Quantum sensing method for cross-class joint detection of tumor markers

Abstract

The invention discloses a quantum sensing method for cross-class combined detection of tumor markers, which utilizes the quantum optical characteristics of an NV color center in a fluorescent nano-diamond as sensing signals to realize cross-class combined detection of two mainstream markers (miRNA and protein) in diagnosis of tumor diseases. The surface of the nano-diamond is functionally modified, and the charge state proportion situation of the NV color center is presented in a spectrum shape, so that the miRNA is detected. By constructing the magnetized gel, the detection of protein markers can be realized by utilizing an optical detection magnetic resonance (ODMR) signal of an NV color center. According to the method, on the basis that NV color center quantum has multiple optical signal characteristics, sensitive detection of miRNA-155 and MUC1 proteins is realized. The two sensing signals of the method are unisource and heterogeneous, so that the two sensing signals do not interfere with each other, and the method is suitable for sensitive and accurate detection of early tumor disease markers.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically involving a quantum sensing method for cross-class joint detection of two mainstream tumor markers (miRNA and protein). Background Technology

[0002] Early cancer diagnosis significantly increases the likelihood of effective treatment and is of paramount importance in saving cancer patients' lives. Therefore, extensive research has focused on developing and exploring technologies and methods for early cancer diagnosis. Currently, various methods exist for detecting tumor biomarkers, including electrochemical methods, surface-enhanced Raman scattering, photoluminescence, and fluorescence analysis. Among these, fluorescence analysis has attracted considerable attention due to its advantages of high sensitivity, accuracy, and ease of operation. However, a single cancer often corresponds to multiple biomarkers, and the expression levels of different biomarkers are closely related to the progression of the tumor. Therefore, developing combined detection technologies targeting mainstream tumor biomarkers (such as miRNAs and proteins) is of significant practical importance for cancer diagnosis and treatment.

[0003] Chinese invention patent CN202510151196.2 discloses a method for detecting multiplex miRNAs based on deoxyribonuclease or CRISPR / Cas12a and its applications. This method leverages the high specificity of ligase recognition to achieve highly specific detection of miRNAs and can simultaneously detect multiple miRNAs by reading changes in multi-channel fluorescence values. However, limited by its signal generation mechanism, this method cannot achieve cross-category joint detection of miRNAs and proteins, resulting in limitations in the scope of applicable diseases and diagnostic value. Furthermore, this method analyzes only a single dimension of fluorescence signal, making it difficult to avoid complementary signal interference generated when different biomarkers are detected separately, thus affecting the final accuracy of the detection results. Summary of the Invention

[0004] To address the aforementioned issues, this invention discloses a quantum sensing method for cross-class joint detection of tumor markers, enabling simultaneous cross-class detection of two core biomarkers for tumor diseases (microRNA and protein). Diamond nitrogen-vacancy (NV) centers, as luminescent defect structures within diamond particles, possess advantages such as high quantum yield, stable luminescence properties, and low cytotoxicity. This novel quantum system can achieve spin polarization and fluorescence signal readout via laser and achieve precise spin state control using microwaves. Given the high sensitivity of NV centers to environmental disturbances (stress, magnetic fields, electric fields, temperature, etc.), they have a natural advantage in the field of physical quantity quantum sensing. Utilizing the single-source heterogeneity of the NV center quantum signal, the sensing signals corresponding to the two biomarkers are completely independent in dimension, effectively avoiding cross-interference during the detection process and providing a core guarantee for the high detection accuracy of this method.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: A quantum sensing method for cross-class joint detection of tumor markers involves pre-preparing a functional gel loaded with functionalized nanodiamonds, with probe-modified magnetic particles adapted to the surface of the functional gel, and finally placing the functional gel on a microwave antenna so that the spin state of the NV color center is modulated by microwaves, thereby generating an ODMR signal.

[0006] When the target miRNA is present in the analyte, the cDNA modified on the surface of the functionalized nanodiamond gel specifically binds to the miRNA and detaches from the diamond particle surface, leading to NV in the ensemble NV center. 0 The change in the proportion of NV⁻ ​​states leads to a change in the shape of the fluorescence spectrum of the NV color center, which is then used to detect miRNAs via a fluorescence collection device. When the target protein is present in the analyte, pulsed microwaves are radiated through a microwave antenna. The probe-modified magnetic particles on the surface of the functionalized nanodiamond gel undergo a chain displacement reaction with the protein and detach from the gel surface, causing a change in the magnetic field strength around the NV color center. This leads to a change in the degree of ODMR signal splitting, and the protein is detected by a fluorescence collection device.

