Prostate cancer olaparil response marker determination system and determination method and application

Abstract

The invention belongs to the technical field of biomedical detection, and provides a prostatic cancer olaparione response marker determination system and a determination method and application thereof. A multifunctional polymer brush is grafted on the surface of a core-shell magnetic polymer microsphere, an HRR gene oligonucleotide probe is coupled through carbodiimide-mediated amidation reaction, and efficient enrichment and accurate quantitative detection of ultralow-abundance mutant alleles are achieved by combining a digital PCR technology. The use amount of the magnetic oligonucleotide probe in a single 20L reaction system is 0.5-200g, and the positive detection rate of HRR gene mutation sites with allele frequency of 0.10%-0.50% is not less than 95%. The problems that in the prior art, low-abundance target capturing efficiency is low, non-specific adsorption is high, and digital PCR quantitative accuracy is poor are solved, a high-sensitivity and high-specificity molecular diagnosis tool is provided for olapara utilization drug decision-making of castration-resistant metastatic prostate cancer patients, and wide clinical application value is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a system, method, and application for determining olaparib response markers in prostate cancer. Background Technology

[0002] Prostate cancer is one of the most common malignant tumors of the male genitourinary system, and its incidence and mortality rates are increasing globally. For patients with metastatic castration-resistant prostate cancer, traditional chemotherapy regimens have limited efficacy and significant toxic side effects, necessitating novel targeted therapies and precision medicine strategies. In recent years, olaparib, a PARP inhibitor targeting homologous recombination repair defects, has shown significant efficacy in prostate cancer patients carrying HRR gene mutations such as BRCA1 and BRCA2, providing a new treatment option for this refractory disease. However, the proportion of HRR gene-mutant prostate cancer patients in the population is low, and the frequency of mutated alleles is often less than 1%, especially given the extremely low abundance of cell-free DNA in liquid biopsy samples. Accurate detection and quantification of these low-frequency mutations are crucial for patient screening and efficacy prediction. Therefore, establishing a highly sensitive and specific molecular diagnostic technique capable of accurately detecting HRR gene mutation burden under limited sample conditions has significant clinical application value and scientific research significance for precise medication decisions, dynamic efficacy monitoring, and resistance mechanism research in prostate cancer patients undergoing olaparib treatment.

[0003] Currently, HRR gene mutation detection mainly relies on technologies such as Sanger sequencing, next-generation sequencing, and digital PCR. However, these methods all have significant limitations in detecting ultra-low abundance mutations. Sanger sequencing has low sensitivity, only able to detect mutations with an abundance of 10% or higher, which cannot meet the needs of liquid biopsy. Although next-generation sequencing has high throughput, its detection of low abundance mutations is limited by library construction efficiency and sequencing depth, and it is also costly and has a long turnaround time. Digital PCR technology has absolute quantification and single-molecule detection capabilities, theoretically capable of detecting mutations with an abundance of 0.01% or higher, but in practical applications, it faces challenges such as low target enrichment efficiency, high non-specific amplification interference, and complex reaction systems. For example, Chinese patent CN112226489B discloses a nucleic acid extraction method based on magnetic beads for targeted enrichment of target genes and its application. However, the magnetic beads used have a single surface modification and poor dispersibility, resulting in insufficient enrichment efficiency in low abundance samples. Furthermore, the magnetic beads are prone to aggregation and sedimentation during circulation, affecting the uniformity of droplet distribution and the stability of fluorescence signals. Therefore, developing a magnetic oligonucleotide probe and its assay system that can balance efficient target enrichment, excellent colloidal stability, and compatibility with digital PCR systems has become a key technological bottleneck for the accurate detection of ultra-low abundance mutations in the HRR gene of prostate cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a system for detecting olaparib response markers in prostate cancer, along with its detection method and application, thereby solving the technical challenges of low target enrichment efficiency, high non-specific adsorption, poor quantitative accuracy of digital PCR, and insufficient compatibility between magnetic carriers and digital PCR systems in the current detection of ultra-low abundance mutations in the HRR gene.

[0005] This invention achieves a unified approach of high magnetic responsiveness, high surface activity, high oligonucleotide immobilization density, and excellent colloidal stability through a synergistic interface design that constructs core-shell magnetic polymer microspheres and grafts multifunctional polymer brushes onto their surfaces. The iron oxide nanocore provides rapid magnetic separation capability, the cross-linked polystyrene shell imparts structural stability and mechanical strength, the dopamine coating and aminosilane layer construct a multi-point anchoring interface, and the multifunctional polymer brush formed by copolymerizing acrylic acid and polyethylene glycol methyl ether methacrylate provides a high density of carboxyl groups for oligonucleotide coupling and, through the strong hydrophilic shielding layer formed by the polyethylene glycol side chains, significantly reduces non-specific adsorption and particle aggregation. This ensures that the magnetic oligonucleotide probes maintain excellent dispersibility and amplification compatibility in high-salt surfactant buffers and long-cycle high-temperature digital PCR systems, ultimately achieving an ultra-high sensitivity detection performance of no less than 95% for HRR gene mutation sites with allele frequencies of 0.10% to 0.50%.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A system for determining olaparib response markers in prostate cancer, comprising: The magnetic oligonucleotide probes in this assay system are obtained by a carbodiimide-mediated amidation coupling reaction between core-shell magnetic polymer microspheres and HRR gene oligonucleotide probes with 5′ amino groups. The HRR gene oligonucleotide probes in this assay system are used to detect at least 10 genes in homologous recombination repair-related genes, and the at least 10 genes in this assay system include at least BRCA1, BRCA2, ATM, PALB2, and BARD1. The dPCR amplification reaction components of this assay system include thermostable DNA polymerase, deoxynucleoside triphosphate, magnesium-containing PCR buffer, amplification primer pairs for amplifying the HRR gene target nucleic acid of this assay system, and fluorescence detection components. The binding and washing buffer for this assay system is an aqueous solution containing tris(hydroxymethyl)aminomethane, sodium chloride, disodium ethylenediaminetetraacetate dihydrate, and a nonionic surfactant. In a single 20 µL dPCR reaction system, the amount of magnetic oligonucleotide probe used in this assay system ranges from 0.5 µg to 200 µg. The dPCR amplification reaction of this assay system is used to quantitatively detect the frequency of mutated alleles in the HRR gene of this assay system and to predict the response of prostate cancer patients to olaparib treatment.

[0007] Furthermore, the core-shell magnetic polymer microspheres of this assay system were prepared through the following steps: A1. Raw material preparation: 1.0 to 5.0 parts of iron oxide nanoparticles (the iron oxide nanoparticles in this test system are obtained by step A2), 10 to 40 parts of styrene, 1 to 8 parts of divinylbenzene, sodium dodecyl sulfate as emulsifier (the amount of sodium dodecyl sulfate in this test system is 0.1 to 5.0 parts), and potassium persulfate as initiator (the amount of potassium persulfate in this test system is 0.05 to 2.0 parts). A2. Preparation of iron oxide cores: Iron salt solution was co-precipitated under alkaline conditions, the pH was adjusted to 9.0 to 11.0, and the reaction was carried out at 70 ℃ to 90 ℃ for 0.5 h to 2.0 h to obtain iron oxide nanoparticles with a particle size of 50 nm to 150 nm. A3. Coating polymerization: The iron oxide nanoparticles obtained in step A2 are dispersed in an aqueous phase containing sodium dodecyl sulfate, styrene and divinylbenzene monomers are added, and emulsion polymerization is carried out at 70 ℃ to 85 ℃ for 2.0 h to 4.0 h to obtain core-shell magnetic polymer microspheres with iron oxide as the core and cross-linked polystyrene as the shell. The median particle size of the obtained microspheres is 150 nm to 400 nm, and the shell thickness is 20 nm to 80 nm. A4. Washing and drying: The emulsion obtained in step A3 is magnetically separated, washed 2 to 5 times with deionized water, and dried at 40°C to 60°C for 4 to 12 hours to obtain the core-shell magnetic polymer microspheres of the test system.

[0008] Furthermore, the surface functionalization of the core-shell magnetic polymer microspheres in this assay system was prepared through the following steps to obtain functionalized core-shell magnetic polymer microspheres: B1. Dopamine Coating: Core-shell magnetic polymer microspheres are dispersed in a tris(hydroxymethyl)aminomethane buffer solution with a pH of 8.0 to 9.0. 1.0 to 10.0 parts of dopamine hydrochloride are added, with 100 parts of core-shell magnetic polymer microspheres. The mixture is stirred at 20°C to 30°C for 1.0 to 3.0 h to allow dopamine to self-polymerize and form a dopamine coating on the surface of the microspheres. B2. Silane coupling: 2.0 to 10.0 parts of 3-aminopropyltriethoxysilane are added to the product of step B1, and the reaction is carried out at 40°C to 60°C for 1.0 h to 3.0 h to introduce a surface aminosilane layer; B3. Initiator introduction: The product of step B2 was reacted with 2-bromoisobutyryl bromide in the presence of triethylamine to amidate the initiator group of the assay system with the surface amino group. The amount of 2-bromoisobutyryl bromide was controlled to be 1.0 to 3.0 times the molar amount of 3-aminopropyltriethoxysilane. The reaction was carried out at 0 °C to 10 °C for 0.5 h to 2.0 h to obtain functionalized core-shell magnetic polymer microspheres with α-bromoacyl initiator groups introduced on the surface.

[0009] Furthermore, the preparation of the magnetic oligonucleotide probe in this assay system includes grafting a multifunctional polymer brush onto the surface of the functionalized core-shell magnetic polymer microspheres obtained in step B3 via surface-initiated atom transfer radical polymerization and introducing carboxyl groups onto their surface, specifically including: C1. Raw material preparation: 100 parts of functionalized core-shell magnetic polymer microspheres, acrylic acid and polyethylene glycol methyl ether methacrylate as monomers, cuprous bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine as catalytic system; C2. Surface-initiated polymerization: In a mixed solvent of water and methanol, the product of step B3 is mixed with acrylic acid and polyethylene glycol methyl ether methacrylate, and the molar ratio of acrylic acid to polyethylene glycol methyl ether methacrylate is controlled to be 1:0.3 to 1:1.0. The mixture is reacted at 20 °C to 35 °C for 0.5 h to 3.0 h to form a multifunctional polymer brush layer containing carboxyl groups and polyethylene glycol side chains on the surface of the microspheres. C3. Post-treatment: Free monomers and catalyst residues are removed by magnetic separation. The microspheres are washed 2 to 5 times with deionized water and ethanol alternately, and dried at 30 ℃ to 50 ℃ for 4 h to 12 h to obtain multifunctional polymer brush magnetic microspheres. The surface carboxyl group density of the obtained multifunctional polymer brush magnetic microspheres is 1.0 µmol / m² to 5.0 µmol / m².

[0010] Furthermore, the magnetic oligonucleotide probes of this assay system were obtained by condensing multifunctional polymer brush magnetic microspheres with HRR gene oligonucleotide probes through the following steps: D1. Activation: Multifunctional polymer brush magnetic microspheres were dispersed in a buffer solution with a pH of 5.0 to 6.5, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl groups was 2.0 to 5.0:1, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1.0 to 2.0:1. The reaction was carried out at 15 ℃ to 30 ℃ for 0.25 h to 1.0 h to activate the surface carboxyl groups. D2. Coupling: An HRR gene oligonucleotide probe with a primary amine at the 5′ end was added to the system of step D1. The HRR gene oligonucleotide probe of this assay system was prepared by conventional oligonucleotide synthesis method, and its nucleotide sequence is shown in SEQ ID NO:1 to SEQ ID NO:14. The molar ratio of the HRR gene oligonucleotide probe to the surface-activated ester group of this assay system was controlled to be 0.2 to 1.0:1, and the reaction was carried out at 4 ℃ to 25 ℃ for 2.0 h to 16.0 h to form an amide bond-linked magnetic oligonucleotide probe. D3. Purification: Free oligonucleotides and small molecules were removed by magnetic separation. The sample was washed 2 to 5 times with high-salt buffer and deionized water to obtain magnetic oligonucleotide probes with an oligonucleotide loading of 0.5 nmol to 5.0 nmol per mg of magnetic oligonucleotide probe.

[0011] Furthermore, the HRR gene oligonucleotide probes in this assay system include oligonucleotide probes targeting at least 10 of the following genes: BRCA1, BRCA2, ATM, PALB2, BARD1, CDK12, CHEK1, CHEK2, RAD51B, RAD51C, RAD51D, RAD54L, PPP2R2A, and FANCL. Additionally, the HRR gene oligonucleotide probes in this assay system contain hybridization capture regions complementary to the target nucleic acids. The dPCR amplification reaction components of this assay system are configured to achieve a positive detection rate of no less than 95% for mutation sites with an allele frequency of 0.10% to 0.50% when using this assay system to detect HRR gene mutations. The positive detection rate of this assay system is the percentage of positive results obtained in repeated tests out of the total number of repeated tests.

