A system and method for detecting oxidized low density lipoprotein

Abstract

The present application relates to the technical field of biological detection, and particularly relates to a system and a method for detecting oxidized low density lipoprotein. The PLA-HCR metallization system provided by the present application constructs an "AND" logic gate based on a proximity effect, realizes direct recognition and signal amplification of oxLDL complete particles in combination with a hybridization chain reaction, and overcomes the limitation in traditional methods that only rely on indirect reflection of lipoprotein particle concentration between single components; the system uses gold-platinum bimetallic nanoparticles to endow oxLDL particles with high-efficiency exogenous peroxidase catalytic activity, and compared with traditional peroxidase, the enzyme catalytic efficiency is significantly improved, and the system has the ability to directly detect undiluted serum samples, avoiding the cumbersome pretreatment process of traditional ELISA methods. The present application has the characteristics of high specificity, wide detection range and simple operation, and has a wide application prospect in early diagnosis, curative effect monitoring and prognosis evaluation of patients with atherosclerotic cardiovascular disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a system and method for detecting oxidized low-density lipoprotein. BACKGROUND

[0002] Proximity Ligation Assay (PLA) is a special immunoassay method, which can be used to detect target proteins, protein interactions, etc. The method uses a pair of single (or multiple) clone antibody or DNA aptamer probes labeled with an oligonucleotide (single-stranded DNA), i.e. PLA probe, to recognize the target protein. When the two probes recognize the same target protein, the distance between the two probes is close, and the so-called proximity effect (proximity) is generated. At this time, by adding a connector oligonucleotide, the DNA on the PLA probe will be complementary to the connector oligonucleotide through base pairing, and the new DNA fragment is quantified by DNA amplification technology, so as to quantify the corresponding target protein.

[0003] Hybridization Chain Reaction (HCR) is an enzyme-free nucleic acid isothermal amplification technology, which can trigger a chain reaction through specific hybridization between DNA or RNA molecules to realize signal amplification. HCR technology is mainly based on two partially complementary DNA hairpin structures (H1 chain and H2 chain). First, the target nucleic acid sequence (initiation chain) is combined with the H1 chain to open the hairpin structure of the H1 chain, and the opened H1 chain exposes a new sequence. The new sequence can be combined with the H2 chain to open the hairpin structure of the H2 chain, and then expose the sequence complementary to the H1 chain, thereby triggering a continuous hybridization reaction. Through repeated hybridization, a long-chain nucleic acid structure is formed to realize signal amplification. HCR technology has wide application prospects in the fields of molecular diagnosis, biological imaging and nanotechnology due to its enzyme-free participation, high sensitivity and strong specificity.

[0004] Oxidized Low-Density Lipoprotein (ox-LDL) is a product formed by the oxidation modification of Low-Density Lipoprotein (LDL) in the body. LDL is a spherical lipoprotein particle composed of lipids and proteins, with a diameter of about 18-25 nm. The main components of its outer layer include a phospholipid layer that maintains particle stability, free cholesterol, and the key structural protein Apolipoprotein B-100 (ApoB-100), which is mainly responsible for transporting cholesterol from the liver to various tissues throughout the body. However, under certain conditions (such as oxidative stress, inflammation, smoking, and high-fat diet), LDL can be oxidized in the body, and its outer layer proteins are oxidized into oxidized Apolipoprotein B-100 (ox-ApoB-100), forming oxidized low-density lipoprotein (ox-LDL).

[0005] Atherosclerotic Cardiovascular Disease (ASCVD) has high morbidity and mortality. Ox-LDL is a product formed by the oxidation modification of Low-Density Lipoprotein (LDL), and has a stronger atherogenic effect than LDL. The level of ox-LDL is positively correlated with the severity of coronary artery disease, and its elevated level is an independent risk factor for ASCVD. In addition, high ox-LDL levels are also associated with increased atherosclerotic plaque volume and stability, as well as an increased incidence of adverse cardiovascular events such as reinfarction and heart failure. Therefore, ox-LDL has a stronger atherogenic effect and is considered a direct pathogenic risk factor for atherosclerotic cardiovascular disease. Therefore, accurate detection of ox-LDL levels is of great significance in predicting the risk of ASCVD, disease progression, and patient prognosis.

