A bimodal biosensor for tumor marker detection and its use

Abstract

The application belongs to the technical field of biosensors, and particularly relates to a dual-mode biosensor for tumor marker detection and application thereof. The dual-mode biosensor is an SPP1 dual-mode biosensor, and the dual-mode is an electrochemical detection mode and a fluorescence detection mode. Test results show that the dual-mode biosensor has the advantages of high sensitivity, good specificity, dual-mode self-correction and suitability for complex serum samples, and has a wide application prospect. In the electrochemical mode, the linear detection range of the dual-mode biosensor for SPP1 is 500 fg / mL to 150 ng / mL, and the detection limit is 167.883 fg / mL. In the fluorescence mode, the linear detection range is 500 fg / mL to 150 ng / mL, and the detection limit is 331.628 fg / mL.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a dual-modal biosensor for tumor marker detection and its applications. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human health. During the development and progression of tumors, biomolecules such as proteins and nucleic acids released by tumor cells and their microenvironment can enter peripheral blood, urine, and other body fluids, forming tumor markers that can be used for liquid biopsy. Liquid biopsy based on tumor markers has advantages such as convenient sampling, repeatability, and the ability to dynamically reflect tumor burden, and has important application value in early tumor screening, efficacy evaluation, and recurrence and metastasis monitoring.

[0003] Currently, tumor marker detection mainly employs methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, and quantitative polymerase chain reaction (qPCR). While ELISA and other immunoassay methods are mature, their sensitivity for detecting low-abundance protein markers still needs improvement. qPCR primarily detects gene or transcript levels and cannot directly reflect the actual content of target proteins in body fluids. Traditional histological methods such as immunohistochemical staining rely on invasive tissue sampling, which is also insufficient for continuous dynamic monitoring. Furthermore, complex biological samples such as serum and plasma contain numerous interfering substances, easily causing matrix effects, non-specific adsorption, and increased background signals. Existing detection platforms typically rely on a single signal channel for quantification, lacking internal cross-correction mechanisms, making them susceptible to operational errors, probe degradation, and instrument signal drift, leading to biased results. This problem is particularly prominent in the detection of low-abundance markers and in multi-batch, multi-center testing scenarios.

[0004] Osteosarcoma is a primary malignant bone tumor that commonly affects adolescents and young adults. It is characterized by its high invasiveness, rapid progression, and tendency to metastasize to the lungs, creating an urgent need for liquid biopsy biomarkers for auxiliary diagnosis and dynamic monitoring. SPP1 (secreted phosphoprotein 1, also known as osteopontin) is a phosphorylated glycoprotein secreted into the extracellular matrix and blood circulation, participating in tumor cell adhesion, migration, and invasion. SPP1 is aberrantly expressed in osteosarcoma and other malignant tumors and is associated with tumor invasion, metastasis, and patient prognosis, thus making it a potential candidate biomarker for tumor liquid biopsy. However, in some detection scenarios, the concentration of tumor markers such as SPP1 in body fluids is low, and their detection is easily affected by complex sample matrices. Existing single-modal detection methods struggle to simultaneously achieve sensitivity, specificity, and stability. Therefore, there is an urgent need to establish a detection platform with dual signal output and cross-validation capabilities to achieve sensitive and accurate detection of tumor markers in complex biological samples.

[0005] To address the aforementioned technical problems, this invention provides a dual-modal biosensor for tumor marker detection and its application, with SPP1 as an exemplary target tumor marker. Summary of the Invention

[0006] The primary objective of this invention is to provide a dual-modal biosensor for the detection of tumor markers.

[0007] The second objective of this invention is to provide the application of a dual-modal biosensor in the preparation of tumor marker detection products.

[0008] The objective of this invention is achieved through the following technical solution: A dual-modal biosensor for tumor marker detection, wherein the dual-modal biosensor is an SPP1 dual-mode biosensor; the dual-modality consists of an electrochemical detection mode and a fluorescence detection mode; The dual-modal biosensor includes: an NH2-ZIF-8@Ab@DNA composite probe, a CRISPR-Cas12a-crRNA complex, an electrochemical mode SPP1 sensing electrode, and a fluorescent reporter molecule FQ; The NH2-ZIF-8@Ab@DNA composite probe uses NH2-ZIF-8 as a nanocarrier, and its surface is co-modified with anti-SPP1 antibody and target DNA. The electrochemical mode SPP1 sensing electrode includes MoS2@AuNPs, DNA tetrahedrons, and an HCR cascade amplification module.

[0009] Furthermore, the preparation method of the NH2-ZIF-8@Ab@DNA complex probe specifically includes the following steps: (1) Prepare an NH2-ZIF-8 solution with a concentration of 2 mg / mL and disperse it by ultrasonication for 20-40 min; (2) Take 4 μg / mL anti-SPP1 antibody solution, mix with 5 mg / mL EDC and 2 mg / mL NHS and activate for 20-40 min. Add the NH2-ZIF-8 solution obtained in step (1), then add 20 μM target DNA. Incubate at 4℃ for 15-18 h. After centrifugation and washing with PBS, resuspend in Tris-HCl buffer to obtain the NH2-ZIF-8@Ab@DNA composite probe.