[0007] Furthermore, the method for preparing the probe-type magnetic particles includes the following steps: Take 50-100 μL of streptavidin-encapsulated magnetic bead solution (10 mg / mL) with a diameter in the range of 10-500 nm, dilute it in 1 mL of deionized water, add biotin-modified aptamer probes at the 3' end, incubate for 4-6 hours, centrifuge, wash and resuspend in 100-400 μL of deionized water to obtain probe-modified magnetic particle solution.

[0008] Furthermore, the preparation method of the functionalized nanodiamond includes the following steps: S1. Preparation of fluorescent nanodiamonds with polyethyleneimine surface modification: Take 50-100 μL of surface-carboxylated fluorescent nanodiamonds with a diameter in the range of 20-800 nm, dilute in 1 mL of deionized water, add 1-3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1-3 mg of N-hydroxysuccinimide, mix well and let stand for 1-3 hours, then add 1-10 mg of polyethyleneimine, incubate at room temperature for 4-6 hours, centrifuge and wash three to five times, and resuspend in 200-500 μL of deionized water to obtain a fluorescent nanodiamond solution with polyethyleneimine surface modification. S2. Take 10-100 μL of fluorescent nanodiamond solution with polyethyleneimine surface modified with 1-2 mL of deionized water, add 10-100 μL of cDNA solution with a concentration in the range of 0.1-100 μM, and incubate on a shaker for 3-5 hours. Wash three to five times using a magnet to obtain the functionalized nanodiamond solution.

[0009] Furthermore, the preparation method of the functional gel includes the following steps: S1. Prepare a matrix gel with good light transmittance and porosity; dissolve 5-15 mg of polyvinyl alcohol in 100 mL of deionized water, heat and stir at 80-95 °C for 3-5 hours, then add 0.5-3 mg of polyethylene glycol and stir at 80-95 °C for 1-3 hours to obtain the basic gel; S2. Prepare a matrix gel loaded with functional nanodiamonds; take 10-100 μL of the prepared functionalized nanodiamond solution, disperse and dilute it in 10-20 mL of the prepared base gel with good light transmittance and porosity, heat and stir at 80-95℃ for 5-8 hours to obtain a matrix gel loaded with functional nanodiamonds. S3. Prepare a matrix gel modified with a magnetic particle probe; take 10-100 μL of DNA probe with an amino group modified at the 3' end at a concentration of 0.1-100 μM and dilute it in 10-20 mL of matrix gel loaded with functional nanodiamonds, stir at 25-30℃ for 3-5 hours to obtain a matrix gel modified with a magnetic particle probe. S4. Prepare functional gel loaded with probe-magnetic particles; take 10-200 μL of functional gel and drop it onto a glass slide, cure it at -5-5℃ for 0.5-2 hours, then drop 50-200 μL of probe-magnetic particle solution onto the gel surface, incubate for 1-3 hours, and rinse the surface with deionized water 3-5 times to obtain functional gel loaded with probe-magnetic particles.

[0010] The quantum sensing method for cross-class joint detection of tumor markers described in this invention requires a fluorescence collection device, which includes an optical lens, a spectrometer, and a single-photon counter; it is used to excite NV color centers and collect fluorescence information, recording the optical information of NV color centers in both spectral and fluorescence intensity forms. The probe-modified magnetic particles of the present invention are used to construct magnetic fields of different intensities and are modified with aptamer probes on their surface. In an environment with MUC1 protein, they bind to and detach from the protein, causing changes in the regional magnetic field strength and thus changing the ODMR signal of the NV color center. The functionalized nanodiamonds described in this invention are used to detect miRNAs. The surface of the particles is specially modified to change the charge population of the ensemble NV center. In the environment where the miRNA to be tested is present, the proportion of zero and negative states of the ensemble NV center changes, thereby changing the shape of the collected fluorescence spectrum. The functional gel described in this invention is used to support and immobilize fluorescent nanodiamonds and probe-magnetized particles. The gel has good light transmittance and porosity, which facilitates the optical reading of sensing signals and the capture of free analytes. The microwave antenna described in this invention is used to radiate microwaves and construct ODMR sensing signals by recording the fluorescence intensity of NV color centers at different microwave frequencies.