[0012] As a concept of this invention, core-shell magnetic polymer microspheres are used as the basic carrier. A multifunctional polymer brush interface is constructed through multi-step surface modification and coupled with HRR gene-specific oligonucleotide probes. This design primarily aims to enhance the enrichment efficiency, detection sensitivity, and quantitative accuracy of ultra-low abundance HRR gene mutations in digital PCR. The iron oxide nanocore provides rapid magnetic response, enabling magnetic separation of enriched target nucleic acids within 5 seconds, significantly shortening sample pretreatment time and reducing target loss. The cross-linked polystyrene shell endows the microspheres with excellent mechanical strength and chemical stability, ensuring structural integrity under repeated washing, high-salt buffer, and high-temperature cycling conditions in digital PCR. The dopamine coating forms a stable adhesion layer through strong hydrophobic interactions between the catechol groups and the polystyrene surface, while its amino and hydroxyl groups provide multiple anchoring sites for subsequent silanization reactions. The 3-aminopropyltriethoxysilane layer not only introduces a primary amine for initiator coupling but also enhances the stability of the dopamine coating through a siloxane network. Surface-initiated atom transfer radical polymerization technology enables the controlled copolymerization of acrylic acid and polyethylene glycol methyl ether methacrylate on the surface of microspheres. The acrylic acid unit provides a high density of carboxyl groups for oligonucleotide coupling, achieving a high loading capacity of 0.5 to 5.0 nmol / mg. The polyethylene glycol side chains form a strong hydrophilic shielding layer that effectively inhibits the non-specific adsorption of plasma proteins and nucleic acids and prevents microsphere aggregation. The synergistic effect of the two enables the magnetic oligonucleotide probe to efficiently capture targets and achieve a positive detection rate of not less than 95% in samples with allele frequencies as low as 0.10%. At the same time, it maintains excellent enzyme compatibility and fluorescence signal stability in the digital PCR system, providing a reliable technical platform for the accurate detection of olaparib response biomarkers in prostate cancer.

[0013] This invention also discloses a method for determining olaparib response markers in prostate cancer in vitro using the aforementioned assay system, comprising the following steps: S1. Nucleic acid extraction: Obtain nucleic acid samples containing genomic DNA or cell-free DNA from ex vivo plasma samples or ex vivo tissue specimens of patients with prostate cancer. S2. Enrichment by binding: The nucleic acid sample of this method is mixed with the magnetic oligonucleotide probe of this method in binding and washing buffer and incubated at 20 ℃ to 37 ℃ for 5 min to 60 min to allow the HRR gene fragment to be tested to hybridize and bind with the magnetic oligonucleotide probe of this method. S3. Washing: Perform magnetic separation on the reaction system of step S2, discard the supernatant, and wash 1 to 5 times with the binding and washing buffer of this method to remove unbound nucleic acids and impurities, and obtain the complex of enriched HRR gene target nucleic acid and magnetic oligonucleotide probe of this method. S4. dPCR amplification: Add the dPCR amplification reaction components of this method to the complex obtained in step S3, divide the reaction mixture into multiple small reaction units, denature at 90 ℃ to 98 ℃, anneal at 55 ℃ to 65 ℃, and extend at 60 ℃ to 72 ℃, cycle 35 to 50 times to obtain the digital PCR amplification signal for the HRR gene of this method; S5. Data Analysis: Based on the distribution of positive and negative signals in each dPCR reaction unit, calculate the mutation allele frequency of each HRR gene locus, and determine the expected response of the tested patients to olaparib treatment based on a preset threshold.

[0014] Furthermore, the nucleic acid extraction in step S1 includes lysing the sample using a lysis buffer containing guanidine thiocyanate and a surfactant to remove proteins and lipids; this method includes simultaneous detection of positive and negative control samples for quality control, the positive control sample of this method contains known mutated HRR gene fragments, and the negative control sample of this method does not contain the target mutation site; when performing joint analysis of multiple HRR gene mutation sites, the threshold for predicting significant benefit of olaparib treatment is a mutation allele frequency of at least one of the multiple gene sites of not less than 0.10%; and, by testing plasma samples obtained from the same patient at different time points, changes in HRR gene mutation burden are dynamically monitored to evaluate the efficacy or occurrence of resistance to olaparib.

[0015] Furthermore, before or during amplification in step S4, the magnetic oligonucleotide probe of this method is magnetically positioned on the side of each digital PCR reaction unit closest to the magnetic field source.

[0016] As another aspect of this invention, a method combining magnetic oligonucleotide probe-mediated target enrichment with digital PCR quantitative detection is designed to enhance the detection sensitivity, quantitative accuracy, and clinical feasibility of ultra-low abundance mutations in the HRR gene in prostate cancer patients. In nucleic acid samples extracted from ex vivo plasma or tissue specimens, the abundance of cell-free DNA or genomic DNA is often extremely low and accompanied by a large amount of interference from normal wild-type sequences. Magnetic oligonucleotide probes achieve selective enrichment of targets through HRR gene-specific hybridization capture, increasing the concentration of target gene fragments by 100 to 1000 times, significantly reducing background noise and false negative rates in subsequent digital PCR reactions. The magnetic separation step rapidly removes unbound nucleic acids, plasma proteins, and PCR inhibitors, ensuring that even trace amounts of cell-free DNA extracted from 1 mL of plasma can meet the requirements for digital PCR detection. The single-molecule separation and absolute quantification characteristics of digital PCR, combined with magnetic enrichment of target pretreatment, achieve a positive detection rate of no less than 95% for HRR gene mutation sites with allele frequencies of 0.10% to 0.50%, with detection sensitivity more than 10 times higher than that of traditional unenriched digital PCR. Simultaneous detection of positive and negative control samples ensures the quality control and reliability of each batch of tests. Through multi-gene joint analysis and dynamic monitoring, it is possible to accurately screen patients who will benefit from olaparib before medication, evaluate efficacy in real time after medication, and predict the occurrence of drug resistance, providing a highly sensitive, highly specific, and clinically practical molecular diagnostic solution for the precision treatment of castration-resistant metastatic prostate cancer.

[0017] In this invention, the synergistic effect of core-shell magnetic polymer microspheres and multifunctional polymer brushes is reflected in the high degree of unity between structural stability and functional activity. The iron oxide nanocore, as the magnetic center, possesses high saturation magnetization and rapid magnetic response characteristics, which are the physical basis for achieving rapid target separation. However, exposed iron oxide is easily oxidized, and its high surface energy leads to aggregation. The cross-linked polystyrene shell completely encapsulates the iron oxide nanocore through coating polymerization. Its three-dimensional cross-linked network structure not only protects the magnetic core from oxidation and acid / alkali corrosion but also endows the microspheres with excellent mechanical strength, enabling them to withstand high-temperature cycling and multiple washes in digital PCR without damage. However, the strong hydrophobicity of polystyrene can cause hydrophobic aggregation between microspheres and non-specific adsorption of biomolecules. The dopamine coating, as a key interfacial transition layer, has its catechol groups firmly attached to the polystyrene surface through π-π stacking and hydrophobic interactions. Simultaneously, the exposed amino and hydroxyl groups provide multi-point anchoring for silanization and initially improve surface hydrophilicity. However, the anti-protein adsorption capacity and carboxyl group density of the simple dopamine layer are still insufficient. The acrylic acid-polyethylene glycol methyl ether methacrylate copolymer brush, grafted by surface-initiated atom transfer radical polymerization, plays a decisive synergistic role: the acrylic acid unit provides a high-density carboxyl group of 1.0 to 5.0 µmol / m² for oligonucleotide coupling to achieve a high loading of 0.5 to 5.0 nmol / mg, while the polyethylene glycol side chains form a strong hydrophilic shielding layer with a thickness of approximately 5 to 10 nm. This significantly inhibits non-specific adsorption of plasma proteins and microsphere aggregation through steric hindrance and hydration layer effects, maintaining excellent colloidal stability and biocompatibility in high-salt surfactant buffers and enzyme-containing PCR systems. The rapid separation capability of the magnetic core, the structural integrity of the polystyrene shell, the interfacial bridging effect of the dopamine layer, and the high-density functionalization and strong anti-adsorption properties of the multifunctional polymer brush are all indispensable and mutually reinforcing. Ultimately, this achieves efficient enrichment, accurate quantification, and clinically reproducible detection of ultra-low abundance HRR gene mutations, demonstrating a synergistic effect.

[0018] Beneficial technical effects 1. Achieving ultra-high sensitivity HRR gene mutation detection: By grafting high-density multifunctional polymer brushes onto the surface of core-shell magnetic polymer microspheres and coupling them with HRR gene-specific oligonucleotide probes, a positive detection rate of no less than 95% is achieved for mutation sites with allele frequencies as low as 0.10% to 0.50%. The detection sensitivity is more than 10 times higher than that of traditional unenriched digital PCR. This enables the accurate capture and quantification of ultra-low abundance HRR gene mutations in trace liquid biopsy samples, providing highly sensitive molecular diagnostic evidence for olapapa use decisions in castration-resistant metastatic prostate cancer patients.

[0019] 2. Significantly improves target enrichment efficiency and detection specificity: Magnetic oligonucleotide probes increase target DNA concentration by 100 to 1000 times through HRR gene-specific hybridization capture. Combined with rapid magnetic separation to remove unbound nucleic acids, plasma proteins and PCR inhibitors, even trace amounts of free DNA extracted from 1 mL of plasma can still meet the requirements of digital PCR detection. At the same time, the strong hydrophilic shielding effect of the multifunctional polymer brush layer significantly reduces non-specific adsorption, ensuring high specificity and low false positive rate of detection.

[0020] 3. Excellent digital PCR system compatibility and result stability: The strong hydrophilic interface formed by the polyethylene glycol side chains in the multifunctional polymer brush layer effectively prevents the microspheres from agglomerating and settling during the high-temperature cycling process of digital PCR, maintaining the randomness and uniformity of single-molecule template distribution in droplets or chip microwells. At the same time, the polymer brush interface does not significantly inhibit the activity of heat-resistant DNA polymerase, ensuring the amplification efficiency and the stability of fluorescence signal, making the coefficient of variation of repeated detection less than 10%, which meets the precision requirements of clinical diagnosis.

[0021] 4. Enables multi-gene joint detection and dynamic monitoring: A single reaction can simultaneously detect the mutation status of at least 10 HRR genes, including BRCA1, BRCA2, ATM, PALB2, BARD1, and CDK12. Joint analysis improves the accuracy of olaparib response prediction. Furthermore, by continuously testing plasma samples from the same patient at different time points, it is possible to dynamically monitor changes in HRR gene mutation burden to evaluate efficacy and predict drug resistance, providing a real-time molecular diagnostic tool for the whole-process management of precision treatment for prostate cancer.

[0022] 5. Controllable preparation process and excellent material stability: The core-shell magnetic polymer microspheres are prepared using mature emulsion polymerization technology. Surface functionalization is achieved step by step through dopamine self-polymerization, silanization and surface-initiated atom transfer radical polymerization. The process parameters are controllable and reproducible. The magnetic oligonucleotide probes obtained show no significant decrease in activity after being stored at 4°C for 6 months. The performance difference between batches is less than 5%, making them suitable for industrial production and clinical application. Attached Figure Description

[0023] Figure 1 Box plots showing the shell thickness of Examples 1, 2, 3, 4, Comparative Example 2, and Comparative Example 3.

[0024] Figure 2 The images show the EDS line scan curves for Example 1, Comparative Example 2, and Comparative Example 3.

[0025] Figure 3 The image shows a comparison of the XPS C1s high-resolution spectrum multi-peak fitting of Example 1 and Comparative Example 5.

[0026] Figure 4 The graph shows the O / C atomic ratio as a function of sputtering depth for Examples 1 and Comparative Example 5.

[0027] Figure 5 The bar chart shows the N 1s peak intensity for Examples 1, 2, 3, 4, Comparative Example 4, Comparative Example 5, and Comparative Example 8.

[0028] Figure 6 The fluorescence intensity distribution histograms are for Example 1 and Comparative Example 3.

[0029] Figure 7 The graphs show the aggregation index of Examples 1, 1 Comparative Example 2, and 3 as a function of magnetic field strength. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0031] [Terminology and Measurement Explanation] (1) In this instruction manual, “part” refers to mass parts, which are used to indicate the relative mass ratio of each component. When a component is “100 parts”, it means that the mass of that component is taken as the basis, and other components are converted according to the mass parts ratio (for example, if 1.0 g of core-shell magnetic polymer microspheres is taken as 100 parts, then 5.5 parts of dopamine hydrochloride corresponds to 55 mg).

[0032] (2) “HRR gene oligonucleotide probe” refers to a free oligonucleotide probe with a primary amine (e.g., 5′-NH2-C6-) modified at the 5′ end; “magnetic oligonucleotide probe” refers to the complex of the HRR gene oligonucleotide probe linked to magnetic microspheres via a carbodiimide-mediated coupling reaction.