[0010] Furthermore, the volume ratio of the anti-SPP1 antibody solution, EDC, and NHS is 100:1:1; the volume ratio of the anti-SPP1 antibody solution to the NH2-ZIF-8 solution is 1:2; the volume ratio of the anti-SPP1 antibody solution to the target DNA is 1:1; and the final concentration of the NH2-ZIF-8@Ab@DNA complex probe is 1-2 mg / mL.

[0011] Furthermore, the nucleotide sequence of the crRNA in the CRISPR-Cas12a-crRNA complex is shown in SEQ ID NO.1; the nucleotide sequence of the target DNA is shown in SEQ ID NO.2; The DNA tetrahedron is formed by the self-assembly of four single strands, DNA1, DNA2, DNA3 and DNA4, through complementary base pairing. The nucleotide sequences of DNA1, DNA2, DNA3 and DNA4 are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively. The nucleotide sequence of the fluorescent reporter molecule FQ is shown in SEQ ID NO.10.

[0012] Furthermore, the HCR cascade amplification module is composed of hairpin probes H1, H2 and H3, and the nucleotide sequences of H1, H2 and H3 are shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0013] Further, the preparation method of the CRISPR-Cas12a-crRNA complex is as follows: add enzyme-free sterile water, 10× NE Buffer 2.1 reaction buffer, crRNA and Cas12a protein to the reaction system, so that the final concentration of crRNA is 20-40 nM and the final concentration of Cas12a protein is 50-70 nM, and incubate at room temperature for 10 min to obtain the CRISPR-Cas12a-crRNA complex.

[0014] Furthermore, the preparation method of the MoS2@AuNPs is as follows: (1) Na2MoO4·2H2O and L-cysteine ​​were dissolved in water at a molar ratio of 1:4. The pH was adjusted to 6.5 with 0.1 mol / L HCl. After hydrothermal reaction at 200℃ for 36 h, the black precipitate was collected by centrifugation, washed with double-distilled water and ethanol in sequence, and dried under vacuum at 80℃ to obtain MoS2 nanomaterials. (2) Dissolve trisodium citrate dihydrate in deionized water, heat to boiling and maintain for 15 min, add HAuCl4 solution for seed synthesis, wherein the molar ratio of trisodium citrate dihydrate to HAuCl4 is (10-15):1; after cooling to 90℃, add trisodium citrate solution and HAuCl4 solution in steps multiple times, wherein the molar ratio of trisodium citrate to HAuCl4 is (2-3):1 each time, repeat the addition 5-10 times; cool naturally to room temperature to obtain gold nanoparticles; (3) Mix the MoS2 nanomaterials obtained in step (1) with the gold nanoparticles obtained in step (2), sonicate at room temperature for 2 hours, centrifuge and wash, and then redisperse in deionized water to obtain the MoS2@AuNPs composite material.

[0015] Application of the dual-modal biosensor described in any of the above claims in the preparation of tumor marker detection products.

[0016] Compared with the prior art, the main advantages of the present invention are as follows: (1) The present invention constructs an NH2-ZIF-8@Ab@DNA integrated composite probe. Using NH2-ZIF-8 as a nanocarrier, SPP1 recognition antibody and DNA functional sequence are co-modified on its surface, so that the probe has both target protein recognition and CRISPR-Cas12a activation functions. The SPP1 protein recognition event is converted into a nucleic acid cleavage signal, which improves the specificity of the detection reaction and the signal conversion efficiency.

[0017] (2) This invention combines the conductive interface of MoS2@AuNPs, the tetrahedral structure of DNA, and the hybridization chain reaction (HCR) amplification module to establish a stable multi-stage signal amplification platform. MoS2@AuNPs can improve electrode conductivity and effective reaction area, DNA tetrahedrons can improve the fixation stability and spatial orientation of nucleic acid probes on the electrode surface, and hybridization chain reaction can further amplify the detection signal, thereby significantly improving the detection sensitivity of low-abundance SPP1.

[0018] (3) The present invention constructs a dual-mode self-calibrating detection system based on the same Cas12a cleavage event. The two opposite changes, namely the decrease in electrochemical signal and the enhancement of fluorescence signal, originate from the same recognition and cleavage process and can verify each other. This effectively reduces the influence of matrix interference, non-specific adsorption and instrument fluctuations on the detection results in complex serum samples, and improves the detection accuracy and reliability.

[0019] (4) Under optimized conditions, the linear detection range of SPP1 in electrochemical mode is 500 fg / mL to 150 ng / mL, with a detection limit of 167.883 fg / mL; and the linear detection range of SPP1 in fluorescence mode is 500 fg / mL to 150 ng / mL, with a detection limit of 331.628 fg / mL. This sensor has a wide detection range and high sensitivity, and can be used for the quantitative detection of low-abundance SPP1.

