substrate 1 is ITO glass or FTO glass.

[0038] The reference electrode 7 is an Ag / AgCl electrode, a saturated calomel electrode, a mercury oxide electrode or a mercurous sulfate electrode. The counter electrode 8 is a platinum electrode or a graphite rod electrode.

[0039] The reference electrode socket 5 and the counter electrode socket 6 are both located directly above the transparent substrate 1 .

[0040] like Figure 1 As shown, the reaction pool 2 is designed to be cylindrical, the transparent substrate 1 is set to be square, and the bottom of the reaction pool 2 is sealed and packaged with the upper surface of the transparent substrate 1 through an O-type gasket; during chemiluminescence, the transparent substrate 1 can ensure the effective operation of the light source and ensure that the chemical reaction reagent will not flow out of the reaction pool 2. In addition, in this case, the optical transparency of the conductive glass electrode can be fully utilized, and the electrolyte can be fully contacted with the electrode, thereby improving the conductivity and stability, thereby ensuring good luminescence efficiency.

[0041] As a specific embodiment, when working, ITO conductive glass is used as the working electrode, silver / silver chloride is used as the reference electrode 7, and platinum wire electrode is used as the counter electrode 8. The photomultiplier tube detector 4 for detecting the light source is placed in the middle of the annular electromagnetic coil 3 for magnetic enrichment enhanced electrochemical luminescence, and the silver / silver chloride and platinum wire electrodes are respectively inserted into the reference electrode jack 5 and the counter electrode jack 6. The reaction cell 2 is filled with electrolyte, and the ITO conductive glass at the bottom of the reaction cell 2 is completely in contact with the electrolyte. An electrochemical system is constructed based on ITO conductive glass, silver / silver chloride, and platinum wire electrodes, and its application in electrochemical luminescence is investigated. Since the ITO glass is in direct contact with the electrolyte, the annular electromagnetic coil 3 can capture functionalized magnetic particles or analytes labeled with magnetic particles in the electrolyte, thereby realizing bottom detection of electrochemical luminescence.

[0042] The present invention uses magnetic nanoparticles to efficiently enrich target molecules and combines them with highly selective luminescent probe labeling to avoid the key problem that chemiluminescence research cannot be directly carried out on opaque samples and pretreatment such as filtration and centrifugation is required. The present invention is specially designed to solve the signal attenuation and background noise problems encountered in the detection of opaque samples. By optimizing the spatial structure design, the absorption and scattering effects of the sample itself are completely avoided, so that the present invention can be directly applied to the detection of opaque samples such as blood and soil.

[0043] The unique design of the annular electromagnetic coil in this application and its efficient magnetic enrichment greatly improve the sensitivity and accuracy of the detection. At the same time, the layout of the detector and the transparent substrate on one side significantly optimizes the spatial structure, produces a synergistic effect in the detection process, further enhances the detection effect, and provides a new and effective solution for the detection of opaque samples. That is, the introduction and optimization of the annular electromagnetic coil 3 not only improves the enrichment efficiency of magnetic nanoparticles, but also ensures the consistency and repeatability of the detection process. This is crucial to improving the accuracy and reliability of the detection results.

[0044] The implementation of the present invention is simple and easy, and does not require complex equipment or high operating costs. In view of the excellent performance of the present invention in detecting opaque samples, it can not only be used for biomedical diagnosis, but also can be extended to environmental monitoring, food safety testing and other fields, and has broad market potential and social value.

[0045] The following experiments are provided below in conjunction with specific examples to illustrate the main performance of the magnetic enrichment optical sensor device for opaque samples of the present invention, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0046] Example 1: Application of magnetic enrichment electrochemiluminescence sensing technology to detect Abeta in blood

[0047] In this embodiment, the ECL (electrochemiluminescence) detection module includes an electrochemical workstation (CHI 660 E, Chenhua, Shanghai, China) and a diamagnetic PMT module (H9319-02, Hamamatsu Photonics Co., Ltd., Japan).

[0048] Synthesis of functional magnetic beads (MB@SA•Bio-Abeta-Ab1): To prepare the capture MB (MB@SA•Bio-Abeta-Ab1) for Abeta bioassay, 50 μL of biotinylated Abeta antibody (bio-Ab1) prepared in 10 mM PBS was added to 400 μL of 10 mg mL -1 MB@SA (2.6×10 8The particles were pre-treated by sonication and washing with washing buffer. After magnetic separation, they were washed five times with 10 mM PBS and finally suspended in 400 μL of 10 mM PBS. The concentration of ECL MBs was expressed as 10 mg mL MB@SA according to the mass of the material used. -1 (6.50×10 8 particles mL -1 ). The ECL MBs suspension was stored at 4°C and its working suspension was diluted with 10 mM PBS.

[0049] Synthesis of ECL probe (Ru-Abeta-Ab2): ECL probe ruthenium complex (Ru1) was labeled with Ru-Abeta-Ab2 targeting Abeta. 1 mg Ru1PF6 (9.85 × 10 -7 1.0 mL of Abeta-Ab2 solution was prepared by diluting the original solution with 10 mM PBS (pH 7.50) and incubated at 4°C for 12 h with stirring. The ECL probe was purified with a 3kd cutoff membrane (Millipore, 10000 rpm, 2 min) to remove excess Ru1 and stored at 4°C. The concentration of the ECL probe solution (1.20 μM) was expressed as the concentration of the antibody used. The purified ECL probe was diluted with 10 mM PBS to prepare the working solution of the ECL probe.