[0006] Aptamer is an oligonucleotide fragment composed of DNA or RNA (mainly DNA), usually obtained from a nucleic acid molecule library using in vitro screening technology—Systematic evolution of ligands by exponential enrichment (SELEX). Aptamer can bind to a variety of targets with high affinity and specificity, such as proteins, peptides, small molecules, metal ions, bacteria, viruses, and whole living cells, with sensitivity comparable to antigen-antibody reactions, easier synthesis, and better stability. Therefore, it is often used as a probe and widely applied in biomedical fields such as biomarker detection, diagnosis, and imaging.

[0007] Traditional detection methods of ox-LDL include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), mass spectrometry (MS) and the like. The above traditional technologies have certain deficiencies in the detection of ox-LDL. For example, the traditional methods generally use ox-LDL specific antibodies to recognize and detect the surface oxidized lipid components, and there is a problem that ox-LDL particle concentration cannot be directly detected; the detection sensitivity of ELISA is low, which affects the assessment of early disease risk; CLIA requires a large amount of samples; MS needs to use complex large-scale instruments and equipment.

[0008] At present, there is no standardization of the detection method of ox-LDL. Therefore, a high specificity method capable of expanding the detection range and directly detecting the concentration of ox-LDL itself is needed to further optimize the ox-LDL detection technology and promote the standardization process. SUMMARY

[0009] The purpose of the present application is to provide a system and a detection method for detecting oxidized low density lipoprotein to solve the problems existing in the prior art.

[0010] To achieve the above purpose, the present application provides the following solutions:

[0011] The present application provides a PLA-HCR metallization system for detecting oxidized low density lipoprotein, which comprises a PLA-HCR system and a metallization system; the PLA-HCR system comprises S1 chain, S2 chain, H1 chain and H2 chain; the nucleotide sequence of the S1 chain is shown in SEQ ID NO. 1; the nucleotide sequence of the S2 chain is shown in SEQ ID NO. 2; the nucleotide sequence of the H1 chain is shown in SEQ ID NO. 3 or SEQ ID NO. 4; the nucleotide sequence of the H2 chain is shown in SEQ ID NO. 5;

[0012] The metallization system comprises L-Proline, HAuCl4 solution, H2PtCl6 solution and ascorbic acid solution.

[0013] The present application provides the application of the above-mentioned PLA-HCR metallization system in the preparation of products for detecting oxidized low density lipoprotein.

[0014] Preferably, the product comprises a reagent, a kit, a chip or a biosensor.

[0015] The present application provides a product for detecting oxidized low density lipoprotein, which comprises the above-mentioned PLA-HCR metallization system.

[0016] Preferably, the product comprises a reagent, a kit, a chip or a biosensor.

[0017] The application provides application of the PLA-HCR metallization system or the product in detection of content of oxidized low density lipoprotein for non-diagnostic and therapeutic purposes.

[0018] The application provides a method for detecting content of oxidized low density lipoprotein based on proximity ligation HCR metallization for non-diagnostic and therapeutic purposes, comprising the following steps:

[0019] The sample to be detected and the PLA-HCR system are mixed, and then mixed with a metallization system to obtain a metallization solution; the PLA-HCR system comprises an S1 chain, an S2 chain, an H1 chain and an H2 chain; the nucleotide sequence of the S1 chain is shown as SEQ ID NO. 1; the nucleotide sequence of the S2 chain is shown as SEQ ID NO. 2; the nucleotide sequence of the H1 chain is shown as SEQ ID NO. 3 or SEQ ID NO. 4; the nucleotide sequence of the H2 chain is shown as SEQ ID NO. 5; the metallization system comprises L-Proline, a HAuCl4 solution, a H2PtCl6 solution and an ascorbic acid solution.