[0020] (5) The sensor of the present invention can detect the changes in SPP1 levels in the serum of different treatment groups of tumor-bearing mice (saline group, cisplatin group, doxorubicin group, and cisplatin combined with doxorubicin group), and can distinguish the difference in SPP1 expression in the serum of healthy controls and osteosarcoma patients. Its detection trend is consistent with the ELISA results, and it has good clinical sample applicability and reliability. Attached Figure Description

[0021] Figure 1 Characterization results of NH2-ZIF-8 and NH2-ZIF-8@Ab@DNA; where A and B are SEM and TEM images of NH2-ZIF-8, respectively; and C and D are SEM and TEM images of NH2-ZIF-8@Ab@DNA, respectively. Figure 2 The results show the feasibility verification of the NH2-ZIF-8@Ab@DNA composite probe; where A is the verification of Cas12a cleavage ability; B is the feasibility verification of the electrochemical mode; and C is the feasibility verification of the fluorescence mode. Figure 3 The results show the optimization of the CRISPR-Cas12a reaction system conditions; where A represents the optimized Cas12a concentration; and B represents the optimized NH2-ZIF-8@Ab@DNA probe concentration. Figure 4 The results show the performance of the SPP1 dual-mode detection analysis; where A is the electrochemical detection response; B is the electrochemical mode calibration curve; C is the fluorescence detection spectrum; and D is the fluorescence mode calibration curve. Figure 5 The results show the detection of SPP1 in serum samples from tumor-bearing mice and the evaluation of drug efficacy. Among them, A is the heatmap of serum SPP1 signal in each group detected by electrochemical mode; B is the heatmap of serum SPP1 signal in each group detected by fluorescence mode; C is the quantitative detection of serum SPP1 level in each group by electrochemical mode and fluorescence mode; D is the comparison between the dual-mode detection results and the ELISA detection results. Figure 6 The results of SPP1 detection and analysis in clinical serum samples are as follows: A is a heatmap of SPP1 signal in serum of healthy control group and osteosarcoma group detected by electrochemical mode; B is a heatmap of SPP1 signal in serum of healthy control group and osteosarcoma group detected by fluorescence mode; C is a quantitative detection of serum SPP1 level in two groups by electrochemical mode and fluorescence mode; D is a comparison of detection results by ELISA, electrochemical mode and fluorescence mode. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0023] Table 1 shows the experimental materials used in the embodiments of the present invention and their sources: Table 1 Experimental Materials and Sources The nucleotide sequences used in the embodiments of this invention are shown in Table 2: Table 2 Sequence List Example 1 Synthesis of NH2-ZIF-8 nanomaterials 1.49 g zinc acetate, 2.9552 g 2-methylimidazole, and 0.5326 g 2-aminobenzimidazole were dissolved in 100 mL methanol. After stirring at room temperature for 1.5 h, the mixture was centrifuged at 10000 rpm for 10 min. The resulting precipitate was washed three times with methanol and then dried for 24 h to obtain NH2-ZIF-8 nanomaterials. The morphology of the synthesized NH2-ZIF-8 was observed using field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the results are as follows. Figure 1 As shown.

[0024] like Figure 1 A and Figure 1 As shown in Figure B, the NH2-ZIF-8 particles have a relatively uniform morphology, exhibiting a typical polyhedral structure with clear particle boundaries and good dispersibility. No obvious aggregation was observed, indicating that the prepared NH2-ZIF-8 has a stable nano-skeleton structure and can be used as a carrier material for subsequent antibody and DNA functional sequence modification.

[0025] Example 2 Preparation of NH2-ZIF-8@Ab@DNA complex probe The NH2-ZIF-8 nanomaterials prepared in Example 1 were resuspended in deionized water to prepare a 2 mg / mL NH2-ZIF-8 solution, which was then ultrasonically dispersed for 30 min. SPP1 antibody (1000 μg / mL) was diluted 250 times to obtain an antibody working solution with a concentration of 4 μg / mL. Before activation, 5 mg of EDC hydrochloride powder and 2.0 mg of NHS powder were accurately weighed and dissolved separately in 1 mL of ultrapure water, and thoroughly vortexed to prepare 5 mg / mL EDC and 2 mg / mL NHS solutions. Immediately afterward, 0.5 μL of each solution was added to the 50 μL antibody working solution, gently mixed by pipetting, and then reacted in a metal bath at 300 rpm for 30 min to activate the antibody. The activated anti-SPP1 antibody was then added to 100 μL of 2 mg / mL NH2-ZIF-8 solution. After gentle mixing, the mixture was vortexed at 300 rpm for 20 min to modify the NH2-ZIF-8 surface with anti-SPP1 antibody. Following antibody modification, 50 μL of 20 μM target DNA sequence (SEQ ID NO. 2) was added to the mixture, gently mixed, and incubated at 4 °C for 16 h. After the reaction, the product was centrifuged at 10,000 rpm for 5 min at 4 °C and washed twice with 10 mM PBS. Finally, the obtained NH2-ZIF-8@Ab@DNA product was resuspended in 200 μL of 10 mM Tris-HCl buffer to a final concentration of 1 mg / mL and stored at 4 °C for later use, yielding the NH2-ZIF-8@Ab@DNA composite probe. The morphology of the obtained NH2-ZIF-8@Ab@DNA composite probe was observed using field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results are as follows: Figure 1 As shown.