[0011] The beneficial effects of this invention are as follows: 1. High-sensitivity detection: This invention utilizes the high sensitivity of NV color centers to environmental charges and magnetic field strength to achieve ultrasensitive detection of cancer biomarkers (miRNA and protein), with a detection limit reaching the fM level (10⁻¹). 5 (mol / L), which is significantly better than traditional fluorescence detection technology.

[0012] 2. Cross-class Joint Detection and Anti-interference: Based on the multi-dimensional signal characteristics of NV color centers, this invention achieves for the first time cross-class simultaneous detection of two mainstream tumor markers (nucleic acid miRNA and protein markers). Since the detection signals of the two markers originate from the responses of the NV color centers to charge (miRNA detection) and magnetic field (protein detection), respectively, the signal dimensions are completely independent, eliminating the risk of cross-interference. This fundamentally avoids the signal complementarity errors that may occur in traditional single-dimensional detection, significantly improving the accuracy and reliability of cancer diagnosis. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of a quantum sensing method for cross-category joint detection of tumor markers according to the present invention.

[0014] List of identifiers in attached diagrams: 1. Fluorescence collection device; 2. Probe-modified magnetic particles; 3. Functionalized nanodiamonds; 4. Functionalized gel; 5. Microwave antenna.

[0015] Figure 2 This is a diagram showing the miRNA detection effect of the method of the present invention.

[0016] Figure 2-1 2-2 represent different concentrations of miRNA-155 (10) -13 10 -12 10 -11 10 -10 and 10 -9Fluorescence and ODMR spectra detected at mol / L.

[0017] Figure 3 This is a diagram showing the MUC1 detection effect of the method of the present invention.

[0018] Figure 3-1 Figures 3 and 3-2 show the ODMR and fluorescence spectra detected at different concentrations of MUC1 (0.1, 1, 10, 100, and 1000 ng / mL), respectively. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. 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.

[0020] Example 1: Combined detection of miRNA-155 and MUC1 I. Experimental Preparation 1. Preparation of Functionalized Nanodiamonds S1: Take 50 μL of surface-carboxylated fluorescent nanodiamond with a diameter of 50 nm (concentration of 10 mg / mL), dilute it in 1 mL of deionized water, add 2 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mg of N-hydroxysuccinimide (NHS), mix well and let stand for 2 hours.

[0021] S2: Add 5 mg of polyethyleneimine (PEI), incubate at room temperature for 5 hours, centrifuge and wash 3 times (8000 rpm, 5 minutes), and resuspend in 300 μL of deionized water to obtain a PEI-modified nanodiamond solution.

[0022] S3: Take 50 μL of the above solution, add 2 mL of deionized water to dilute, then add 50 μL of 10 μM miRNA-155 complementary cDNA solution, incubate on a shaker at 100 rpm for 4 hours, wash 3 times using a magnet, and resuspend in 200 μL of deionized water to obtain a functionalized nanodiamond solution.

[0023] Preparation of functional gels S1: Dissolve 10 mg of polyvinyl alcohol (PVA) in 100 mL of deionized water, heat and stir at 90°C for 4 hours, add 1 mg of polyethylene glycol (PEG), and continue stirring for 2 hours to obtain the basic gel.

[0024] S2: Take 80 μL of functionalized nanodiamond solution, disperse it in 15 mL of base gel, heat and stir at 90℃ for 6 hours to obtain a matrix gel loaded with nanodiamond.

[0025] S3: Take 50 μL of 10 μM 3' amino-modified DNA probe (used to immobilize magnetic particle probes), add it to 15 mL of matrix gel, stir at 25℃ for 4 hours to obtain gel modified with magnetic particle probes.

[0026] S4: Take 80 μL of streptavidin magnetic bead solution (10 mg / mL) with a diameter of 200 nm, dilute it in 1 mL of deionized water, add 3' biotin-modified MUC1 aptamer probe (10 μM), incubate for 5 hours, centrifuge and wash (5000 rpm, 3 min), and resuspend in 200 μL of deionized water to obtain probe-modified magnetic particle solution.