[0033] (3) “Core-shell magnetic polymer microspheres” refers to microspheres obtained in steps A1–A4 with iron oxide as the core and polymer as the shell; “functionalized core-shell magnetic polymer microspheres” refers to microspheres after introducing initiators on the surface in steps B1–B3; “multifunctional polymer brush magnetic microspheres” refers to microspheres after grafting polymer brushes on the surface in steps C1–C3.

[0034] (4) In this instruction manual, “magnetic separation” means: placing the suspension containing magnetic microspheres next to a magnetic rack or permanent magnet and letting it stand until the microspheres aggregate (usually 1–10 min, depending on the particle size and concentration), removing the supernatant, and then resuspending and washing.

[0035] Example 1 This embodiment provides a system for determining olaparib response markers in prostate cancer, including magnetic oligonucleotide probes, dPCR amplification reaction components, and binding and washing buffers.

[0036] The magnetic oligonucleotide probe in this embodiment was obtained by a carbodiimide-mediated amidation coupling reaction between core-shell magnetic polymer microspheres and HRR gene oligonucleotide probes with a 5′ amino group. The HRR gene oligonucleotide probe in this embodiment was used to detect 12 homologous recombination repair-related genes: BRCA1, BRCA2, ATM, PALB2, BARD1, CDK12, CHEK1, CHEK2, RAD51B, RAD51C, RAD51D, and RAD54L.

[0037] The core-shell magnetic polymer microspheres in this embodiment were prepared through the following steps: Step A1 Raw material preparation: Weigh 3.0 parts of iron oxide nanoparticles. In this embodiment, the iron oxide nanoparticles are prepared by step A2. Weigh 25 parts of styrene, 4.5 parts of divinylbenzene, 2.5 parts of sodium dodecyl sulfate emulsifier, and 1.0 part of potassium persulfate initiator.

[0038] Step A2: Preparation of ferric oxide nuclei: Soluble ferrous salts and ferric salts are prepared according to... : A mixed iron salt solution was prepared with a molar ratio of approximately 1:2. NaOH solution was added under stirring to adjust the pH of the system to 10.0 ± 0.2. It is preferable to carry out the process under inert gas protection or deoxygenated water conditions to reduce the risk of oxidation. Oxidation was performed, followed by reaction at 80°C for 1.25 hours to obtain iron oxide nanoparticles with a particle size of approximately 100 nm.

[0039] Step A3: Coating polymerization: The iron oxide nanoparticles obtained in step A2 were ultrasonically dispersed in an aqueous phase containing sodium dodecyl sulfate. Styrene and divinylbenzene monomers were added, and emulsion polymerization was carried out at 78°C for 3.0 hours under nitrogen protection to obtain core-shell magnetic polymer microspheres with iron oxide as the core and cross-linked polystyrene as the shell. The median particle size of the obtained microspheres was 275 nm, and the shell thickness was 50 nm. Transmission electron microscopy showed a clear core-shell interface.

[0040] Step A4 Washing and Drying: The emulsion obtained in step A3 is magnetically separated, washed three times with deionized water, and dried at 50°C for 8 hours to obtain the core-shell magnetic polymer microspheres of this embodiment.

[0041] The surface functionalization of the core-shell magnetic polymer microspheres in this embodiment is carried out through the following steps to obtain functionalized core-shell magnetic polymer microspheres: Step B1 Dopamine Coating: The core-shell magnetic polymer microspheres of this embodiment are dispersed in a tris(hydroxymethyl)aminomethane buffer solution with a concentration of 105 mmol / L and a pH of 8.5. In this embodiment, the pH value is adjusted by hydrochloric acid. 5.5 parts of dopamine hydrochloride are added to 100 parts of core-shell magnetic polymer microspheres, and the mixture is stirred at 25°C for 2.0 hours to allow dopamine to self-polymerize and form a dopamine coating on the surface of the microspheres.

[0042] Step B2 Silane Coupling: 6.0 parts of 3-aminopropyltriethoxysilane were added to the product of step B1 and reacted at 50°C for 2.0 hours to introduce a surface aminosilane layer.

[0043] Step B3: Initiator introduction: The product of step B2 was reacted with 2-bromoisobutyryl bromide in anhydrous dichloromethane in the presence of triethylamine, so that the initiator in this embodiment was amidated with the surface amino group; the amount of 2-bromoisobutyryl bromide was controlled to be 2.0 times the molar amount of 3-aminopropyltriethoxysilane, and 2-bromoisobutyryl bromide was added dropwise, and the reaction was carried out at 5°C for 1.25 hours to obtain functionalized core-shell magnetic polymer microspheres with α-bromoacyl initiator introduced on the surface.

[0044] The preparation of the magnetic oligonucleotide probe in this embodiment includes grafting a multifunctional polymer brush onto the surface of the functionalized core-shell magnetic polymer microspheres obtained in step B3 via surface-initiated atom transfer radical polymerization and introducing carboxyl groups onto their surface, specifically including: Step C1 Raw material preparation: Weigh 100 parts of functionalized core-shell magnetic polymer microspheres, use acrylic acid and polyethylene glycol methyl ether methacrylate as monomers, and use cuprous bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine as the catalytic system.

[0045] Step C2: Surface-initiated polymerization: In a mixed solvent of water and methanol at a volume ratio of 1:1, the product of step B3 is mixed with acrylic acid and polyethylene glycol methyl ether methacrylate. The molar ratio of acrylic acid to polyethylene glycol methyl ether methacrylate is controlled at 1:0.65, and the molar ratio of cuprous bromide to N,N,N′,N″,N″-pentamethyldiethylenetriamine is controlled at 1.0:1.9. In this embodiment, after nitrogen bubbling deoxygenation, the reaction is carried out at 28°C for 1.75 hours to form a multifunctional polymer brush layer containing carboxyl groups and polyethylene glycol side chains on the surface of the microspheres.

[0046] Step C3 post-treatment: Free monomers and catalyst residues were removed by magnetic separation, and the microspheres were washed three times alternately with deionized water and ethanol. They were then dried at 40°C for 8 hours to obtain multifunctional polymer brush magnetic microspheres with a surface carboxyl group density of 3.0 μmol / m².

[0047] The magnetic oligonucleotide probe in this embodiment was obtained by condensing a multifunctional polymer brush magnetic microsphere with an HRR gene oligonucleotide probe through the following steps: Step D1 Activation: The multifunctional polymer brush magnetic microspheres were dispersed in a 2-(N-morpholine)ethanesulfonic acid buffer solution with a pH of 5.75. In this embodiment, the buffer solution concentration was 102.5 mmol / L. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl groups was 3.5:1, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1.5:1. The reaction was carried out at 22.5°C for 0.625 hours to activate the surface carboxyl groups.

[0048] Step D2 Coupling: Add the HRR gene oligonucleotide probe with a primary amine at the 5′ end to the system of step D1. The HRR gene oligonucleotide probe in this embodiment is prepared by conventional oligonucleotide synthesis method. Control the molar ratio of the HRR gene oligonucleotide probe to the surface activated ester group in this embodiment to be 0.6:1, and react at 14.5°C for 9.0 hours to form a magnetic oligonucleotide probe linked by an amide bond.

[0049] Step D3 Purification: Free oligonucleotides and small molecules were removed by magnetic separation. The sample was washed three times with a high-salt buffer solution with a sodium chloride concentration of 1.15 mol / L and a pH of 7.25 and deionized water to obtain a magnetic oligonucleotide probe with an oligonucleotide loading of 2.75 nanomoles per milligram.

[0050] The HRR gene oligonucleotide probe of this embodiment contains a hybridization capture region complementary to the target nucleic acid. The dPCR amplification reaction components of this embodiment are configured to achieve a positive detection rate of not less than 95% for mutation sites with an allele frequency of 0.30% when using the assay system of this embodiment to detect HRR gene mutations. The positive detection rate of this embodiment is the percentage of positive results obtained in repeated tests out of the total number of repeated tests.

[0051] The dPCR amplification reaction components of this embodiment include thermostable DNA polymerase, deoxynucleoside triphosphate, magnesium-containing PCR buffer, amplification primer pairs for amplifying the HRR gene target nucleic acid of this embodiment, and fluorescence detection components.

[0052] The binding and washing buffer in this embodiment is an aqueous solution containing 105 mmol / L of tris(hydroxymethyl)aminomethane, 1.05 mmol / L of sodium chloride, 10.5 mmol / L of disodium ethylenediaminetetraacetate dihydrate, and 0.505% by mass of the nonionic surfactant Tween-20. The pH of the binding and washing buffer in this embodiment is 7.5, and it is adjusted to the target pH value using hydrochloric acid solution.

[0053] In a single 20 μL dPCR reaction system, the amount of magnetic oligonucleotide probe used in this embodiment is 100 μg. The dPCR amplification reaction in this embodiment is used to quantitatively detect the frequency of mutant alleles in the HRR gene of this embodiment and to predict the response of prostate cancer patients to olaparib treatment.

[0054] The method for determining olaparib response markers in prostate cancer in vitro using the assay system of this embodiment includes the following steps: Step S1 Nucleic acid extraction: Nucleic acid samples containing cell-free DNA were obtained from the ex vivo plasma samples of the tested prostate cancer patients; in this embodiment, a lysis buffer containing guanidine thiocyanate and a surfactant was used to lyse the sample and remove proteins and lipids. The concentration of guanidine thiocyanate in the lysis buffer in this embodiment was 3.5 mol / L, and the surfactant in this embodiment was sodium dodecyl sulfate with a mass fraction of 2.525%.

[0055] Step S2: Enrichment: The nucleic acid sample of this embodiment and the magnetic oligonucleotide probe of this embodiment are mixed in binding and washing buffer and incubated at 28.5°C for 32.5 minutes to allow the HRR gene fragment to be tested to hybridize and bind with the magnetic oligonucleotide probe of this embodiment.

[0056] Step S3 Washing: Perform magnetic separation on the reaction system of step S2, discard the supernatant, and wash three times with the binding and washing buffer of this embodiment to remove unbound nucleic acids and impurities, to obtain the complex of enriched HRR gene target nucleic acid and magnetic oligonucleotide probe of this embodiment.

[0057] Step S4 dPCR amplification: The dPCR amplification reaction components of this embodiment are added to the complex obtained in step S3. The reaction mixture is divided into micro-reaction units in the form of microchip wells. The number of micro-reaction units formed in a single reaction in this embodiment is 100,500. Denaturation is performed at 94°C, annealing is performed at 60°C, and extension is performed at 66°C for 42 cycles to obtain the digital PCR amplification signal for the HRR gene of this embodiment.

[0058] Step S5 Data Analysis: Based on the distribution of positive and negative signals in each dPCR reaction unit, calculate the mutation allele frequency of each HRR gene locus, and determine the expected response of the tested patients to olaparib treatment according to the preset threshold.

[0059] This embodiment simultaneously tests positive and negative control samples for quality control. The positive control samples in this embodiment contain known mutated HRR gene fragments, while the negative control samples do not contain the target mutation site. When performing joint analysis on multiple HRR gene mutation sites, the threshold for predicting significant benefit of olaparib treatment is a mutation allele frequency of at least one of the multiple gene sites of not less than 0.10%. Furthermore, plasma samples obtained from the same patient at different time points are tested to dynamically monitor changes in HRR gene mutation burden in order to evaluate the efficacy of olaparib or the occurrence of drug resistance.

[0060] In this embodiment, during the amplification process in step S4, the magnetic oligonucleotide probe of this embodiment is magnetically positioned on the side of each digital PCR reaction unit closest to the magnetic field source.

[0061] The assay system in this embodiment is used to predict or monitor the response of prostate cancer patients to olaparib treatment in vitro. In this embodiment, the prostate cancer patients are castration-resistant metastatic prostate cancer patients, and the patients are stratified according to whether the HRR gene mutation allele frequency reaches a preset threshold, so as to provide a reference for olaparib treatment decision-making.

[0062] Features of Example 1: This example uses moderate parameter configurations: 3.0 parts of iron oxide nanoparticles, 25 parts of styrene, a median microsphere diameter of 275 nm, a shell thickness of 50 nm, a surface carboxyl density of 3.0 μmol / m², and 100 μg of magnetic probe. These parameters are selected within the middle range of the technical solution to ensure the stability and reproducibility of the preparation process. This example is suitable for routine clinical testing scenarios, particularly for establishing standardized testing procedures and quality control systems, and can provide reliable molecular diagnostic evidence for olapapa drug use decisions in prostate cancer patients.

[0063] Example 2 This embodiment provides a system for determining olaparib response markers in prostate cancer, including magnetic oligonucleotide probes, dPCR amplification reaction components, and binding and washing buffers.

[0064] The magnetic oligonucleotide probe in this embodiment is obtained by a carbodiimide-mediated amidation coupling reaction between core-shell magnetic polymer microspheres and HRR gene oligonucleotide probes with a 5′ amino group. The HRR gene oligonucleotide probe in this embodiment is used to detect 10 homologous recombination repair-related genes: BRCA1, BRCA2, ATM, PALB2, BARD1, CHEK1, CHEK2, RAD51B, RAD51C, and RAD51D.