[0026] like Figure 1 As shown in Figure C, compared to NH2-ZIF-8, the NH2-ZIF-8@Ab@DNA particles retain their original polyhedral structure, but their surface roughness is significantly increased, suggesting that antibody and DNA functional sequences have been modified onto the NH2-ZIF-8 surface. Figure 1As shown in Figure D, the TEM image of NH2-ZIF-8@Ab@DNA reveals a clear outer coating structure and dotted modification signals on the particle surface, further indicating that the antibody and DNA functional sequences were successfully immobilized on the surface of NH2-ZIF-8 nanoparticles. SEM and TEM characterization results of NH2-ZIF-8 and NH2-ZIF-8@Ab@DNA show that the modification with antibody and DNA functional sequences did not disrupt the original nanoframework structure of NH2-ZIF-8, while successfully endowing it with target recognition and CRISPR-Cas12a signal activation functions. Therefore, the preparation of the NH2-ZIF-8@Ab@DNA composite probe is successful and can be used as a probe in the subsequent SPP1 detection system.

[0027] Example 3 Construction of electrochemical and fluorescence model biosensors (1) Preparation of MoS2@AuNPs dispersion: 0.25 g Na2MoO4·2H2O was dissolved in 25 mL of water, sonicated for 5 minutes, and the pH of the solution was adjusted to 6.5 with 0.1 mol / L HCl. Then, 0.5 g L-cysteine ​​and 50 mL of water were added, and sonication was continued for 10 min. The mixture was transferred to a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene and reacted at 200 °C for 36 h. After the reaction was completed, the mixture was cooled, and the black precipitate was collected by centrifugation. It was washed with double-distilled water and ethanol in sequence, and dried in a vacuum oven at 80 °C for 24 h. The resulting precipitate was redispersed in deionized water and stored in a refrigerator at 4 °C for later use. Then, 51.76 mg of trisodium citrate dihydrate was added to 80 mL of deionized water, and the mixture was placed in a three-necked flask and heated to boiling with continuous stirring for 15 min. Subsequently, 533 μL of HAuCl4 solution (25 mM) was slowly added. The solution color changed from light yellow to bright red within 10 min, indicating successful synthesis of gold seeds. After cooling the above solution to 90°C, 553 μL of trisodium citric acid solution (60 mM) was added, followed by another 533 μL of HAuCl4 solution (25 mM) after 2 min. This step was repeated 7 times. The synthesized gold nanoparticles were allowed to cool naturally to room temperature and stored at 4°C. Finally, the obtained MoS2 and AuNPs were uniformly mixed and sonicated at room temperature for 2 h. The resulting black dispersion was centrifuged at 10,000 rpm for 10 min, washed three times with deionized water, and finally redispersed in deionized water and stored at 4°C for later use.

[0028] (2) Construction of electrochemical mode sensing electrode: Before modification, the glassy carbon electrode (GCE) was polished to a mirror finish on a chamois coated with ultrafine alumina powder. Then, it was ultrasonically cleaned for 3 minutes each in ultrapure water, anhydrous ethanol, and ultrapure water to thoroughly remove residual polishing powder. Finally, it was dried with high-purity nitrogen to obtain the pretreated glassy carbon electrode. The washed and centrifuged MoS2@AuNPs precipitate was resuspended in deionized water to prepare a dispersion with a concentration of 2.0 mg / mL. 6 μL of the dispersion was accurately pipetted onto the pretreated GCE surface and dried in a 37°C oven for 2 h to form a uniform modified film. Subsequently, DNA tetrahedral self-assembly was performed: four strands (DNA1, DNA2, DNA3, and DNA4) were diluted with enzyme-free sterile water to prepare a 50 μM stock solution. 1 μL of each strand was thoroughly mixed with 46 μL of TCEP buffer (resulting in a final concentration of 1 μM per strand). The mixture was heated in a 95°C metal bath for 10 min, then rapidly transferred to a 0°C ice-water mixture for cooling for 2 min, and finally incubated at 4°C for 6 h to complete assembly. 6 μL of the assembled DNA tetrahedral solution was added to the electrode surface and incubated at 4°C for 12 h to fix it to the electrode surface. Non-specific sites were then blocked with MCH. After blocking, 10 μL of a mixture containing 0.5 μM H1 hairpins, 1 μM H2 hairpins, and 1 μM H3 hairpins was added to the electrode surface and incubated at 37°C for 2 h. The electrode was then washed with distilled water to remove unbound hairpin structures, forming a DNA tetrahedral-HCR single-point anchored multi-site cascade amplification module. Finally, methylene blue solution was added and incubated for 30 min to allow the signal molecules to bind to the nucleic acid amplification structure, thus obtaining the electrochemical mode SPP1 sensing electrode.

[0029] (3) Preparation of CRISPR-Cas12a-crRNA complex: Add 53 μL of enzyme-free sterile water, 3 μL of 10× NE Buffer 2.1 reaction buffer, 3 μL of 600 nM crRNA and 1 μL of 3.6 μM Cas12a to a centrifuge tube, gently aspirate and mix well, and then incubate at room temperature for 10 minutes to form CRISPR-Cas12a-crRNA complex.