[0027] S5: Take 100 μL of gel modified with magnetic particle probe and drop it onto a glass slide. Cure at 0℃ for 1 hour. Add 100 μL of probe-modified magnetic particle solution and incubate for 2 hours. Rinse three times with deionized water to obtain the functional gel.

[0028] II. Detection Experiment 1. miRNA detection experiment: Different concentrations of miRNA-155 (10...) were used to detect miRNA. -13 10 -12 10 -11 10 -10 and 10 -9 The prepared solution (mol / L) was incubated with the functional gel for 30 minutes.

[0029] 2. Protein detection experiment: The prepared solutions containing different concentrations of MUC1 (0.1, 1, 10, 100 and 1000 ng / mL) were incubated with the functional gel for 30 minutes.

[0030] 3. Signal Acquisition: A fluorescence collection device (excitation wavelength 532 nm) was used to record the fluorescence spectra of the NV centers, with a focus on analyzing the zero state (NV). 0 The ratio of the fluorescence peak intensity of the positive state (NV⁻) to that of the negative state (NV⁻).

[0031] When miRNA-155 binds to and detaches from cDNA on the nanodiamond surface, the proportion of NV⁻ ​​states increases, and the intensity of the 638 nm peak (NV⁻) in the spectrum increases relative to the 575 nm peak (NV⁻). 0 () significantly reduced.

[0032] A microwave antenna radiates 2-3 GHz pulsed microwaves, and the fluorescence intensity of NV color centers at different frequencies is recorded to construct ODMR spectra.

[0033] When the MUC1 protein binds to and detaches from the aptamer on the surface of the magnetic particle, the local magnetic field strength of the magnetic particle decreases, and the splitting distance (Δf) of the ODMR signal decreases.

[0034] III. Experimental Results 1. Figure 2 To obtain test results by only changing the concentration of miRNA in the prepared solution, Figure 2-1 Figures 2 and 2-2 show the fluorescence spectra and ODMR spectra at various corresponding concentrations, respectively. As the miRNA concentration increases, the shape of the fluorescence spectrum changes, with a peak at 575 nm (NV). 0 The corresponding increase was observed in the ODMR. Meanwhile, the degree of ODMR splitting did not change significantly.

[0035] 2. Figure 3 To change the test results by only altering the concentration of MUC1 in the prepared solution, Figure 3-1 Figures 3 and 3-2 show the ODMR and fluorescence spectra at various corresponding concentrations, respectively. As the concentration of MUC1 increases, the degree of splitting in the ODMR spectrum changes, with the bimodal splitting frequency decreasing accordingly. Meanwhile, the shape of the fluorescence spectrum does not change significantly.

[0036] 3. The combined results of the two experiments show that the two signals of the method of the present invention can respond independently to the corresponding markers, and there is no complementarity or interference between them.

[0037] 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 sensing method for cross-class joint detection of tumor markers, characterized in that: A functional gel loaded with functionalized nanodiamonds was prepared in advance, and probe-modified magnetic particles were adapted to the surface of the functional gel. Finally, the functional gel was placed on a microwave antenna so that the spin state of the NV color center was modulated by microwaves, thereby generating an ODMR signal. When the target miRNA is present in the analyte, the cDNA modified on the surface of the functionalized nanodiamond gel specifically binds to the miRNA and detaches from the diamond particle surface, leading to NV in the ensemble NV center. 0 The change in the proportion of NV⁻ ​​states leads to a change in the shape of the fluorescence spectrum of the NV color center, which is then used to detect miRNAs via a fluorescence collection device. When the target protein is present in the analyte, pulsed microwaves are radiated through a microwave antenna. The probe-modified magnetic particles on the surface of the functionalized nanodiamond gel undergo a chain displacement reaction with the protein and detach from the gel surface, causing a change in the magnetic field strength around the NV color center. This, in turn, leads to a change in the degree of ODMR signal splitting, and the protein is detected by a fluorescence collection device.