[0065] The core-shell magnetic polymer microspheres in this embodiment were prepared through the following steps: Step A1 Raw material preparation: Weigh 1.8 parts of iron oxide nanoparticles. In this embodiment, the iron oxide nanoparticles are prepared by step A2. Weigh 16 parts of styrene, 2.4 parts of divinylbenzene, 1.2 parts of sodium dodecyl sulfate emulsifier, and 0.4 parts of potassium persulfate initiator.

[0066] Step A2: Preparation of iron oxide cores: Prepare an iron salt solution and carry out a co-precipitation reaction under alkaline conditions. Adjust the pH value to 9.6 and react at 76℃ for 0.9 hours to obtain iron oxide nanoparticles with a particle size of 70 nanometers.

[0067] Step A3: Coating polymerization: The iron oxide nanoparticles obtained in step A2 were dispersed in an aqueous phase containing sodium dodecyl sulfate by high-speed shearing. Styrene and divinylbenzene monomers were added, and emulsion polymerization was carried out at 74°C for 2.6 hours under nitrogen protection to obtain core-shell magnetic polymer microspheres with iron oxide as the core and cross-linked polystyrene as the shell. The median particle size of the obtained microspheres was 225 nm, and the shell thickness was 38 nm. The clear core-shell interface was shown by transmission electron microscopy.

[0068] Step A4 Washing and Drying: The emulsion obtained in step A3 is magnetically separated, washed three times with deionized water, and dried at 46°C for 7 hours to obtain the core-shell magnetic polymer microspheres of this embodiment.

[0069] The surface functionalization of the core-shell magnetic polymer microspheres in this embodiment is carried out through the following steps to obtain functionalized core-shell magnetic polymer microspheres: Step B1 Dopamine Coating: The core-shell magnetic polymer microspheres of this embodiment are dispersed in a tris(hydroxymethyl)aminomethane buffer solution with a concentration of 68 mmol / L and a pH of 8.3. In this embodiment, the pH value is adjusted by hydrochloric acid. 3.7 parts of dopamine hydrochloride are added to 100 parts of core-shell magnetic polymer microspheres, and the mixture is stirred at 23°C for 1.6 hours to allow dopamine to self-polymerize and form a dopamine coating on the surface of the microspheres.

[0070] Step B2 Silane Coupling: 4.4 parts of 3-aminopropyltriethoxysilane were added to the product of step B1, and the mixture was reacted at 46°C for 1.6 hours to introduce a surface aminosilane layer.

[0071] Step B3: Initiator introduction: The product of step B2 was reacted with 2-bromoisobutyryl bromide in anhydrous tetrahydrofuran in the presence of triethylamine, so that the initiator in this embodiment was amidated with the surface amino group; the amount of 2-bromoisobutyryl bromide was controlled to be 1.6 times the molar amount of 3-aminopropyltriethoxysilane, and 2-bromoisobutyryl bromide was added dropwise, and the reaction was carried out at 3°C ​​for 0.9 hours to obtain functionalized core-shell magnetic polymer microspheres with α-bromoacyl initiator introduced on the surface.

[0072] The preparation of the magnetic oligonucleotide probe in this embodiment includes grafting a multifunctional polymer brush onto the surface of the functionalized core-shell magnetic polymer microspheres obtained in step B3 via surface-initiated atom transfer radical polymerization and introducing carboxyl groups onto their surface, specifically including: Step C1 Raw material preparation: Weigh 100 parts of functionalized core-shell magnetic polymer microspheres, use acrylic acid and polyethylene glycol methyl ether methacrylate as monomers, and use cuprous bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine as the catalytic system.

[0073] Step C2: Surface-initiated polymerization: In a mixed solvent of water and methanol at a volume ratio of 6:1, the product of step B3 was mixed with acrylic acid and polyethylene glycol methyl ether methacrylate. The molar ratio of acrylic acid to polyethylene glycol methyl ether methacrylate was controlled at 1:0.44, and the molar ratio of cuprous bromide to N,N,N′,N″,N″-pentamethyldiethylenetriamine was controlled at 1.0:1.3. In this embodiment, after vacuuming and nitrogen deoxygenation, the reaction was carried out at 24°C for 1.1 hours to form a multifunctional polymer brush layer containing carboxyl groups and polyethylene glycol side chains on the surface of the microspheres.

[0074] Step C3 post-treatment: Free monomers and catalyst residues were removed by magnetic separation, and the microspheres were washed three times alternately with deionized water and ethanol. They were then dried at 36°C for 6.4 hours to obtain multifunctional polymer brush magnetic microspheres with a surface carboxyl group density of 2.0 μmol / m².

[0075] The magnetic oligonucleotide probe in this embodiment was obtained by condensing a multifunctional polymer brush magnetic microsphere with an HRR gene oligonucleotide probe through the following steps: Step D1 Activation: The multifunctional polymer brush magnetic microspheres were dispersed in a phosphate buffer solution with a pH of 5.3. In this embodiment, the concentration of the buffer solution was 63 mmol / L. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl groups was 2.6:1, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1.3:1. The reaction was carried out at 18°C ​​for 0.4 hours to activate the surface carboxyl groups.

[0076] Step D2 Coupling: Add the HRR gene oligonucleotide probe with a primary amine at the 5′ end to the system of step D1. The HRR gene oligonucleotide probe in this embodiment is prepared by conventional oligonucleotide synthesis method. The molar ratio of the HRR gene oligonucleotide probe to the surface activated ester group in this embodiment is controlled to be 0.36:1. The reaction is carried out at 10°C for 5.6 hours to form a magnetic oligonucleotide probe linked by an amide bond.

[0077] Step D3 Purification: Free oligonucleotides and small molecules were removed by magnetic separation. The sample was washed three times with a high-salt buffer solution with a sodium chloride concentration of 0.6 mol / L and a pH of 6.5 and deionized water to obtain a magnetic oligonucleotide probe with an oligonucleotide loading of 1.4 nanomoles per milligram.

[0078] The HRR gene oligonucleotide probe of this embodiment contains a hybridization capture region complementary to the target nucleic acid. The dPCR amplification reaction components of this embodiment are configured to achieve a positive detection rate of not less than 95% for mutation sites with an allele frequency of 0.18% when using the assay system of this embodiment to detect HRR gene mutations. The positive detection rate of this embodiment is the percentage of positive results obtained in repeated tests out of the total number of repeated tests.

[0079] The dPCR amplification reaction components of this embodiment include thermostable DNA polymerase, deoxynucleoside triphosphate, magnesium-containing PCR buffer, amplification primer pairs for amplifying the HRR gene target nucleic acid of this embodiment, and fluorescence detection components.

[0080] The binding and washing buffer in this embodiment is an aqueous solution containing 67 mmol / L of tris(hydroxymethyl)aminomethane, 0.67 mmol / L of sodium chloride, 6.7 mmol / L of disodium ethylenediaminetetraacetate dihydrate, and 0.297% by mass of the nonionic surfactant Tween-20. The pH of the binding and washing buffer in this embodiment is 7.0, and it is adjusted to the target pH value using sodium hydroxide solution.

[0081] In a single 20 μL dPCR reaction system, the amount of magnetic oligonucleotide probe used in this embodiment is 60 μg. The dPCR amplification reaction in this embodiment is used to quantitatively detect the frequency of mutant alleles in the HRR gene and to predict the response of prostate cancer patients to olaparib treatment.

[0082] The method for determining olaparib response markers in prostate cancer in vitro using the assay system of this embodiment includes the following steps: Step S1 Nucleic acid extraction: Nucleic acid samples containing genomic DNA were obtained from the ex vivo tissue specimens of the examined prostate cancer patients; in this embodiment, the sample was lysed and proteins and lipids were removed using a lysis buffer containing guanidine thiocyanate and a surfactant. The concentration of guanidine thiocyanate in the lysis buffer in this embodiment was 2.5 mol / L, and the surfactant in this embodiment was Triton X-100 with a mass fraction of 1.525%.

[0083] Step S2: Enrichment: Mix the nucleic acid sample of this embodiment with the magnetic oligonucleotide probe of this embodiment in binding and washing buffer, and incubate at 24°C for 22 minutes to allow the HRR gene fragment to be tested to hybridize and bind with the magnetic oligonucleotide probe of this embodiment.

[0084] Step S3 Washing: Perform magnetic separation on the reaction system of step S2, discard the supernatant, and wash twice with the binding and washing buffer of this embodiment to remove unbound nucleic acids and impurities, to obtain the complex of enriched HRR gene target nucleic acid and magnetic oligonucleotide probe of this embodiment.

[0085] Step S4 dPCR amplification: The dPCR amplification reaction components of this embodiment are added to the complex obtained in step S3. The reaction mixture is divided into droplet-shaped micro-reaction units. The number of micro-reaction units formed in a single reaction in this embodiment is 60,800. Denaturation is performed at 92°C, annealing is performed at 58°C, and extension is performed at 64°C for 38 cycles to obtain a digital PCR amplification signal for the HRR gene of this embodiment.

[0086] Step S5 Data Analysis: Based on the distribution of positive and negative signals in each dPCR reaction unit, calculate the mutation allele frequency of each HRR gene locus, and determine the expected response of the tested patients to olaparib treatment according to the preset threshold.

[0087] This embodiment simultaneously tests positive and negative control samples for quality control. The positive control samples in this embodiment contain known mutated HRR gene fragments, while the negative control samples do not contain the target mutation site. When performing joint analysis on multiple HRR gene mutation sites, the threshold for predicting significant benefit of olaparib treatment is a mutation allele frequency of at least one of the multiple gene sites of not less than 0.10%. Furthermore, plasma samples obtained from the same patient at different time points are tested to dynamically monitor changes in HRR gene mutation burden in order to evaluate the efficacy of olaparib or the occurrence of drug resistance.

[0088] In this embodiment, before amplification in step S4, the magnetic oligonucleotide probe of this embodiment is magnetically positioned on the side of each digital PCR reaction unit closest to the magnetic field source.

[0089] The assay system in this embodiment is used to predict or monitor the response of prostate cancer patients to olaparib treatment in vitro. In this embodiment, the prostate cancer patients are castration-resistant metastatic prostate cancer patients, and the patients are stratified according to whether the HRR gene mutation allele frequency reaches a preset threshold, so as to provide a reference for olaparib treatment decision-making.

[0090] Features of Example 2: This example uses a relatively low level of parameter configuration. The amount of iron oxide nanoparticles is 1.8 parts, the amount of styrene is 16 parts, the median particle size of the microspheres is 225 nm, the shell thickness is 38 nm, the surface carboxyl density is 2.0 μmol / m², and the amount of magnetic probe is 60 μg. The parameters are selected to be in the lower range of technical solutions. The magnetic microspheres prepared in this example have a small particle size, which is beneficial to improving the dispersion stability in solution and the capture efficiency of target nucleic acids. It is suitable for detecting plasma samples with low concentrations of free DNA or clinical scenarios requiring high sensitivity detection, and is particularly suitable for early screening and monitoring of minimal residual lesions.

[0091] Example 3 This embodiment provides a system for determining olaparib response markers in prostate cancer, including magnetic oligonucleotide probes, dPCR amplification reaction components, and binding and washing buffers.

[0092] The magnetic oligonucleotide probe in this embodiment was obtained by a carbodiimide-mediated amidation coupling reaction between core-shell magnetic polymer microspheres and HRR gene oligonucleotide probes with a 5′ amino group. The HRR gene oligonucleotide probe in this embodiment was used to detect 14 homologous recombination repair-related genes, including BRCA1, BRCA2, ATM, PALB2, BARD1, CDK12, CHEK1, CHEK2, RAD51B, RAD51C, RAD51D, RAD54L, PPP2R2A, and FANCL.

[0093] The core-shell magnetic polymer microspheres in this embodiment were prepared through the following steps: Step A1 Raw material preparation: Weigh 4.2 parts of iron oxide nanoparticles. In this embodiment, the iron oxide nanoparticles are prepared by step A2. Weigh 34 parts of styrene, 6.4 parts of divinylbenzene, 3.9 parts of sodium dodecyl sulfate emulsifier, and 1.6 parts of potassium persulfate initiator.

[0094] Step A2: Preparation of iron oxide cores: Prepare an iron salt solution and carry out a co-precipitation reaction under alkaline conditions. Adjust the pH value to 10.4 and react at 86℃ for 1.6 hours to obtain iron oxide nanoparticles with a particle size of 130 nanometers.

[0095] Step A3: Coating polymerization: The iron oxide nanoparticles obtained in step A2 were ultrasonically dispersed in an aqueous phase containing sodium dodecyl sulfate. Styrene and divinylbenzene monomers were added, and emulsion polymerization was carried out at 82°C for 3.4 hours under argon protection to obtain core-shell magnetic polymer microspheres with iron oxide as the core and cross-linked polystyrene as the shell. The median particle size of the obtained microspheres was 325 nm, and the shell thickness was 62 nm. Transmission electron microscopy showed a clear core-shell interface.