[0030] (4) Construction of the fluorescence detection system: First, the anti-SPP1 antibody was activated. 55 μL of 5 μg / mL Anti-SPP1 antibody solution was added, along with 0.55 μL of 5 mg / mL EDC and 0.55 μL of 2 mg / mL NHS. After gentle mixing, the solution was activated by shaking at 300 rpm for 30 min in a metal bath. The activated antibody was added to each well of a 96-well plate and incubated at 4℃ for 12 h to fix it on the plate surface. After blocking with BSA, 50 μL of different concentrations of the SPP1 protein sample to be tested were added to each well and incubated at 37℃ for 1 h. Then, 55 μL of the 1 mg / mL NH2-ZIF-8@Ab@DNA composite probe prepared in Example 2 was added and incubated for further incubation to allow it to bind to SPP1. Finally, add 60 μL of the CRISPR-Cas12a-crRNA complex (crRNA sequence as shown in SEQ ID NO.1) prepared in step (3) and 3 μL of 10 μM fluorescent reporter molecule FQ (SEQ ID NO.10), react at 37 °C for 20 min, and use it for fluorescence signal detection.

[0031] Example 4 Feasibility verification of NH2-ZIF-8@Ab@DNA complex probe To verify the feasibility of the SPP1 dual-modal biosensor constructed in this invention, experiments were conducted to verify both the CRISPR-Cas12a trans-cleavage activity and the SPP1 response detection.

[0032] (1) Verification of CRISPR-Cas12a trans-cleavage activity: To verify the necessary components for the trans-cleavage activity of CRISPR-Cas12a, a complete reaction system and multiple control experiments were constructed: The complete system was constructed by using the CRISPR-Cas12a-crRNA complex (crRNA sequence as shown in SEQ ID NO.1), the NH2-ZIF-8@Ab@DNA composite probe prepared in Example 2, and the fluorescent reporter molecule FQ (SEQ ID NO.10); the control group was replaced with an equal volume of enzyme-free water to replace crRNA, Cas12a, or NH2-ZIF-8@Ab@DNA probe (when crRNA or Cas12a was missing, it was replaced during the preparation stage of the CRISPR-Cas12a-crRNA complex; when ZIF-8@Ab@DNA composite probe was missing, it was replaced during the final reaction system construction stage). All reaction systems were incubated at 37 °C in the dark for 20 min, and then fluorescence spectroscopy was performed using a multi-functional microplate reader (excitation wavelength 485 nm, scanning range 500–650 nm).

[0033] The results are as follows Figure 2As shown in Figure A, the complete system exhibits a significant fluorescence peak at approximately 520 nm. However, when crRNA, Cas12a, or NH2-ZIF-8@Ab@DNA is missing, the fluorescence signal is significantly reduced, indicating that the above components are all essential for the Cas12a cleavage reaction. This demonstrates that the probe can effectively activate the trans-cleavage activity of Cas12a.

[0034] (2) Electrochemical Model Validation: The modified sensing electrode was inverted in a 96-well plate, avoiding contact with the bottom of the plate. The SPP1 sample to be tested was added to each well, and the plate was incubated at 37 °C for 20 min under light-shielding conditions. After the reaction was complete, the electrode was rinsed three times with 10 μL of distilled water. All electrochemical measurements were performed in a standard three-electrode system, including the working electrode (different modified glassy carbon electrodes), the reference electrode (Ag / AgCl electrode saturated with KCl), and the counter electrode (1 mm diameter Pt wire). Electrochemical impedance spectroscopy (EIS) was used to verify the assembly process of the sensing interface under open-circuit conditions, with a frequency range of 100 kHz to 0.1 Hz and an amplitude signal of 5 mV. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were used to measure the electrochemical response of the electrode at a scan rate of 50 mV·s. -1 The potential range was -0.2 to +0.6 V, and the detection environment was a 0.1 M KCl solution containing 10 mM K3[Fe(CN)6]. Additionally, the detection was performed at 50 mV·s in 10 mM PBS buffer (pH 7.4). -1 DPV was performed at a scan rate in the potential range of -0.6 to 0 V to study the electrochemical signal of methylene blue (MB).

[0035] The results are as follows Figure 2 As shown in Figure B, without SPP1, the amplification module on the electrode surface remains intact, and the electrochemical signal is strong. After adding SPP1, the probe recognizes SPP1 and activates Cas12a, causing the nucleic acid amplification module on the electrode surface to be cleaved and released, and the electrochemical peak current is significantly reduced, confirming that the electrochemical mode detection is feasible.

[0036] (3) Fluorescence pattern verification: The SPP1 sample to be tested was added to a 96-well plate, followed by the addition of NH2-ZIF-8@Ab@DNA complex probe, CRISPR-Cas12a-crRNA complex and FQ fluorescent reporter molecule, and reacted at 37 °C for 20 min. The fluorescence signal in the 96-well plate was detected using a multi-functional microplate reader at an excitation wavelength of 610 nm, and the fluorescence spectrum of each well was obtained by scanning in the 600~700 nm band with a step size of 1 nm.

