Quantum method for alpha fetoprotein detection

Abstract

The invention relates to a quantum method for alpha fetoprotein detection, and belongs to the technical field of biosensing and quantum. The invention provides an alpha fetoprotein sensing strategy based on nano-catalysis enhanced quantum detection aiming at the limitation of a traditional quantum detection method on the magnetism of a target molecule. The method comprises the following steps: modifying alpha fetoprotein on a substrate; the method comprises the following steps: modifying a quantitative FND (at) Au-Pd compound onto alpha fetoprotein; the excessive FND (at) Au-Pd compound which is not combined is collected; catalyzing hydrogen peroxide to generate hydroxyl radicals by using the catalyst; measuring the magnetic noise of the hydroxyl radicals through the NV color center quantum relaxation time (T1); and determining the alpha fetoprotein concentration according to the magnetic noise variation. The invention breaks through the limitation of the traditional magnetic resonance technology, realizes high-sensitivity and high-specificity alpha fetoprotein detection, and is suitable for clinical scenes such as early diagnosis of liver cancer and the like.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and quantum, specifically relating to a quantum method for detecting alpha-fetoprotein. Background Technology

[0002] Nitrogen-vacancy (NV)-centered nanodiamonds possess significant spin quantum state characteristics. NV centers exhibit millisecond-level spin coherence times at room temperature, enabling them to stably capture weak signals in complex biological environments. Spin relaxation time (T1) detection technology based on NV centers achieves all-optical quantum sensing without microwave excitation, a technological breakthrough demonstrating significant application value in biological systems. The signal transduction of NV centers essentially relies on local magnetic field perturbations generated by the target molecule. This mechanism shows significant inadequacy for non-magnetic biomolecules (such as over 80% of protein biomarkers). Achieving high-fidelity quantum detection of non-magnetic biomolecules has become a critical technological barrier to be overcome in this field. Alpha-fetoprotein (AFP) is a crucial and specific tumor marker for primary hepatocellular carcinoma. Significantly elevated AFP levels strongly indicate the risk of liver cancer, testicular cancer, ovarian cancer, etc. Close monitoring of AFP is beneficial for the early diagnosis, early treatment, and early recovery of related cancers.

[0003] Chinese patent CN202011414549.7 introduces a single-spin quantum diamond precision magnetic measurement system, which generates microwaves through a microwave module and accurately radiates them onto the sample to achieve quantitative and non-destructive magnetic detection. Chinese patent CN202210627312.X reports a magnetic detection method for biomolecular interactions, which uses superparamagnetic particles to label biomolecular interaction pairs in biological samples, achieving high sensitivity, low background, high stability, and high specificity for detecting biomolecular interactions. However, despite the introduction of superparamagnetic particles, the cytotoxicity and immunogenicity risks associated with such paramagnetic materials severely limit their application potential in in vivo research and clinical practice. Therefore, there is an urgent need for a quantum detection method for alpha-fetoprotein (AFP) without relying on exogenous magnetic labeling. Summary of the Invention

[0004] To address the aforementioned issues, this invention discloses a quantum method for detecting alpha-fetoprotein (AFP). Plasma nanodiamonds catalyze hydrogen peroxide to generate hydroxyl radicals, and non-magnetic biomolecule detection is achieved through changes in relaxation time (T1).

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: A quantum method for detecting alpha-fetoprotein includes the following steps: (1) Modify the alpha-fetoprotein probe onto the gold substrate; (2) Alpha-fetoprotein was modified onto a gold substrate; (3) The quantitative plasma nanodiamond complex (FND@Au-Pd) was modified onto the alpha-fetoprotein, and then the unbound FND@Au-Pd complex was washed away. (4) Collect unbound FND@Au-Pd complexes from step (3) after washing; (5) The FND@Au-Pd complex collected in step (4) is used as a catalyst to catalyze hydrogen peroxide into hydroxyl radicals (•OH) during the catalytic process; (6) Measure the magnetic noise generated by the •OH through the quantum relaxation time (T1) of the NV color center; (7) Determine the concentration of alpha-fetoprotein based on the change in the magnetic noise.

[0006] The total amount of plasma nanodiamond composite (FND@Au-Pd) remains constant. A portion of it is used for the modification of alpha-fetoprotein in step (3). After modification, the unbound composite is removed by a washing step. These remaining unbound FND@Au-Pd composites are then used to catalyze the hydrogen peroxide reaction. Their molar concentration directly determines the relaxation time of the magnetic noise generated by ·OH measured by quantum relaxation time (T1) in step (6). Finally, the concentration of alpha-fetoprotein in the sample can be quantitatively determined based on the change in the magnetic noise relaxation time.