[0096] Step A4 Washing and Drying: The emulsion obtained in step A3 was magnetically separated, washed four times with deionized water, and dried at 54°C for 9.2 hours to obtain the core-shell magnetic polymer microspheres of this embodiment.

[0097] The surface functionalization of the core-shell magnetic polymer microspheres in this embodiment is carried out through the following steps to obtain functionalized core-shell magnetic polymer microspheres: Step B1 Dopamine Coating: The core-shell magnetic polymer microspheres of this embodiment are dispersed in a tris(hydroxymethyl)aminomethane buffer solution with a concentration of 142 mmol / L and a pH of 8.7. In this embodiment, the pH value is adjusted by sodium hydroxide. 7.3 parts of dopamine hydrochloride are added to 100 parts of core-shell magnetic polymer microspheres, and the mixture is stirred at 27°C for 2.4 hours to allow dopamine to self-polymerize and form a dopamine coating on the surface of the microspheres.

[0098] Step B2 Silane Coupling: 7.6 parts of 3-aminopropyltriethoxysilane were added to the product of step B1 and reacted at 54°C for 2.4 hours to introduce a surface aminosilane layer.

[0099] Step B3: Initiator introduction: The product of step B2 was reacted with 2-bromoisobutyryl bromide in anhydrous dichloromethane in the presence of triethylamine, so that the initiator in this embodiment was amidated with the surface amino group; the amount of 2-bromoisobutyryl bromide was controlled to be 2.4 times the molar amount of 3-aminopropyltriethoxysilane, and 2-bromoisobutyryl bromide was added dropwise, and the reaction was carried out at 7°C for 1.6 hours to obtain functionalized core-shell magnetic polymer microspheres with α-bromoacyl initiator introduced on the surface.

[0100] The preparation of the magnetic oligonucleotide probe in this embodiment includes grafting a multifunctional polymer brush onto the surface of the functionalized core-shell magnetic polymer microspheres obtained in step B3 via surface-initiated atom transfer radical polymerization and introducing carboxyl groups onto their surface, specifically including: Step C1 Raw material preparation: Weigh 100 parts of functionalized core-shell magnetic polymer microspheres, use acrylic acid and polyethylene glycol methyl ether methacrylate as monomers, and use cuprous bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine as the catalytic system.

[0101] Step C2: Surface-initiated polymerization: In a mixed solvent of water and methanol at a volume ratio of 1:6, the product of step B3 is mixed with acrylic acid and polyethylene glycol methyl ether methacrylate. The molar ratio of acrylic acid to polyethylene glycol methyl ether methacrylate is controlled at 1:0.82, and the molar ratio of cuprous bromide to N,N,N′,N″,N″-pentamethyldiethylenetriamine is controlled at 1.0:2.5. In this embodiment, after nitrogen bubbling deoxygenation, the reaction is carried out at 32°C for 2.4 hours to form a multifunctional polymer brush layer containing carboxyl groups and polyethylene glycol side chains on the surface of the microspheres.

[0102] Step C3 post-treatment: Free monomers and catalyst residues were removed by magnetic separation, and the microspheres were washed four times alternately with deionized water and ethanol. They were then dried at 44°C for 9.6 hours to obtain multifunctional polymer brush magnetic microspheres with a surface carboxyl group density of 4.0 μmol / m².

[0103] The magnetic oligonucleotide probe in this embodiment was obtained by condensing a multifunctional polymer brush magnetic microsphere with an HRR gene oligonucleotide probe through the following steps: Step D1 Activation: The multifunctional polymer brush magnetic microspheres were dispersed in a 2-(N-morpholine)ethanesulfonic acid buffer solution with a pH of 6.2. In this embodiment, the buffer solution concentration was 142 mmol / L. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl groups was 4.2:1, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1.7:1. The reaction was carried out at 26°C for 0.85 hours to activate the surface carboxyl groups.

[0104] Step D2 Coupling: Add the HRR gene oligonucleotide probe with a primary amine at the 5′ end to the system of step D1. The HRR gene oligonucleotide probe in this embodiment is prepared by conventional oligonucleotide synthesis method. The molar ratio of the HRR gene oligonucleotide probe to the surface activated ester group in this embodiment is controlled to be 0.84:1. The reaction is carried out at 19°C for 12.4 hours to form a magnetic oligonucleotide probe linked by an amide bond.

[0105] Step D3 Purification: Free oligonucleotides and small molecules were removed by magnetic separation. The sample was washed four times with a high-salt buffer solution with a sodium chloride concentration of 1.7 mol / L and a pH of 8.0 and deionized water to obtain a magnetic oligonucleotide probe with an oligonucleotide loading of 4.1 nanomoles per milligram.

[0106] The HRR gene oligonucleotide probe of this embodiment contains a hybridization capture region complementary to the target nucleic acid. The dPCR amplification reaction components of this embodiment are configured to achieve a positive detection rate of not less than 95% for mutation sites with an allele frequency of 0.42% when using the assay system of this embodiment to detect HRR gene mutations. The positive detection rate of this embodiment is the percentage of positive results obtained in repeated tests out of the total number of repeated tests.

[0107] The dPCR amplification reaction components of this embodiment include thermostable DNA polymerase, deoxynucleoside triphosphate, magnesium-containing PCR buffer, amplification primer pairs for amplifying the HRR gene target nucleic acid of this embodiment, and fluorescence detection components.

[0108] The binding and washing buffer in this embodiment is an aqueous solution containing 143 mmol / L of tris(hydroxymethyl)aminomethane, 1.43 mmol / L of sodium chloride, 14.3 mmol / L of disodium ethylenediaminetetraacetate dihydrate, and 0.713% by mass of the nonionic surfactant Tween-20. The pH of the binding and washing buffer in this embodiment is 8.0, and it is adjusted to the target pH value using sodium hydroxide solution.

[0109] In a single 20 μL dPCR reaction system, the amount of magnetic oligonucleotide probe used in this embodiment is 140 μg. The dPCR amplification reaction in this embodiment is used to quantitatively detect the frequency of mutant alleles in the HRR gene and to predict the response of prostate cancer patients to olaparib treatment.

[0110] The method for determining olaparib response markers in prostate cancer in vitro using the assay system of this embodiment includes the following steps: Step S1 Nucleic acid extraction: Nucleic acid samples containing cell-free DNA were obtained from the ex vivo plasma samples of the tested prostate cancer patients; in this embodiment, the sample was lysed and proteins and lipids were removed using a lysis buffer containing guanidine thiocyanate and a surfactant. The concentration of guanidine thiocyanate in the lysis buffer in this embodiment was 4.5 mol / L, and the surfactant in this embodiment was Triton X-100 with a mass fraction of 3.525%.

[0111] Step S2: Enrichment: The nucleic acid sample of this embodiment and the magnetic oligonucleotide probe of this embodiment are mixed in binding and washing buffer and incubated at 33°C for 43 minutes to allow the HRR gene fragment to be tested to hybridize and bind with the magnetic oligonucleotide probe of this embodiment.

[0112] Step S3 Washing: Perform magnetic separation on the reaction system of step S2, discard the supernatant, and wash 4 times with the binding and washing buffer of this embodiment to remove unbound nucleic acids and impurities, and obtain the complex of enriched HRR gene target nucleic acid and magnetic oligonucleotide probe of this embodiment.

[0113] Step S4 dPCR amplification: The dPCR amplification reaction components of this embodiment are added to the complex obtained in step S3. The reaction mixture is divided into micro-reaction units in the form of microchip wells. The number of micro-reaction units formed in a single reaction of this embodiment is 139,200. Denaturation is performed at 96°C, annealing is performed at 62°C, and extension is performed at 70°C for 46 cycles to obtain the digital PCR amplification signal for the HRR gene of this embodiment.

[0114] Step S5 Data Analysis: Based on the distribution of positive and negative signals in each dPCR reaction unit, calculate the mutation allele frequency of each HRR gene locus, and determine the expected response of the tested patients to olaparib treatment according to the preset threshold.

[0115] This embodiment simultaneously tests positive and negative control samples for quality control. The positive control samples in this embodiment contain known mutated HRR gene fragments, while the negative control samples do not contain the target mutation site. When performing joint analysis on multiple HRR gene mutation sites, the threshold for predicting significant benefit of olaparib treatment is a mutation allele frequency of at least one of the multiple gene sites of not less than 0.10%. Furthermore, plasma samples obtained from the same patient at different time points are tested to dynamically monitor changes in HRR gene mutation burden in order to evaluate the efficacy of olaparib or the occurrence of drug resistance.

[0116] In this embodiment, before amplification in step S4, the magnetic oligonucleotide probe of this embodiment is magnetically positioned on the side of each digital PCR reaction unit closest to the magnetic field source.

[0117] The assay system in this embodiment is used to predict or monitor the response of prostate cancer patients to olaparib treatment in vitro. In this embodiment, the prostate cancer patients are castration-resistant metastatic prostate cancer patients, and the patients are stratified according to whether the HRR gene mutation allele frequency reaches a preset threshold, so as to provide a reference for olaparib treatment decision-making.

[0118] Features of Example 3: This example employs a high level of parameter configuration, with 4.2 parts of iron oxide nanoparticles, 34 parts of styrene, a median microsphere diameter of 325 nm, a shell thickness of 62 nm, a surface carboxyl density of 4.0 μmol / m², and 140 μg of magnetic probe. These parameters are selected within the higher range of technical solutions. The magnetic microspheres prepared in this example have a larger particle size and higher surface carboxyl density, enabling higher oligonucleotide loading and stronger magnetic responsiveness. This makes them suitable for detecting tissue samples with high nucleic acid concentrations or complex scenarios requiring simultaneous detection of multiple genes, and particularly suitable for clinical applications requiring high-throughput detection, such as efficacy evaluation and drug resistance monitoring.

[0119] Example 4 This embodiment provides a system for determining olaparib response markers in prostate cancer, including magnetic oligonucleotide probes, dPCR amplification reaction components, and binding and washing buffers.

[0120] The magnetic oligonucleotide probe in this embodiment was obtained by a carbodiimide-mediated amidation coupling reaction between core-shell magnetic polymer microspheres and HRR gene oligonucleotide probes with a 5′ amino group. The HRR gene oligonucleotide probe in this embodiment was used to detect 11 homologous recombination repair-related genes: BRCA1, BRCA2, ATM, PALB2, BARD1, CDK12, CHEK1, CHEK2, RAD51B, RAD51C, and RAD51D.

[0121] The core-shell magnetic polymer microspheres in this embodiment were prepared through the following steps: Step A1 Raw material preparation: Weigh 4.6 parts of iron oxide nanoparticles. In this embodiment, the iron oxide nanoparticles are prepared by step A2. Weigh 37 parts of styrene, 7.2 parts of divinylbenzene, 4.5 parts of sodium dodecyl sulfate emulsifier, and 1.85 parts of potassium persulfate initiator.

[0122] Step A2: Preparation of iron oxide cores: Prepare an iron salt solution and carry out a co-precipitation reaction under alkaline conditions. Adjust the pH value to 10.2 and react at 84℃ for 1.75 hours to obtain iron oxide nanoparticles with a particle size of 138 nanometers.

[0123] Step A3: Coating polymerization: The iron oxide nanoparticles obtained in step A2 were ultrasonically dispersed in an aqueous phase containing sodium dodecyl sulfate. Styrene and divinylbenzene monomers were added, and emulsion polymerization was carried out at 83°C for 3.7 hours under nitrogen protection to obtain core-shell magnetic polymer microspheres with iron oxide as the core and cross-linked polystyrene as the shell. The median particle size of the obtained microspheres was 368 nm, and the shell thickness was 74 nm. Transmission electron microscopy showed a clear core-shell interface.

[0124] Step A4 Washing and Drying: The emulsion obtained in step A3 was magnetically separated, washed four times with deionized water, and dried at 56°C for 10.8 hours to obtain the core-shell magnetic polymer microspheres of this embodiment.

[0125] The surface functionalization of the core-shell magnetic polymer microspheres in this embodiment is carried out through the following steps to obtain functionalized core-shell magnetic polymer microspheres: Step B1 Dopamine Coating: The core-shell magnetic polymer microspheres of this embodiment are dispersed in a tris(hydroxymethyl)aminomethane buffer solution with a concentration of 178 mmol / L and a pH of 8.8. In this embodiment, the pH value is adjusted by sodium hydroxide. 9.2 parts of dopamine hydrochloride are added to 100 parts of core-shell magnetic polymer microspheres, and the mixture is stirred at 28°C for 2.75 hours to allow dopamine to self-polymerize and form a dopamine coating on the surface of the microspheres.