[0037] The results are as follows Figure 2As shown in Figure C, the fluorescence signal is weak without SPP1; after the addition of SPP1, Cas12a is activated and cleaved to release the amplification module, resulting in a significant enhancement of the fluorescence signal in the solution. These results demonstrate that the SPP1 sensor constructed in this invention can achieve dual-mode detection through electrochemical signal reduction and fluorescence signal enhancement, proving the feasibility of this detection strategy.

[0038] Example 5 Optimization of key parameters in the CRISPR-Cas12a reaction system To achieve optimal sensing performance, this study optimized key parameters in the CRISPR-Cas12a reaction system (the specific method is the same as the construction of the fluorescence mode detection system in Example 3 (4), where the concentration of the SPP1 sample to be tested was 100 pg / mL). First, under the condition of fixing the concentration of NH2-ZIF-8@Ab@DNA probe (1.0 mg / mL), Cas12a solution with a concentration of 0.6–6.0 μM (i.e., the concentration of Cas12a solution added when preparing the CRISPR-Cas12a-crRNA complex) was added to the reaction system along with the probe and the fluorescent reporter molecule, and the effect on the change value ΔI of the electrochemical signal was observed; then, under the condition of fixing the concentration of Cas12a to 3.6 μM, the effect of the concentration of NH2-ZIF-8@Ab@DNA probe in the range of 0.1~2.0 mg / mL (the final concentration after resuspension in Example 2) on ΔI was investigated, and the results are as follows. Figure 3 As shown.

[0039] like Figure 3 As shown in Figure A, the electrochemical signal change ΔI gradually increased as the Cas12a concentration increased from 0.6 μM to 3.6 μM, indicating that increasing the Cas12a concentration is beneficial to enhancing the efficiency of the trans-cleavage reaction. When the Cas12a concentration continued to increase to 4.8 μM and 6.0 μM, ΔI did not increase significantly further, suggesting that the reaction system was approaching saturation. Therefore, 3.6 μM was selected as the optimal reaction concentration of Cas12a.

[0040] like Figure 3 As shown in Figure B, ΔI increased significantly with increasing NH2-ZIF-8@Ab@DNA concentration from 0.1 mg / mL to 1.0 mg / mL, indicating that increasing the probe concentration improves SPP1 recognition and Cas12a activation efficiency. When the NH2-ZIF-8@Ab@DNA concentration was further increased to 1.5 mg / mL and 2.0 mg / mL, the change in ΔI was not significant, indicating that the system had basically reached the optimal response state. Therefore, 1.0 mg / mL was selected as the optimal concentration of the NH2-ZIF-8@Ab@DNA probe.

[0041] The above results indicate that both the Cas12a concentration and the NH2-ZIF-8@Ab@DNA probe concentration affect the detection performance of the SPP1 sensor. Considering both signal intensity and reagent dosage, this invention preferably uses a Cas12a concentration of 3.6 μM and an NH2-ZIF-8@Ab@DNA concentration of 1.0 mg / mL for subsequent SPP1 detection experiments.

[0042] Example 6 SPP1 Dual-Modal Biosensor Detection and Analysis Performance Evaluation To evaluate the analytical performance of the constructed SPP1 dual-mode biosensor under the optimal experimental conditions described in Example 5, the sensor was used to detect a series of SPP1 standards at different concentrations (500 fg / mL to 150 ng / mL). The signal response was measured sequentially using electrochemical and fluorescence modes. The results are as follows: Figure 4 As shown.

[0043] like Figure 4 As shown in Figure A, the electrochemical response signal exhibits a regular change as the SPP1 concentration increases from 500 fg / mL to 150 ng / mL. Further linear fitting was performed on the electrochemical signal change ΔI against the logarithm of the SPP1 concentration, as shown in Figure A. Figure 4 As shown in B, the two exhibit a good linear relationship in the range of 500 fg / mL to 150 ng / mL, with the linear regression equation being y = 1.391 ×lg C. SPP1 - 2.199, correlation coefficient R 2 = 0.995. Calculated according to the 3SD / m standard (where SD is the standard deviation obtained from multiple measurements of blank samples, and m is the slope of the calibration curve), the detection limit of SPP1 in electrochemical mode is 167.883 fg / mL.

[0044] like Figure 4 As shown in Figure C, the fluorescence signal gradually increases with increasing SPP1 concentration, indicating that SPP1 can trigger probe activation of Cas12a and cause more amplification modules to be cleaved and released into the solution. Figure 4 As shown in Figure D, the fluorescence intensity showed a good linear relationship with the logarithm of SPP1 concentration in the range of 500 fg / mL to 150 ng / mL, and the linear regression equation was y = 55.484 × lg C. SPP1 - 66.967, correlation coefficient R 2 = 0.994. Based on the 3SD / m standard, the detection limit of SPP1 in fluorescence mode is 331.628 fg / mL.

[0045] The above results demonstrate that the SPP1 dual-mode biosensor constructed in this invention exhibits a wide linear detection range and low detection limit in both electrochemical and fluorescence modes, making it suitable for sensitive quantitative detection of low-abundance SPP1. Furthermore, since the electrochemical and fluorescence signals originate from the same recognition and cleavage process, they can mutually verify each other, contributing to improved accuracy and reliability of the detection results.