[0007] Furthermore, the preparation method of FND@Au-Pd in ​​step (3) includes the following steps: Mix 5-15 mL of palladium nitrate (Pd(NO3)2, 1 mM), 1-10 mL of chloroauric acid (HAuCl4, 0.5 mM), and 10 mL of NH2OH·HCl together and stir for 20-60 min to obtain Au-Pd complex. After surface carboxylation treatment, 50-100 nm nanodiamonds (FND) containing NV color centers are modified with polyethyleneimine (PEI) through covalent interaction to obtain FND-PEI composite materials. Au-Pd complexes are combined with FND-PEI by electrostatic adsorption to form FND@Au-Pd complexes.

[0008] Furthermore, in step (5), the concentration of hydrogen peroxide is 10-50 mM, the catalytic reaction is carried out at a pH of 6-8, the temperature of the catalytic reaction is 25-30°C, and the time of the catalytic reaction is 5-15 minutes.

[0009] Furthermore, the measurement of the quantum relaxation time (T1) of the NV color center in step (6) includes the following steps: (1) The NV color center is excited using a laser with a wavelength of 532 nm; (2) Collect red fluorescence signals at wavelengths of 637~800nm; (3) The NV color center is manipulated using microwave pulses at a frequency of 2.87 GHz; (4) Measurements were performed within a magnetic field range of 0 to 60 Gauss; (5) The quantum relaxation time of the NV color center was measured at room temperature.

[0010] Furthermore, the molar ratio of the FND@Au-Pd complex in step (3) to step (4) is 4:1 to 4:3.

[0011] The beneficial effects of this invention are as follows: 1. This invention provides a quantum method for detecting alpha-fetoprotein (AFP), which can be used to detect non-magnetic cancer biomarkers (such as AFP). Traditional magnetic resonance (MR) detection typically relies on the paramagnetism of target molecules (such as metal ions and free radicals). This method ingeniously utilizes the strongly oxidizing ·OH generated by catalysis as a "molecular probe," converting information about non-magnetic biomolecules (presence, concentration, conformational changes, etc.) into measurable signals affecting the relaxation time of quantum sensors through chemical reactions. This overcomes the limitations of traditional MR technology on the magnetism of target molecules, greatly expanding the range of detectable biomolecules.

[0012] 2. This invention provides a quantum method for the detection of alpha-fetoprotein (AFP), employing plasma-enhanced nanodiamond (FND@Au-Pd) with NV color centers as a catalyst. The FND@Au-Pd consists of spherical Au-Pd nanoparticles (50–70 nm in shape) encapsulating spherical nanodiamond (FND) particles (90–120 nm in size). As a highly efficient catalyst, FND@Au-Pd significantly improves the efficiency and local concentration of hydroxyl radicals (·OH) generated by the decomposition of hydrogen peroxide. The physicochemical changes in the microenvironment caused by the ·OH reaction (such as local pH, ionic strength, and paramagnetic substance concentration) subsequently affect the relaxation time of the quantum sensor.

[0013] 3. This invention provides a quantum method for the detection of alpha-fetoprotein (AFP), in which the total amount of plasma nanodiamond composite (FND@Au-Pd) is kept constant. A portion of it is used to modify AFP. After modification, the unbound composite is removed by a washing step. The remaining unbound FND@Au-Pd composite is then used to catalyze a hydrogen peroxide reaction, and its molar concentration directly determines the quantum relaxation time (T1) of the relaxation time of the magnetic noise generated by ·OH. Finally, based on the change in this magnetic noise relaxation time, the concentration of AFP in the sample can be quantitatively determined. Attached Figure Description

[0014] Figure 1This is a schematic diagram of the quantum method for alpha-fetoprotein detection according to the present invention.

[0015] Figure 2 This is a comparison graph of the experimental results of the examples and the comparative examples. Detailed Implementation

[0016] The following description, in conjunction with the accompanying drawings and specific embodiments, further clarifies the quantum method for alpha-fetoprotein detection according to the present invention. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0017] Detection steps: (1) Modify the alpha-fetoprotein probe onto the gold substrate; (2) Modify the alpha-fetoprotein onto the gold substrate; (3) Modify the alpha-fetoprotein onto the quantitative plasma nanodiamond complex (FND@Au-Pd), and then wash the unbound FND@Au-Pd complex; (4) Collect the unbound FND@Au-Pd complex after washing in step (3); (5) Use the FND@Au-Pd complex collected in step (4) as a catalyst to catalyze hydrogen peroxide into hydroxyl radicals (•OH) during the catalytic process; (6) Measure the magnetic noise generated by the •OH by the quantum relaxation time (T1) of the NV color center; (7) Determine the concentration of the alpha-fetoprotein based on the change in the magnetic noise.