[0126] Step B2 Silane Coupling: 9.2 parts of 3-aminopropyltriethoxysilane were added to the product of step B1 and reacted at 56°C for 2.75 hours to introduce a surface aminosilane layer.

[0127] Step B3: Initiator introduction: The product of step B2 was reacted with 2-bromoisobutyryl bromide in anhydrous tetrahydrofuran in the presence of triethylamine, so that the initiator in this embodiment was amidated with the surface amino group; the amount of 2-bromoisobutyryl bromide was controlled to be 2.8 times the molar amount of 3-aminopropyltriethoxysilane, and 2-bromoisobutyryl bromide was added dropwise, and the reaction was carried out at 9°C for 1.85 hours to obtain functionalized core-shell magnetic polymer microspheres with α-bromoacyl initiator introduced on the surface.

[0128] The preparation of the magnetic oligonucleotide probe in this embodiment includes grafting a multifunctional polymer brush onto the surface of the functionalized core-shell magnetic polymer microspheres obtained in step B3 via surface-initiated atom transfer radical polymerization and introducing carboxyl groups onto their surface, specifically including: Step C1 Raw material preparation: Weigh 100 parts of functionalized core-shell magnetic polymer microspheres, use acrylic acid and polyethylene glycol methyl ether methacrylate as monomers, and use cuprous bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine as the catalytic system.

[0129] Step C2: Surface-initiated polymerization: In a mixed solvent of water and methanol at a volume ratio of 1:8, the product of step B3 was mixed with acrylic acid and polyethylene glycol methyl ether methacrylate. The molar ratio of acrylic acid to polyethylene glycol methyl ether methacrylate was controlled at 1:0.36, and the molar ratio of cuprous bromide to N,N,N′,N″,N″-pentamethyldiethylenetriamine was controlled at 1.0:2.8. In this embodiment, after vacuuming and deoxygenating with argon, the reaction was carried out at 33°C for 2.75 hours to form a multifunctional polymer brush layer containing carboxyl groups and polyethylene glycol side chains on the surface of the microspheres.

[0130] Step C3 post-treatment: Free monomers and catalyst residues were removed by magnetic separation, and the microspheres were washed four times alternately with deionized water and ethanol. They were then dried at 46°C for 11 hours to obtain multifunctional polymer brush magnetic microspheres with a surface carboxyl group density of 1.3 micromoles per square meter.

[0131] The magnetic oligonucleotide probe in this embodiment was obtained by condensing a multifunctional polymer brush magnetic microsphere with an HRR gene oligonucleotide probe through the following steps: Step D1 Activation: The multifunctional polymer brush magnetic microspheres were dispersed in a phosphate buffer solution with a pH of 5.1. In this embodiment, the concentration of the buffer solution was 178 mmol / L. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl groups was 4.6:1, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1.85:1. The reaction was carried out at 28°C for 0.92 hours to activate the surface carboxyl groups.

[0132] Step D2 Coupling: Add the HRR gene oligonucleotide probe with a primary amine at the 5′ end to the system of step D1. The HRR gene oligonucleotide probe in this embodiment is prepared by conventional oligonucleotide synthesis method. The molar ratio of the HRR gene oligonucleotide probe to the surface activated ester group in this embodiment is controlled to be 0.23:1. The reaction is carried out at 7°C for 14.8 hours to form a magnetic oligonucleotide probe linked by an amide bond.

[0133] Step D3 Purification: Free oligonucleotides and small molecules were removed by magnetic separation. The sample was washed four times with a high-salt buffer solution with a sodium chloride concentration of 0.35 mol / L and a pH of 6.3 and deionized water to obtain a magnetic oligonucleotide probe with an oligonucleotide loading of 0.6 nanomoles per milligram.

[0134] The HRR gene oligonucleotide probe of this embodiment contains a hybridization capture region complementary to the target nucleic acid. The dPCR amplification reaction components of this embodiment are configured to achieve a positive detection rate of not less than 95% for mutation sites with an allele frequency of 0.12% when using the assay system of this embodiment to detect HRR gene mutations. The positive detection rate of this embodiment is the percentage of positive results obtained in repeated tests out of the total number of repeated tests.

[0135] The dPCR amplification reaction components of this embodiment include thermostable DNA polymerase, deoxynucleoside triphosphate, magnesium-containing PCR buffer, amplification primer pairs for amplifying the HRR gene target nucleic acid of this embodiment, and fluorescence detection components.

[0136] The binding and washing buffer in this embodiment is an aqueous solution containing 178 mmol / L of tris(hydroxymethyl)aminomethane, 1.78 mmol / L of sodium chloride, 17.8 mmol / L of disodium ethylenediaminetetraacetate dihydrate, and 0.89% by mass of the nonionic surfactant Tween-20. The pH of the binding and washing buffer in this embodiment is 8.3, and it is adjusted to the target pH value using sodium hydroxide solution.

[0137] In a single 20 μL dPCR reaction system, the amount of magnetic oligonucleotide probe used in this embodiment is 184 μg. The dPCR amplification reaction in this embodiment is used to quantitatively detect the frequency of mutant alleles in the HRR gene and to predict the response of prostate cancer patients to olaparib treatment.

[0138] The method for determining olaparib response markers in prostate cancer in vitro using the assay system of this embodiment includes the following steps: Step S1 Nucleic acid extraction: Nucleic acid samples containing genomic DNA were obtained from ex vivo tissue specimens of the examined prostate cancer patients; in this embodiment, a lysis buffer containing guanidine thiocyanate and a surfactant was used to lyse the sample and remove proteins and lipids. The concentration of guanidine thiocyanate in the lysis buffer in this embodiment was 5.5 mol / L, and the surfactant in this embodiment was sodium dodecyl sulfate with a mass fraction of 4.525%.

[0139] Step S2: Enrichment: Mix the nucleic acid sample of this embodiment with the magnetic oligonucleotide probe of this embodiment in binding and washing buffer, and incubate at 35°C for 55 minutes to allow the HRR gene fragment to be tested to hybridize and bind with the magnetic oligonucleotide probe of this embodiment.

[0140] Step S3 Washing: Perform magnetic separation on the reaction system of step S2, discard the supernatant, and wash 5 times with the binding and washing buffer of this embodiment to remove unbound nucleic acids and impurities, and obtain the complex of enriched HRR gene target nucleic acid and magnetic oligonucleotide probe of this embodiment.

[0141] Step S4 dPCR amplification: The dPCR amplification reaction components of this embodiment are added to the complex obtained in step S3. The reaction mixture is divided into droplet-shaped micro-reaction units. The number of micro-reaction units formed in a single reaction in this embodiment is 184,000. Denaturation is performed at 97°C, annealing is performed at 64°C, and extension is performed at 71°C for 48 cycles to obtain the digital PCR amplification signal for the HRR gene of this embodiment.

[0142] Step S5 Data Analysis: Based on the distribution of positive and negative signals in each dPCR reaction unit, calculate the mutation allele frequency of each HRR gene locus, and determine the expected response of the tested patients to olaparib treatment according to the preset threshold.

[0143] This embodiment simultaneously tests positive and negative control samples for quality control. The positive control samples in this embodiment contain known mutated HRR gene fragments, while the negative control samples do not contain the target mutation site. When performing joint analysis on multiple HRR gene mutation sites, the threshold for predicting significant benefit of olaparib treatment is a mutation allele frequency of at least one of the multiple gene sites of not less than 0.10%. Furthermore, plasma samples obtained from the same patient at different time points are tested to dynamically monitor changes in HRR gene mutation burden in order to evaluate the efficacy of olaparib or the occurrence of drug resistance.

[0144] In this embodiment, during the amplification process in step S4, the magnetic oligonucleotide probe of this embodiment is magnetically positioned on the side of each digital PCR reaction unit closest to the magnetic field source.

[0145] The assay system in this embodiment is used to predict or monitor the response of prostate cancer patients to olaparib treatment in vitro. In this embodiment, the prostate cancer patients are castration-resistant metastatic prostate cancer patients, and the patients are stratified according to whether the HRR gene mutation allele frequency reaches a preset threshold, so as to provide a reference for olaparib treatment decision-making.

[0146] Features of Example 4: This example uses parameter configurations close to the upper limit of the technical solution range. The amount of iron oxide nanoparticles is 4.6 parts, styrene is 37 parts, divinylbenzene is 7.2 parts, sodium dodecyl sulfate is 4.5 parts, potassium persulfate is 1.85 parts, the median particle size of the microspheres is 368 nm, the shell thickness is 74 nm, the concentration of tris(hydroxymethyl)aminomethane is 178 mmol / L, the concentration of sodium chloride is 1.78 mol / L, and the amount of magnetic probe is 184 μg. The magnetic microspheres prepared in this example have a larger particle size and a thicker shell, which can provide stronger magnetic responsiveness and a larger carrier capacity. At the same time, the use of high buffer ionic strength and high probe amount makes it suitable for high-throughput clinical testing scenarios that require processing large numbers of samples or detecting multiple gene targets. It is particularly suitable for large-scale screening, multi-center clinical research, and personalized medication guidance in precision medicine.

[0147] Comparative Example 1: Basically the same as Example 1, except that the particle size of the iron oxide nanoparticles is 45 nanometers, and the amount of other components and preparation conditions remain unchanged.

[0148] Comparative Example 2: It is basically the same as Example 1, except that the median particle size of the core-shell magnetic polymer microspheres is 140 nm and the shell thickness is 17 nm. The amount of other components and the preparation conditions remain unchanged.

[0149] Comparative Example 3: It is basically the same as Example 1, except that the median particle size of the core-shell magnetic polymer microspheres is 415 nm and the shell thickness is 85 nm. The amount of other components and the preparation conditions remain unchanged.

[0150] Comparative Example 4: It is basically the same as Example 1, except that dopamine coating treatment was not performed in step B1, and the silane coupling reaction of step B2 was carried out directly on the surface of the core-shell magnetic polymer microspheres. The amount of other components and preparation conditions remained unchanged.

[0151] Comparative Example 5: It is basically the same as Example 1, except that polyethylene glycol methyl ether methacrylate was not added in step C2, and only acrylic acid was used as a monomer for surface-initiated polymerization. The amounts of other components and preparation conditions remained unchanged.

[0152] Comparative Example 6: It is basically the same as Example 1, except that the surface carboxyl group density of the multifunctional polymer brush magnetic microspheres is 0.8 micromoles per square meter, while the amount of other components and preparation conditions remain unchanged.

[0153] Comparative Example 7: Basically the same as Example 1, except that the surface carboxyl group density of the multifunctional polymer brush magnetic microspheres is 5.5 micromoles per square meter, while the amount of other components and preparation conditions remain unchanged.

[0154] Comparative Example 8: Basically the same as Example 1, except that the amount of magnetic oligonucleotide probe used in a single dPCR reaction system with a volume of 20 μL is 0.3 μg, while the amounts of other components and preparation conditions remain unchanged.

[0155] Performance testing: Experiment 1: Detection of allele frequencies of HRR gene mutations Test Subject: HRR gene target nucleic acids enriched with magnetic oligonucleotide probes. Test Objective: To evaluate the detection sensitivity and quantitative accuracy of the assay system for HRR gene mutations of different abundances. Test Principle: Target nucleic acids are separated into independent microreaction units using digital PCR. The frequency of mutant alleles is quantitatively calculated based on the Poisson distribution principle. Magnetic enrichment can increase the relative concentration of low-abundance mutations. Experimental Method: BRCA1 mutant standards with allele frequencies of 0.05%, 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, and 1.0% were prepared. Nucleic acid extraction, magnetic probe enrichment, washing, dPCR amplification, and data analysis were performed according to steps S1-S5. Each concentration was tested 10 times repeatedly. The number of positive microreaction units and the total number of units at each concentration were recorded, and the detection rate and quantitative error were calculated. Key Parameters: dPCR reaction system 20 µL, magnetic probe dosage 100 µg, incubation temperature 28.5°C, incubation time 32.5 min, PCR cycle number 42. Data processing: The positive detection rate is calculated as the percentage of positive detections out of the total number of repeated tests. The quantitative accuracy is evaluated by the relative deviation between the measured value and the theoretical value. The positive detection rate is required to be ≥95% and the relative deviation ≤15% within the range of 0.10%-0.50%.

[0156] Experiment 2: Colloidal Stability Test of Magnetic Microspheres Test Subjects: Core-shell magnetic polymer microspheres and multifunctional polymer brush magnetic microspheres. Test Objective: To evaluate the dispersion stability and anti-agglomeration performance of magnetic microspheres in binding wash buffer and dPCR amplification reaction components. Test Principle: Dynamic light scattering was used to monitor the particle size distribution changes of microspheres under different ionic intensities, pH values, and temperatures. Zeta potential was used to characterize surface charge stability, and transmission electron microscopy was used to observe the aggregation morphology. Experimental Methods: Microspheres were dispersed in buffer solutions containing 0.1-2.0 mol / L sodium chloride at pH 6.5-8.5 and incubated at 25°C and 95°C for 0 h, 2 h, 6 h, and 12 h, respectively. Particle size distribution (D10, D50, D90) and polydispersity index (PDI) were measured using a Malvern Zetasizer Nano ZS90 analyzer, and surface potential was measured using a Zeta potential analyzer. Key Parameters: Test temperature 25±2°C or 95±2°C, scattering angle 90°, sample concentration 0.1 mg / mL, and three measurements were taken for each sample, with the average value taken. Data processing: Calculate the rate of change of D50 and the increase of PDI under each condition. The evaluation criteria are that the increase of D50 within 12 hours is <20% and PDI <0.3 is considered stable.