[0046] Example 7 Detection of SPP1 in serum samples from tumor-bearing mice using a dual-modal biosensor and evaluation of drug efficacy (1) To explore the applicability of the constructed SPP1 dual-modal biosensor in actual sample detection and its feasibility for evaluating the efficacy of osteosarcoma drugs, this study first established an osteosarcoma-bearing mouse model: 1×10 8 143B cells (suspended in 100 μL PBS) were subcutaneously injected into the right flank of 6-week-old female BALB / c nude mice. After tumor formation, the mice were randomly divided into 4 groups (n=5 per group): saline group, cisplatin group, doxorubicin group, and cisplatin combined with doxorubicin group. The administration regimen was intraperitoneal injection every 5 days (i.e., days 15, 20, and 25 after tumor inoculation). The concentration of cisplatin and doxorubicin was 2 mg / kg, and the saline group received an equal volume of saline.

[0047] (2) At the end of the experiment, blood samples were collected from each group by collecting blood from the orbital cavity of mice. The samples were allowed to stand at 4 °C for 1 hour to coagulate, and then the supernatant was collected by centrifugation at 3000 rpm for 10 minutes. These were the serum samples. All serum samples were aliquoted and stored in a -80 °C freezer for later use. Before testing, the serum samples were placed on ice to thaw slowly. The expression level of SPP1 in the serum of mice in each group was detected using the electrochemical mode, fluorescence mode, and ELISA method constructed in this invention, respectively. For electrochemical detection, 10 μL of serum sample was added to the surface of the modified sensing electrode and incubated at 37 °C under light-shielding conditions for 20 min to complete probe binding and CRISPR-Cas12a activation cleavage. After washing, DPV was detected in 0.1 M KCl solution. For fluorescence detection, 55 μL of serum sample was added to a 96-well plate and incubated at 37 °C for 1 h. After washing, 55 μL of the NH2-ZIF-8@Ab@DNA complex probe prepared in Example 2, 60 μL of the CRISPR-Cas12a-crRNA complex, and 3 μL of 10 μM FQ fluorescent reporter molecule were added sequentially, and the fluorescence intensity was detected using a microplate reader. ELISA detection was performed using Human OPNELISA. Kit (E-EL-H1347), strictly following the kit instructions, was used to measure the SPP1 concentration in each group of serum samples. The detection results of the two modes of this invention were compared with the ELISA detection results. The results are as follows: Figure 5 As shown.

[0048] like Figure 5 As shown in Figures A and 5B, both electrochemical and fluorescence modes detected differences in SPP1 signals in serum samples from different treatment groups. The saline group showed a higher SPP1 response signal, indicating higher SPP1 levels in the serum of untreated tumor-bearing mice. After treatment with cisplatin or doxorubicin, the SPP1 response signal decreased, with the lowest signal observed in the cisplatin combined with doxorubicin group, indicating the most significant decrease in SPP1 levels after combined treatment.

[0049] like Figure 5 As shown in Figure C, the trends in SPP1 concentration measured in electrochemical and fluorescence modes are basically consistent, showing the highest concentration in the saline group, a decrease in the monotherapy group, and the lowest concentration in the combination therapy group. These results indicate that the dual-mode sensor constructed in this invention can stably detect SPP1 in complex serum samples and can be used to distinguish differences in SPP1 expression under different treatment regimens.

[0050] like Figure 5 As shown in Figure D, the trend of SPP1 changes obtained by ELISA is consistent with the results of electrochemical and fluorescence modes, further demonstrating that the SPP1 dual-mode biosensor constructed in this invention has good reliability and applicability to practical samples. The above results indicate that serum SPP1 levels can reflect the response of osteosarcoma-bearing mice to different drug treatments, and the sensor of this invention can be used for evaluating the efficacy of osteosarcoma drugs and monitoring the treatment process.

[0051] Example 8 SPP1 detection and analysis in clinical serum samples To verify the applicability of the constructed SPP1 dual-modal biosensor in clinical sample detection, serum samples from four healthy controls and four osteosarcoma patients were analyzed. The expression levels of SPP1 in each group of serum samples were detected using electrochemical, fluorescence, and ELISA methods, respectively. The consistency of the results obtained by different methods was compared, and the detection results of this invention were compared with the ELISA detection results. The results are as follows: Figure 6 As shown.

[0052] like Figure 6 As shown in Figures A and 6B, both electrochemical and fluorescence modes detected differences in SPP1 signal between serum samples from the healthy control group and the osteosarcoma group. Compared with the healthy control group, the SPP1 response signal in the serum samples of osteosarcoma patients was significantly increased, indicating that the dual-mode biosensor constructed in this invention can identify changes in SPP1 expression levels in clinical serum samples.