2. The quantum sensing method for cross-class joint detection of tumor markers according to claim 1, characterized in that: The method for preparing the probe-magnetized particles includes the following steps: Take 50-100 μL of streptavidin-encapsulated 10 mg / mL magnetic beads with diameters in the range of 10-500 nm, dilute them in 1 mL of deionized water, add biotin-modified aptamer probes at the 3' end, incubate for 4-6 hours, centrifuge, wash, and resuspend in 100-400 μL of deionized water to obtain probe-modified magnetic particle solution.

3. The quantum sensing method for cross-class joint detection of tumor markers according to claim 2, characterized in that: The method for preparing the functionalized nanodiamond includes the following steps: S1. Preparation of fluorescent nanodiamonds with polyethyleneimine surface modification: Take 50-100 μL of surface-carboxylated fluorescent nanodiamonds with a diameter in the range of 20-800 nm, dilute them in 1 mL of deionized water, add 1-3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1-3 mg of N-hydroxysuccinimide, mix well and let stand for 1-3 hours, then add 1-10 mg of polyethyleneimine, incubate at room temperature for 4-6 hours, centrifuge and wash three to five times, and resuspend in 200-500 μL of deionized water to obtain a fluorescent nanodiamond solution with polyethyleneimine surface modification. S2. Take 10-100 μL of fluorescent nanodiamond solution with polyethyleneimine surface modified with 1-2 mL of deionized water, add 10-100 μL of cDNA solution with a concentration in the range of 0.1-100 μM, and incubate on a shaker for 3-5 hours. Wash three to five times using a magnet to obtain the functionalized nanodiamond solution.

4. The quantum sensing method for cross-class joint detection of tumor markers according to claim 3, characterized in that: The preparation method of the functional gel includes the following steps: S1. Prepare a matrix gel with good light transmittance and porosity; dissolve 5-15 mg of polyvinyl alcohol in 100 mL of deionized water, heat and stir at 80-95 °C for 3-5 hours, then add 0.5-3 mg of polyethylene glycol and stir at 80-95 °C for 1-3 hours to obtain the basic gel; S2. Prepare a matrix gel loaded with functional nanodiamonds; take 10-100 μL of the prepared functionalized nanodiamond solution, disperse and dilute it in 10-20 mL of the prepared base gel with good light transmittance and porosity, heat and stir at 80-95℃ for 5-8 hours to obtain a matrix gel loaded with functional nanodiamonds. S3. Prepare a matrix gel modified with a magnetic particle probe; take 10-100 μL of DNA probe with an amino group modified at the 3' end at a concentration of 0.1-100 μM and dilute it in 10-20 mL of matrix gel loaded with functional nanodiamonds, stir at 25-30 ℃ for 3-5 hours to obtain a matrix gel modified with a magnetic particle probe. S4. Prepare functional gel loaded with probe-magnetic particles; take 10-200 μL of functional gel and drop it onto a glass slide, cure it at -5-5℃ for 0.5-2 hours, then drop 50-200 μL of probe-magnetic particle solution onto the gel surface, incubate for 1-3 hours, and rinse the surface with deionized water 3-5 times to obtain functional gel loaded with probe-magnetic particles.

5. The quantum sensing method for cross-class joint detection of tumor markers according to claim 1, characterized in that: The fluorescence collection device includes an optical lens, a spectrometer, and a single-photon counter; it is used to excite NV color centers and collect fluorescence information, recording the optical information of NV color centers in both spectral and fluorescence intensity forms. The probe-modified magnetic particles are used to construct magnetic fields of different intensities and are modified with aptamer probes on their surface. In an environment with MUC1 protein, they bind to and detach from the protein, causing changes in the regional magnetic field strength and thus altering the ODMR signal of the NV color center. The functionalized nanodiamonds are used to detect miRNAs. The surface of the particles is specially modified to change the charge population of the ensemble NV center. In the environment where the miRNA to be tested is present, the proportion of zero and negative states of the ensemble NV center changes, thereby changing the shape of the collected fluorescence spectrum. The functional gel is used to support and immobilize fluorescent nanodiamonds and probe-magnetized particles. The gel has good light transmittance and porosity, which facilitates the optical reading of sensing signals and the capture of free analytes. The microwave antenna is used to radiate microwaves, and ODMR sensing signals are constructed by recording the fluorescence intensity of NV color centers at different microwave frequencies.

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