[0018] The preparation method of FND@Au-Pd includes the following steps: 5 mL of palladium nitrate (Pd(NO3)2, 1 mM), 1 mL of chloroauric acid (HAuCl4, 0.5 mM), and 10 mL of NH2OH·HCl are mixed together and stirred for 20 min to obtain an Au-Pd composite. After surface carboxylation treatment, 50 nm nanodiamonds (FND) containing NV centers are covalently modified with polyethyleneimine (PEI) to obtain an FND-PEI composite material. The Au-Pd composite and FND-PEI are then combined using electrostatic adsorption to finally form the FND@Au-Pd composite.

[0019] The concentration of hydrogen peroxide is 10 mM, the catalytic reaction is carried out at a pH of 6, the temperature of the catalytic reaction is 25°C, and the time of the catalytic reaction is 5 min.

[0020] The quantum relaxation time (T1) measurement of the NV color center includes the following steps: (1) exciting the NV color center with a laser with a wavelength of 532 nm; (2) collecting a red fluorescence signal with a wavelength of 637 nm; (3) manipulating the NV color center with a microwave pulse with a frequency of 2.87 GHz; (4) measuring in a magnetic field range of 0 to 60 Gauss; and (5) measuring the quantum relaxation time of the NV color center at room temperature. Example

[0021] Detection steps: (1) Modify the alpha-fetoprotein probe onto the gold substrate; (2) Modify the alpha-fetoprotein onto the gold substrate; (3) Modify the alpha-fetoprotein onto the quantitative plasma nanodiamond complex (FND@Au-Pd), and then wash the unbound FND@Au-Pd complex; (4) Collect the unbound FND@Au-Pd complex after washing in step (3); (5) Use the FND@Au-Pd complex collected in step (4) as a catalyst to catalyze hydrogen peroxide into hydroxyl radicals (•OH) during the catalytic process; (6) Measure the magnetic noise generated by the •OH by the quantum relaxation time (T1) of the NV color center; (7) Determine the concentration of the alpha-fetoprotein based on the change in the magnetic noise.

[0022] The preparation method of FND@Au-Pd includes the following steps: 10 mL of palladium nitrate (Pd(NO3)2, 1 mM), 5 mL of chloroauric acid (HAuCl4, 0.5 mM), and 10 mL of NH2OH·HCl are mixed together and stirred for 40 min to obtain an Au-Pd composite. After surface carboxylation treatment, 80 nm nanodiamonds (FND) containing NV centers are covalently modified with polyethyleneimine (PEI) to obtain an FND-PEI composite material. The Au-Pd composite and FND-PEI are then combined using electrostatic adsorption to finally form the FND@Au-Pd composite.

[0023] The concentration of hydrogen peroxide is 10 mM, the catalytic reaction is carried out at a pH of 7, the temperature of the catalytic reaction is 30°C, and the time of the catalytic reaction is 10 min.

[0024] The quantum relaxation time (T1) measurement of the NV color center includes the following steps: (1) exciting the NV color center with a laser with a wavelength of 532 nm; (2) collecting a red fluorescence signal with a wavelength of 750 nm; (3) manipulating the NV color center with a microwave pulse with a frequency of 2.87 GHz; (4) measuring in a magnetic field range of 0 to 60 Gauss; and (5) measuring the quantum relaxation time of the NV color center at room temperature. Example

[0025] Detection steps: (1) Modify the alpha-fetoprotein probe onto the gold substrate; (2) Modify the alpha-fetoprotein onto the gold substrate; (3) Modify the alpha-fetoprotein onto the quantitative plasma nanodiamond complex (FND@Au-Pd), and then wash the unbound FND@Au-Pd complex; (4) Collect the unbound FND@Au-Pd complex after washing in step (3); (5) Use the FND@Au-Pd complex collected in step (4) as a catalyst to catalyze hydrogen peroxide into hydroxyl radicals (•OH) during the catalytic process; (6) Measure the magnetic noise generated by the •OH by the quantum relaxation time (T1) of the NV color center; (7) Determine the concentration of the alpha-fetoprotein based on the change in the magnetic noise.

[0026] The preparation method of FND@Au-Pd includes the following steps: 15 mL of palladium nitrate (Pd(NO3)2, 1 mM), 10 mL of chloroauric acid (HAuCl4, 0.5 mM), and 10 mL of NH2OH·HCl are mixed and stirred for 60 min to obtain an Au-Pd composite. After surface carboxylation treatment, 100 nm nanodiamonds (FND) containing NV centers are covalently modified with polyethyleneimine (PEI) to obtain an FND-PEI composite material. The Au-Pd composite and FND-PEI are then combined using electrostatic adsorption to finally form the FND@Au-Pd composite.