[0050] Electrochemiluminescence detection: For ECL detection of Abeta in blood, 50 μL 1.0 mg mL −1 MB@SA• Bio-Abeta-Ab1 was added to a 5.0 mL test tube containing 3 mL of a fixed concentration of antigen (Abeta) prepared with 0.10 M PBS (50 mM TPA) buffer and incubated at room temperature for 10 minutes at 50 rpm. Next, 10 μL of ECL probe (0.27 μM Ru-Abeta-Ab2) was added to the above solution and incubated for a further 10 minutes. The mixture was completely transferred to the ECL reaction pool 2, and when the circular electromagnetic coil 3 was working, the MB was magnetically enriched to the surface of the conductive glass electrode within 90 seconds. The ECL measurement was performed on an ECL analyzer with a scan rate of 100 mV / s and a potential range of 0 to -1.60 V. The concentration of the analyte Abeta was quantified by increasing the ECL intensity.

[0051] For Figure 2As shown, the experiment investigated the luminescence intensity of the ECL device of the present invention when the annular electromagnetic coil 3 generates a magnetic field or not. Curve a is the electrochemical luminescence intensity-voltage curve when the annular electromagnetic coil 3 generates a magnetic field, and curve b is the electrochemical luminescence intensity-voltage curve when the annular electromagnetic coil 3 does not generate a magnetic field. The experimental results show that when the annular electromagnetic coil 3 is working, the device of the present invention has the best enhancement effect on the luminescence system, and the luminescence intensity is enhanced by about 10 times, and the luminescence intensity is significantly improved.

[0052] The electrochemiluminescence signal obtained is Figure 3 As shown, as the Abeta antigen concentration increases, the electrochemiluminescence response also increases; in the present invention, the linear range of the electrochemiluminescence intensity and the Abeta antigen concentration is 0.1-500 ug / mL.

[0053] Example 2 Magnetic Enrichment Electrochemical Optical Sensing Technology for Detecting P-tau in Blood

[0054] In this example, some steps in Example 1 were repeated, but the differences were that functionalized magnetic beads (MB@SA•Bio- p-tau-apt) were synthesized, the antibody solution in the synthesis of the ECL probe in Example 1 was replaced with p-tau-Ab1, and the antigen in the electrochemiluminescence detection was replaced with p-tau.

[0055] Synthesis of functionalized magnetic beads (MB@SA•Bio-p-tau apt). TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and binding wash buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 7.50) were added to a 2 mL centrifuge tube containing 400 μL of 10 mg mL -1 MB@SA (2.6×10 8 The captured MB concentration is expressed as 10 mg mL -1 (6.50×10 8 particles mL -1 ).

[0056] The electrochemiluminescence signal obtained is Figure 4 As shown, the linear range of electrochemiluminescence intensity and p-tau antigen concentration is 0.05-800 ug / mL.

[0057] In the electrochemiluminescence technology, the detection method of the present invention can detect a minimum of 0.1ug / mL of Abeta and 0.05ug / mL of p-tau protein. This result shows that the ring-shaped electromagnetic coil 3 structure used in the present invention can directly detect the target with high sensitivity without the need for sample transparency treatment. It is precisely by using the ring-shaped electromagnet of the present invention that the functionalized magnetic beads are aggregated on the electrode surface, and the resulting magnetic enrichment enhancement effect improves the sensitivity of the electrochemiluminescence detection technology by four orders of magnitude.

[0058] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0059] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only used to help understand the method and core technology of the present invention. At the same time, for those skilled in the art, according to the technology of the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A magnetic enrichment luminescence sensing device for opaque samples, characterized in that: The device comprises: a transparent substrate, a reaction cell, a reference electrode, a counter electrode, a ring-shaped electromagnetic coil and a photomultiplier tube detector; The transparent substrate serves as a working electrode; the transparent substrate contacts an electrolyte; The reaction pool is used to contain electrolyte and functionalized magnetic beads; The bottom of the reaction pool is sealed and packaged using the transparent substrate; The lower surface of the transparent substrate is arranged in contact with the annular electromagnetic coil; The photomultiplier tube detector is arranged at the center of the annular electromagnetic coil and is arranged in contact with the lower surface of the transparent substrate; the annular electromagnetic coil has a built-in photomultiplier tube detector, and after power is turned on, the photomultiplier tube detector and the annular electromagnetic coil form a longitudinal magnetic field, which pulls the functional magnetic beads in the reaction pool to the upper surface of the transparent substrate and obtains a light signal through a chemical reaction; The top cover of the reaction cell is provided with a reference electrode jack and a counter electrode jack; the reference electrode is inserted into the reaction cell through the reference electrode jack; the counter electrode is inserted into the reaction cell through the counter electrode jack; The reference electrode socket and the counter electrode socket are both located directly above the transparent substrate.

2. A magnetic enrichment luminescence sensing device for opaque samples according to claim 1, characterized in that: The transparent substrate is ITO glass or FTO glass.

3. A magnetic enrichment luminescence sensing device for opaque samples according to claim 1, characterized in that: The reference electrode is an Ag / AgCl electrode, a saturated calomel electrode, a mercury oxide electrode or a mercurous sulfate electrode.

4. A magnetic enrichment luminescence sensing device for opaque samples according to claim 1, characterized in that: The counter electrode is a platinum electrode or a graphite rod electrode.

5. A magnetic enrichment luminescence sensing device for opaque samples according to claim 1, characterized in that: The bottom of the reaction pool is sealed and packaged with the upper surface of the transparent substrate through an O-ring.

6. A magnetic enrichment luminescence sensing device for opaque samples according to any one of claims 1 to 5, characterized in that: The transparent substrate is square; the reaction pool is cylindrical; and the reaction pool is tangent to four sides of the transparent substrate.

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