[0053] like Figure 6As shown in Figure C, the trends in SPP1 concentration measured in electrochemical and fluorescence modes are basically consistent, both showing that the osteosarcoma group has higher concentrations than the healthy control group, indicating that the dual-mode detection method of this invention has good stability and consistency in clinical samples. Figure 6 As shown in Figure D, the SPP1 expression trends detected by ELISA, electrochemical mode, and fluorescence mode were consistent, all indicating that the SPP1 level in the serum of osteosarcoma patients was significantly higher than that in healthy controls.

[0054] The above results indicate that the SPP1 dual-mode biosensor constructed in this invention can be used to detect SPP1 in clinical serum samples and can help distinguish between healthy controls and osteosarcoma patients, demonstrating good applicability to clinical samples.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A dual-modal biosensor for tumor marker detection, characterized in that, The dual-modal biosensor is the SPP1 dual-mode biosensor; the dual-modality consists of an electrochemical detection mode and a fluorescence detection mode. The dual-modal biosensor includes: an NH2-ZIF-8@Ab@DNA composite probe, a CRISPR-Cas12a-crRNA complex, an electrochemical mode SPP1 sensing electrode, and a fluorescent reporter molecule FQ; The NH2-ZIF-8@Ab@DNA composite probe uses NH2-ZIF-8 as a nanocarrier, and its surface is co-modified with anti-SPP1 antibody and target DNA. The electrochemical mode SPP1 sensing electrode includes MoS2@AuNPs, DNA tetrahedrons, and an HCR cascade amplification module.

2. The dual-modal biosensor according to claim 1, characterized in that, The preparation method of the NH2-ZIF-8@Ab@DNA complex probe specifically includes the following steps: (1) Prepare an NH2-ZIF-8 solution with a concentration of 2 mg / mL and disperse it by ultrasonication for 20-40 min; (2) Take 4 μg / mL anti-SPP1 antibody solution, mix with 5 mg / mL EDC and 2 mg / mL NHS and activate for 20-40 min. Add the NH2-ZIF-8 solution obtained in step (1), then add 20 μM target DNA. Incubate at 4℃ for 15-18 h. After centrifugation and washing with PBS, resuspend in Tris-HCl buffer to obtain the NH2-ZIF-8@Ab@DNA composite probe.

3. The dual-modal biosensor according to claim 2, characterized in that, The volume ratio of the anti-SPP1 antibody solution, EDC, and NHS is 100:1:1; the volume ratio of the anti-SPP1 antibody solution to the NH2-ZIF-8 solution is 1:2; the volume ratio of the anti-SPP1 antibody solution to the target DNA is 1:1; and the final concentration of the NH2-ZIF-8@Ab@DNA complex probe is 1-2 mg / mL.

4. The dual-modal biosensor according to claim 1, characterized in that, The nucleotide sequence of the crRNA in the CRISPR-Cas12a-crRNA complex is shown in SEQ ID NO.1; the nucleotide sequence of the target DNA is shown in SEQ ID NO.

2. The DNA tetrahedron is composed of DNA1, DNA2, DNA3 and DNA4, and the nucleotide sequences of DNA1, DNA2, DNA3 and DNA4 are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively. The nucleotide sequence of the fluorescent reporter molecule FQ is shown in SEQ ID NO.

10.

5. The dual-modal biosensor according to claim 1, characterized in that, The HCR cascade amplification module consists of hairpin probes H1, H2, and H3, and the nucleotide sequences of H1, H2, and H3 are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

6. The dual-modal biosensor according to claim 1, characterized in that, The CRISPR-Cas12a-crRNA complex was prepared by adding enzyme-free sterile water, 10× NE Buffer 2.1 reaction buffer, crRNA, and Cas12a protein to the reaction system, so that the final concentration of crRNA was 20-40 nM and the final concentration of Cas12a protein was 50-70 nM. The mixture was incubated at room temperature for 10 min to obtain the CRISPR-Cas12a-crRNA complex.

7. The dual-modal biosensor according to claim 1, characterized in that, The preparation method of the MoS2@AuNPs is as follows: (1) Na2MoO4·2H2O and L-cysteine ​​were dissolved in water at a molar ratio of 1:

4. The pH was adjusted to 6.5 with 0.1 mol / L HCl. After hydrothermal reaction at 200℃ for 36 h, the black precipitate was collected by centrifugation, washed with double-distilled water and ethanol in sequence, and dried under vacuum at 80℃ to obtain MoS2 nanomaterials. (2) Dissolve trisodium citrate dihydrate in deionized water, heat to boiling and maintain for 15 min, add HAuCl4 solution for seed synthesis, wherein the molar ratio of trisodium citrate dihydrate to HAuCl4 is (10-15):1; after cooling to 90℃, add trisodium citrate solution and HAuCl4 solution in steps multiple times, wherein the molar ratio of trisodium citrate to HAuCl4 is (2-3):1 each time, repeat the addition 5-10 times; cool naturally to room temperature to obtain gold nanoparticles; (3) Mix the MoS2 nanomaterials obtained in step (1) with the gold nanoparticles obtained in step (2), sonicate at room temperature for 2 h, centrifuge and wash, and then redisperse in deionized water to obtain the MoS2@AuNPs composite material.

8. The application of the dual-modal biosensor according to any one of claims 1-7 in the preparation of tumor marker detection products.

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