[0027] The concentration of hydrogen peroxide is 50 mM, the catalytic reaction is carried out at a pH of 8, the temperature of the catalytic reaction is 30°C, and the time of the catalytic reaction is 15 min.

[0028] The quantum relaxation time (T1) measurement of the NV color center includes the following steps: (1) exciting the NV color center with a laser with a wavelength of 532 nm; (2) collecting a red fluorescence signal with a wavelength of 800 nm; (3) manipulating the NV color center with a microwave pulse with a frequency of 2.87 GHz; (4) measuring in a magnetic field range of 0 to 60 Gauss; and (5) measuring the quantum relaxation time of the NV color center at room temperature.

[0029] Example 4 (Comparative Example) Detection steps: (1) Add the samples from the commercial ELISA kit to the cuvettes; (2) Record the absorbance value A1 at 10s; (3) Quickly place in a 37℃ water bath for 3min, remove and quickly wipe dry, then measure the absorbance value A2 at 3min 10s; (4) Calculate A = A2 - A1; (5) Based on the amount of alpha-fetoprotein in the commercial ELISA kit, the concentration of alpha-fetoprotein in the sample can be determined. Experimental results are as follows: Figure 2As shown.

[0030] The above embodiments illustrate in detail a quantum method for the detection of alpha-fetoprotein. By comparing the actual concentration with the concentration detected by commercial ELISA kits, the method in Example 1 is deemed optimal.

[0031] The detection method described in this application overcomes the inherent limitations of traditional quantum sensing systems in detecting non-magnetic biomarkers without relying on external magnetic labeling. It enables precise detection of substances such as alpha-fetoprotein, and the proposed method has broad application prospects in quantum measurement of non-magnetic molecules.

[0032] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A quantum method for detecting alpha-fetoprotein, characterized in that: Includes the following steps: (1) Modify the alpha-fetoprotein probe onto the gold substrate; (2) Alpha-fetoprotein was modified onto a gold substrate; (3) The quantitative plasma nanodiamond composite FND@Au-Pd was modified onto the alpha-fetoprotein, and then the unbound FND@Au-Pd composite was washed away. (4) Collect unbound FND@Au-Pd complexes from step (3) after washing; (5) The FND@Au-Pd complex collected in step (4) is used as a catalyst to catalyze hydrogen peroxide into hydroxyl radicals (•OH) during the catalytic process; (6) Measure the magnetic noise generated by the •OH through the quantum relaxation time (T1) of the NV color center; (7) Determine the concentration of alpha-fetoprotein based on the change in the magnetic noise.

2. The quantum method for detecting alpha-fetoprotein according to claim 1, characterized in that: The preparation method of FND@Au-Pd in ​​step (3) includes the following steps: Mix 5-15 mL of palladium nitrate (Pd(NO3)2, 1 mM), 1-10 mL of chloroauric acid (HAuCl4, 0.5 mM), and 10 mL of NH2OH·HCl together and stir for 20-60 min to obtain Au-Pd complex. After surface carboxylation treatment, 50-100 nm nanodiamonds containing NV color centers (FND) are modified with polyethyleneimine (PEI) through covalent interaction to obtain FND-PEI composite material. Au-Pd complexes are combined with FND-PEI by electrostatic adsorption to form FND@Au-Pd complexes.

3. The quantum method for detecting alpha-fetoprotein according to claim 1, characterized in that: In step (5), the concentration of hydrogen peroxide is 10-50 mM, the catalytic reaction is carried out at a pH of 6-8, the temperature of the catalytic reaction is 25-30°C, and the time of the catalytic reaction is 5-15 minutes.

4. The quantum method for detecting alpha-fetoprotein according to claim 1, characterized in that: Step (6) involves measuring the quantum relaxation time (T1) of the NV color center, which includes the following steps: (1) The NV color center is excited using a laser with a wavelength of 532 nm; (2) Collect red fluorescence signals at wavelengths of 637~800nm; (3) The NV color center is manipulated using microwave pulses at a frequency of 2.87 GHz; (4) Measurements were performed within a magnetic field range of 0 to 60 Gauss; (5) The quantum relaxation time of the NV color center was measured at room temperature.

5. The quantum method for detecting alpha-fetoprotein according to claim 1, characterized in that: The molar ratio of the FND@Au-Pd complex in steps (3) and (4) is 4:1 to 4:3.

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