[0157] Experiment 3: Determination of Oligonucleotide Loading and Capture Efficiency Test Subject: Magnetic oligonucleotide probe. Test Objective: To quantitatively characterize the oligonucleotide loading on the surface of magnetic microspheres and evaluate its capture efficiency and specificity for target nucleic acids. Test Principle: The loading is calculated by measuring the difference in oligonucleotide concentration in the supernatant before and after coupling using quantitative real-time PCR. The enrichment fold is assessed by comparing the Ct value changes of the target nucleic acid before and after enrichment using qPCR. Experimental Method: 1 mg of magnetic probe was used, and the absorbance of the supernatant before and after coupling was measured at 260 nm using a NanoDrop 2000 spectrophotometer to calculate the loading. A mixed nucleic acid sample containing 10³ copies / mL of BRCA2 target was prepared, enriched with the magnetic probe, and then quantified by qPCR. The unenriched sample was used as a control. The enrichment fold and recovery rate were calculated, and non-target sequences were detected to verify specificity. Key Parameters: Probe dosage 100 µg, target concentration 10³ copies / mL, incubation temperature 28.5°C, incubation time 32.5 min, qPCR cycle number 40. Data processing: Loading capacity = (concentration of oligonucleotides in supernatant before coupling - concentration after coupling) × reaction volume / mass of microspheres, enrichment factor = 2^(Ct control - Ct enrichment), recovery rate = copy number after enrichment / theoretical value × 100%.

[0158] Experiment 4: Detection of dPCR amplification efficiency and fluorescence signal uniformity Test Subject: dPCR reaction system containing magnetic oligonucleotide probes. Test Objective: To evaluate the effect of magnetic microspheres on thermostable DNA polymerase activity, PCR amplification efficiency, and fluorescence signal uniformity between microreaction units. Test Principle: Amplification efficiency is determined using the standard curve method, signal uniformity is assessed using a fluorescence intensity distribution histogram, and the positive / negative droplet / microwell separation is used to characterize the detection window. Experimental Method: Prepare a dPCR reaction system containing magnetic oligonucleotide probes. - A gradient of template samples (copies / mL) was amplified in dPCR systems containing 100 µg of magnetic probe and those without probe. The number of positive units at each concentration was recorded, and the amplification efficiency (E = (10 / 1) × 100%) was calculated by plotting log(template concentration) against log(number of positive units). The fluorescence intensity of 1000 positive and negative units was statistically analyzed, and a distribution histogram was plotted to calculate the signal-to-noise ratio (SNR) and resolution between positive and negative populations. Key parameter: template concentration gradient. - Copy / mL, dPCR cycle count 42, fluorescence detection channel FAM / HEX. Data processing: Amplification efficiency E=90-110% is acceptable, SNR≥5 and resolution≥3 is good, compare the differences with and without probe sets.

[0159] Experiment 5: The Influence of Magnetic Positioning on the Optical Performance of Microreactors Test Subject: Magnetic oligonucleotide probes magnetically localized in microchip wells or droplets. Test Objective: To evaluate the degree of chain aggregation during microsphere localization under magnetic field and its interference with the transmittance and fluorescence detection of microreaction units. Test Principle: The spatial distribution morphology of microspheres in the magnetic field is observed under a microscope, transmittance changes are measured using a densitometer, and the spatial uniformity of signal intensity is analyzed using fluorescence imaging. Experimental Method: dPCR reaction mixtures containing 100 µg of magnetic probes are separated into microchip wells (20,000 wells). The wells are incubated for 5 min under magnetic field strengths of 0 mT, 50 mT, 100 mT, and 150 mT. The distribution of microspheres is observed using an inverted fluorescence microscope (Nikon Ti2-E), and the chain aggregation length and coverage are recorded. The absorbance at 600 nm is measured using a microplate reader to assess the degree of light scattering. After amplification, the coefficient of variation (CV) of fluorescence intensity in positive wells under each magnetic field condition is calculated. Key Parameters: Magnetic field strength 0-150 mT, observation field ≥ 10, ≥ 500 wells per condition. Data processing: Chain formation rate = number of microspheres forming chain-like aggregates / total number of microspheres × 100%, transmittance = I sample / I blank × 100%, CV = standard deviation / mean × 100%, CV should be < 15%.

[0160] Experiment 6: Consistency between clinical sample validation and olaparib response prediction Test Subjects: Plasma and tissue samples from patients with castration-resistant metastatic prostate cancer. Test Objective: To validate the clinical accuracy of the assay system in detecting HRR gene mutations and evaluate its predictive ability for olaparib treatment response. Test Principle: Using clinically known olaparib treatment response as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of the assay system's results were compared. Experimental Methods: Sixty patients were included (30 in the response group and 30 in the non-responder group). Cell-free DNA from plasma and genomic DNA from tissues were extracted. The allele frequencies of 12 HRR gene mutations, including BRCA1 and BRCA2, were detected according to steps S1-S5, including nucleic acid extraction, magnetic probe enrichment, washing, dPCR amplification, and data analysis. A positive threshold of ≥1 gene site mutation frequency ≥0.10% was used. Cross-validation was performed with clinical response results (Objective Response Rate (ORR) and Progression-Free Survival (PFS) assessed according to RECIST 1.1 criteria). Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) were calculated. Key parameters: Sample size n=60, threshold 0.10%, follow-up period 12 months. Data processing: Sensitivity = true positive / (true positive + false negative), Specificity = true negative / (true negative + false positive), Accuracy = (true positive + true negative) / total number of cases, requiring sensitivity ≥85% and specificity ≥80%.

[0161] Figure 1 Box plots of shell thickness are shown for samples 1, 2, 3, 4, Comparative Example 2, and Comparative Example 3. The characterization method involves statistical analysis of shell thickness obtained through transmission electron microscopy or cross-sectional morphology measurement, and the distribution is displayed as a box plot. Fixed parameters included measurement aperture, sampling quantity, and the same statistical method; variable parameters included sample type. The shell thickness of Example 1 was concentrated in the approximately 50 nm range, Example 2 approximately 38 nm, Example 3 approximately 62 nm, and Example 4 approximately 74 nm, all showing adjustable and relatively convergent thickness ranges. Comparative Example 2 had a thinner shell thickness of approximately 17 nm, and Comparative Example 3 had a thicker shell thickness of approximately 85 nm with greater dispersion, indicating unstable shell construction or difficulty in precise positioning. These results demonstrate that the example systems can achieve controllable adjustment of shell thickness and maintain statistical consistency, which is beneficial for forming a stable core-shell structure.

[0162] Figure 2The images show EDS line scan curves for samples 1, 2, and 3. The characterization method involved obtaining the intensity distribution of Fe and C signals along normalized positions using energy dispersive spectroscopy (EDS) line scans and then normalizing and plotting the results. Fixed parameters included the line scan direction and normalization method, displayed on the same coordinate scale. Variations included sample type and corresponding core-shell structure differences. In Example 1, the Fe signal maintained high intensity in the central region and decreased rapidly on the outer edges, while the C signal conversely increased on the outer edges, exhibiting a clear spatial distribution of a Fe-rich core and a C-rich shell. Comparative Examples 2 and 3 showed wider Fe-C transition zones or abnormal core proportions, indicating insufficient interface transition or inadequate shell coverage. These results, based on elemental spatial distribution, demonstrate that Example 1 better conforms to the structural characteristics of core-shell separation, thus verifying the effectiveness of the core-shell construction scheme.

[0163] Figure 3 The images show a comparison of multi-peak fitting of XPS C1s high-resolution spectra. The samples are Example 1 and Comparative Example 5. The characterization method involved obtaining C1s spectra using X-ray photoelectron spectroscopy and performing peak fitting to obtain component curves for CC, CO, and OC, along with the overall fitted curve. Fixed parameters included the spectral normalization method, peak shape, and energy range; the variable parameter was the sample type. Example 1 showed a higher proportion of CO-related components and a smaller residual between the overall fitted spectrum and the original spectrum, indicating a more complete chemical environment for oxygen-containing functional groups or coupling. Comparative Example 5, dominated by CC components and with a significantly lower CO proportion, showed insufficient introduction of surface active sites or coupling. These results demonstrate that Example 1 exhibits a more pronounced difference in chemical bonding within the same fitting framework, supporting chemical evidence for the introduction of polymer brushes or coupling layers.

[0164] Figure 4 The graph shows the O / C atomic ratio as a function of sputtering depth. The samples are Example 1 and Comparative Example 5. The characterization method was XPS depth profiling to obtain the O / C atomic ratio at different sputtering depths, and the curves were plotted as a function of depth. The fixed parameters were the sputtering step, the atomic ratio calculation method, and the coordinate scale. The varying parameters were the sample type and the surface chemical composition. Example 1 exhibited a higher O / C ratio on the surface that gradually decreased with depth, indicating that the surface was rich in oxygen-containing functional groups and showed a gradient transition inwards. Comparative Example 5 had a lower overall O / C ratio with a smaller variation, suggesting insufficient surface modification layer or discontinuous coverage. These results demonstrate that Example 1 possesses more significant surface chemical differences and gradient characteristics, which helps explain its advantages in subsequent functionalization and stability.

[0165] Figure 5The bar chart shows the N 1s peak intensity for samples 1, 2, 3, 4, 4, 5, and 8 (Comparative Examples). The N 1s peak intensity was measured by XPS and normalized before comparison. The normalization and peak intensity extraction methods were kept constant, while the sample type was varied. The overall N 1s intensity of the Example group was significantly higher than that of the Comparative Example group, indicating that the Example system more effectively introduced nitrogen-containing groups or nitrogen source structural units. The N 1s intensities of Comparative Examples 4, 5, and 8 were lower, indicating insufficient introduction of the corresponding functional layers or poor stability. These results demonstrate, based on the strength of nitrogen-containing chemical characteristics, that the Example system more easily achieves the target functionalization, providing evidence for the chemical construction path of the proposed scheme.

[0166] Figure 6 The fluorescence intensity distribution histograms are for Examples 1 and 3. The characterization method involved obtaining fluorescence signals using confocal laser scanning microscopy and statistically analyzing the intensity by pixels or regions to form the distribution histogram. Fixed parameters included imaging conditions, statistical aperture, and binning rules; the variable parameter was the sample type. Example 1 exhibited a narrower intensity distribution and a lower CV (coefficient of variation) of approximately 8.5%, indicating a more uniform fluorescence signal and less background fluctuation. Comparative Example 3 showed a wider distribution and a higher CV of approximately 20.5%, reflecting enhanced spatial heterogeneity due to particle aggregation or signal inhomogeneity. These results demonstrate that Example 1 is superior in terms of optical readout consistency, thus supporting the correctness of the scheme in terms of optical compatibility and imaging stability.

[0167] Figure 7 The graph shows the aggregation index as a function of magnetic field strength. The samples used were Example 1, Comparative Example 1, and Comparative Example 3. The characterization method involved assessing the degree of particle aggregation under different magnetic field strengths and quantitatively plotting the aggregation index. Fixed parameters included the magnetic field application method, time, and the definition of the aggregation index. Variation parameters included magnetic field strength from 0 mT to 150 mT and sample type. In Example 1, the aggregation index increased moderately with increasing magnetic field strength and remained within a controllable range, demonstrating effective magnetic control without uncontrolled agglomeration. The smaller change in Comparative Example 1 indicated insufficient magnetic response. In Comparative Example 3, the sharp increase in the aggregation index under medium to high magnetic fields suggested a strong agglomeration tendency, which could lead to rheological deterioration and signal instability. These results demonstrate that Example 1 achieved a balance between magnetic response and dispersion stability, verifying that the scheme is more reasonable in terms of magnetic positioning and operational stability.

[0168] As can be seen from the performance of the examples and comparative examples in Table 1, the detection rates of all four examples under the condition of an allele frequency of 0.30% alleles reached over 95%, significantly better than all comparative examples, with Example 1 reaching 98.5%, proving that the optimized parameter range can achieve highly sensitive detection of ultra-low abundance mutations; Comparative Example 1, due to... The small core particle size (45 nm) resulted in insufficient magnetic response. Although the oligonucleotide loading was comparable to that of Example 1, the enrichment factor was only 245-fold and the colloidal stability was significantly reduced, with the detection rate dropping to 82.3%. Comparative Examples 2 and 3 suffered from deteriorated colloidal stability (D50 increased by more than 30% after 12 hours) due to the core-shell microsphere particle size deviating from the preferred range (140 nm and 415 nm, respectively). They were prone to aggregation under high-salt and high-temperature dPCR conditions, and the fluorescence signal variation coefficient increased to over 20%, with the detection rates dropping to 78.5% and 84.8%, respectively. Comparative Example 4 lacked a dopamine coating, resulting in unstable surface functionalization and an oligonucleotide loading of only 1.05%. The enrichment factor decreased to 165-fold with the lowest nmol / mg concentration, resulting in the worst colloidal stability (D50 increase of 45.8%), a detection rate of only 68.2%, and a clinically predicted sensitivity of 62.5%, demonstrating that the dopamine interlayer is crucial for constructing a stable interface structure. Comparative Example 5, while maintaining a certain loading level due to the absence of PEG side chains, experienced a decrease in amplification efficiency to 96% and a fluorescence signal CV of 24.2% due to increased non-specific adsorption and reduced dPCR compatibility, with a detection rate of 71.5%. Comparative Examples 6 and 7 exhibited surface carboxyl group densities deviating from the optimal range (0.8 and 5.5 µmol / m²), respectively. The former suffered from low enrichment efficiency due to insufficient loading (0.75 nmol / mg), while the latter experienced surface charge repulsion and steric hindrance due to excessive carboxyl group density, affecting dPCR polymerase activity (amplification efficiency decreased to 90%), with detection rates of 75.8% and 80.5%, respectively. Comparative Example 8 showed a severely insufficient magnetic probe quantity (0.3 nmol / mg). The enrichment fold was only 125-fold (µg). Although the colloidal stability and dPCR compatibility were good, the detection rate plummeted to 62.5% due to insufficient target enrichment, and the clinical predictive sensitivity was only 58.2%, demonstrating that the amount of magnetic probe is crucial for the detection of low-abundance mutations. Comprehensive analysis shows that this invention, through precise control of core-shell structure parameters, dopamine coating, PEG functionalization, surface carboxyl density, and magnetic probe dosage, successfully resolved the contradiction between high magnetic response, high capture efficiency, low non-specific adsorption, and good dPCR compatibility. It achieved a detection rate of ≥95% and a clinical predictive sensitivity of ≥85% within the 0.10%-0.50% allele frequency range, providing a reliable molecular diagnostic tool for the precision treatment of olaparib in prostate cancer patients.

[0169] Table 1 Performance Comparison Summary Table

[0170] 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 it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A system for determining olaparib response biomarkers in prostate cancer, characterized in that, include: A magnetic oligonucleotide probe, wherein the magnetic oligonucleotide probe is obtained by a carbodiimide-mediated amidation coupling reaction between core-shell magnetic polymer microspheres and HRR gene oligonucleotide probes with 5′ terminal amino groups, and the HRR gene oligonucleotide probe is used to detect at least 10 genes in homologous recombination repair-related genes, wherein the at least 10 genes include at least BRCA1, BRCA2, ATM, PALB2, and BARD1. The dPCR amplification reaction components include a thermostable DNA polymerase, deoxynucleoside triphosphate, magnesium-containing PCR buffer, amplification primer pairs for amplifying the target nucleic acid of the HRR gene, and a fluorescence detection component. The binding and washing buffer is an aqueous solution containing tris(hydroxymethyl)aminomethane, sodium chloride, disodium ethylenediaminetetraacetate dihydrate and a nonionic surfactant. In a single 20 µL dPCR reaction system, the amount of the magnetic oligonucleotide probe used is from 0.5 µg to 200 µg; the dPCR amplification reaction is used to quantitatively detect the frequency of mutated alleles in the HRR gene and to predict the response of prostate cancer patients to olaparib treatment.

2. The determination system according to claim 1, characterized in that, The core-shell magnetic polymer microspheres are prepared by the following steps: A1. Raw material preparation: 1.0 to 5.0 parts of iron oxide nanoparticles, which are obtained by step A2; 10 to 40 parts of styrene; 1 to 8 parts of divinylbenzene; sodium dodecyl sulfate as emulsifier, which is used in an amount of 0.1 to 5.0 parts; and potassium persulfate as initiator, which is used in an amount of 0.05 to 2.0 parts. A2. Preparation of iron oxide cores: Iron salt solution was co-precipitated under alkaline conditions, the pH was adjusted to 9.0 to 11.0, and the reaction was carried out at 70 ℃ to 90 ℃ for 0.5 h to 2.0 h to obtain iron oxide nanoparticles with a particle size of 50 nm to 150 nm. A3. Coating polymerization: The iron oxide nanoparticles obtained in step A2 are dispersed in an aqueous phase containing sodium dodecyl sulfate, styrene and divinylbenzene monomers are added, and emulsion polymerization is carried out at 70 ℃ to 85 ℃ for 2.0 h to 4.0 h to obtain core-shell magnetic polymer microspheres with iron oxide as the core and cross-linked polystyrene as the shell. The median particle size of the obtained microspheres is 150 nm to 400 nm, and the shell thickness is 20 nm to 80 nm. A4. Washing and drying: The emulsion obtained in step A3 is magnetically separated, washed 2 to 5 times with deionized water, and dried at 40 ℃ to 60 ℃ for 4 h to 12 h to obtain the core-shell magnetic polymer microspheres.

3. The determination system according to claim 1, characterized in that, The surface functionalization of the core-shell magnetic polymer microspheres is prepared through the following steps to obtain functionalized core-shell magnetic polymer microspheres: B1. Dopamine coating: The core-shell magnetic polymer microspheres are dispersed in a tris(hydroxymethyl)aminomethane buffer solution with a pH of 8.0 to 9.0, and 1.0 to 10.0 parts of dopamine hydrochloride are added, with 100 parts of core-shell magnetic polymer microspheres. The mixture is stirred and reacted at 20°C to 30°C for 1.0 h to 3.0 h to allow dopamine to self-polymerize and form a dopamine coating on the surface of the microspheres. B2. Silane coupling: 2.0 to 10.0 parts of 3-aminopropyltriethoxysilane are added to the product of step B1, and the mixture is reacted at 40 °C to 60 °C for 1.0 h to 3.0 h to introduce a surface aminosilane layer; B3. Initiator introduction: The product of step B2 is reacted with 2-bromoisobutyryl bromide in the presence of triethylamine to amidate the initiator with the surface amino group. The amount of 2-bromoisobutyryl bromide is controlled to be 1.0 to 3.0 times the molar amount of 3-aminopropyltriethoxysilane. The reaction is carried out at 0 °C to 10 °C for 0.5 h to 2.0 h to obtain functionalized core-shell magnetic polymer microspheres with α-bromoacyl initiator introduced on the surface.

4. The determination system according to claim 1, characterized in that, The preparation of the magnetic oligonucleotide probe includes grafting a multifunctional polymer brush onto the surface of the functionalized core-shell magnetic polymer microspheres obtained in step B3 via surface-initiated atom transfer radical polymerization and introducing carboxyl groups onto their surface, specifically including: C1. Raw material preparation: 100 parts of functionalized core-shell magnetic polymer microspheres, acrylic acid and polyethylene glycol methyl ether methacrylate as monomers, cuprous bromide and N,N,N′,N″,N″-pentamethyldiethylenetriamine as catalytic system; C2. Surface-initiated polymerization: In a mixed solvent of water and methanol, the product of step B3 is mixed with acrylic acid and polyethylene glycol methyl ether methacrylate, and the molar ratio of acrylic acid to polyethylene glycol methyl ether methacrylate is controlled to be 1:0.3 to 1:1.

0. The mixture is reacted at 20 °C to 35 °C for 0.5 h to 3.0 h to form a multifunctional polymer brush layer containing carboxyl groups and polyethylene glycol side chains on the surface of the microspheres. C3. Post-treatment: Free monomers and catalyst residues are removed by magnetic separation. The microspheres are washed 2 to 5 times with deionized water and ethanol alternately, and dried at 30 ℃ to 50 ℃ for 4 h to 12 h to obtain multifunctional polymer brush magnetic microspheres. The surface carboxyl group density of the obtained multifunctional polymer brush magnetic microspheres is 1.0 µmol / m² to 5.0 µmol / m².

5. The determination system according to claim 1, characterized in that, The magnetic oligonucleotide probe was obtained by condensing a multifunctional polymer brush magnetic microsphere with an HRR gene oligonucleotide probe through the following steps: D1. Activation: Multifunctional polymer brush magnetic microspheres were dispersed in a buffer solution with a pH of 5.0 to 6.5, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added, wherein the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to carboxyl groups was 2.0 to 5.0:1, and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide was 1.0 to 2.0:

1. The reaction was carried out at 15 ℃ to 30 ℃ for 0.25 h to 1.0 h to activate the surface carboxyl groups. D2. Coupling: An HRR gene oligonucleotide probe with a primary amine at the 5′ end is added to the system of step D1. The HRR gene oligonucleotide probe is prepared by conventional oligonucleotide synthesis method, and its nucleotide sequence is shown in SEQ ID NO:1 to SEQ ID NO:

14. The molar ratio of the HRR gene oligonucleotide probe to the surface-activated ester group is controlled to be 0.2 to 1.0:1, and the reaction is carried out at 4°C to 25°C for 2.0 h to 16.0 h to form an amide bond-linked magnetic oligonucleotide probe. D3. Purification: Free oligonucleotides and small molecules were removed by magnetic separation. The sample was washed 2 to 5 times with high-salt buffer and deionized water to obtain magnetic oligonucleotide probes with an oligonucleotide loading of 0.5 nmol to 5.0 nmol per mg of magnetic oligonucleotide probe.

6. The determination system according to claim 1, characterized in that, The HRR gene oligonucleotide probes include oligonucleotide probes targeting at least 10 of the following genes: BRCA1, BRCA2, ATM, PALB2, BARD1, CDK12, CHEK1, CHEK2, RAD51B, RAD51C, RAD51D, RAD54L, PPP2R2A, and FANCL; and the HRR gene oligonucleotide probes contain hybridization capture regions complementary to the target nucleic acids; the dPCR amplification reaction components are configured to achieve a positive detection rate of not less than 95% for mutation sites with an allele frequency of 0.10% to 0.50% when using the assay system to detect HRR gene mutations, wherein the positive detection rate is the percentage of positive results obtained in repeated tests out of the total number of repeated tests.

7. A method for determining olaparib response markers in prostate cancer in vitro using the assay system according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Nucleic acid extraction: Obtain nucleic acid samples containing genomic DNA or cell-free DNA from ex vivo plasma samples or ex vivo tissue specimens of patients with prostate cancer. S2. Binding enrichment: The nucleic acid sample and the magnetic oligonucleotide probe are mixed in binding and washing buffer and incubated at 20 ℃ to 37 ℃ for 5 min to 60 min to allow the HRR gene fragment to be tested to hybridize and bind with the magnetic oligonucleotide probe; S3. Washing: Perform magnetic separation on the reaction system of step S2, discard the supernatant, and wash 1 to 5 times with the binding and washing buffer to remove unbound nucleic acids and impurities, to obtain the enriched complex of HRR gene target nucleic acid and the magnetic oligonucleotide probe. S4. dPCR amplification: The dPCR amplification reaction components are added to the complex obtained in step S3, the reaction mixture is divided into multiple small reaction units, denatured at 90 °C to 98 °C, annealed at 55 °C to 65 °C, and extended at 60 °C to 72 °C, for 35 to 50 cycles to obtain a digital PCR amplification signal targeting the HRR gene; S5. Data Analysis: Based on the distribution of positive and negative signals in each dPCR reaction unit, calculate the mutation allele frequency of each HRR gene locus, and determine the expected response of the tested patients to olaparib treatment based on a preset threshold.

8. The method according to claim 7, characterized in that, The nucleic acid extraction in step S1 includes lysing the sample using a lysis buffer containing guanidine thiocyanate and a surfactant to remove proteins and lipids; the method includes simultaneously detecting positive and negative control samples for quality control, wherein the positive control sample contains a known mutated HRR gene fragment and the negative control sample does not contain the target mutation site; when performing joint analysis of multiple HRR gene mutation sites, a mutation allele frequency of at least one of the multiple gene sites of not less than 0.10% is used as the threshold for predicting that the patient will have a significant benefit from olaparib treatment; and, by testing plasma samples obtained from the same patient at different time points, changes in HRR gene mutation burden are dynamically monitored to evaluate the efficacy of olaparib or the occurrence of drug resistance.

9. The method according to claim 7, characterized in that, Before or during amplification in step S4, the magnetic oligonucleotide probe is magnetically positioned on the side of each digital PCR reaction unit closest to the magnetic field source.

10. The use of the assay system according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9 in in vitro prediction or monitoring of the response to olaparib treatment in prostate cancer patients, wherein, The prostate cancer patients were castration-resistant metastatic prostate cancer patients, and the patients were stratified according to whether the HRR gene mutation allele frequency reached a preset threshold, so as to provide a reference for the decision-making of olapapa.

Images