[0020] The metallization solution is detected.

[0021] Preferably, the final concentration of HAuCl4 in the HAuCl4 solution is 2 mM; and the final concentration of H2PtCl6 in the H2PtCl6 solution is 2 mM.

[0022] Preferably, in the metallization solution, the final concentration of L-Proline is 50 mM.

[0023] The application discloses the following technical effects:

[0024] 1. The PLA-HCR metallization system provided by the application realizes direct recognition and signal amplification of oxLDL complete particles by combining proximity effect and hybrid chain reaction, that is, the PLA-HCR metallization system provided by the application constructs an “AND” logic gate based on proximity effect, and realizes direct recognition and signal amplification of oxLDL complete particles in combination with hybrid chain reaction. Compared with a traditional method in which only a single component (apolipoprotein or cholesterol content) is used to indirectly reflect the concentration of ox-LDL particles, the PLA-HCR metallization system provided by the application constructs an “AND” logic gate based on proximity effect, and simultaneously recognizes two main components of ox-LDL particles by using double probes, so that the detection specificity is significantly improved. In addition, the HCR reaction greatly amplifies the metal ion binding sites, and further realizes signal amplification.

[0025] 2、The PLA-HCR metallization system provided by the application is based on a biomimetic metallization strategy, and converts oxLDL particles into a biomimetic metal complex with brand-new catalytic function. The system introduces gold-platinum bimetallic nanoparticles by using the high affinity of thiol groups for metal ions, endows the oxLDL particles with excellent exogenous peroxidase catalytic activity, and significantly improves the enzyme catalytic efficiency. Compared with the traditional ELISA method, the method has a wider detection range (0.01-10 μg / mL), a detection limit as low as 4.14 ng / mL, and can directly detect high concentrations of oxLDL in patient serum (has the ability to directly detect undiluted serum samples), without the need for pre-dilution treatment, avoiding the cumbersome pretreatment process of the traditional ELISA method, and has high specificity, wide detection range, simple operation and clinical application potential.

[0026] 3、The PLA-HCR metallization system provided by the application can distinguish ASCVD patients from healthy people by detecting the concentration of oxLDL particles, has actual diagnostic ability, and has important significance for the early detection and disease development monitoring of ASCVD. As can be seen, the PLA-HCR metallization system provided by the application has wide application prospects in the early diagnosis, efficacy monitoring and prognosis evaluation of patients with atherosclerotic cardiovascular disease. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0028] Figure 1 It is a schematic diagram of oxLDL specific recognition process based on proximity effect (PLA); B is a schematic diagram of HCR amplification reaction process; C is a schematic diagram of oxLDL metallization process based on PLA-HCR system;

[0029] Figure 2 It is a PLA system feasibility verification diagram (fluorescence spectrum, A) and a HCR system feasibility verification diagram (fluorescence intensity, B);

[0030] Figure 3 It is a PLA-HCR system feasibility verification diagram (PAGE gel electrophoresis);

[0031] Figure 4 It is a metallized ox-LDL peroxidase activity verification diagram;

[0032] Figure 5 Optimized graph for metal ion concentration ratio condition;

[0033] Figure 6 UV-Vis spectrum graph under ox-LDL concentration gradient;

[0034] Figure 7 Standard curve graph of ox-LDL concentration and absorbance;

[0035] Figure 8 Actual sample detection result graph. DETAILED DESCRIPTION

[0036] The detailed description set forth below of various illustrative embodiments explains the principles of the application and the best mode presently contemplated by the inventors for carrying out the application. It will be understood that the detailed description is merely meant to illustrate certain aspects of the application, and is not meant to limit the scope of the application.

[0037] It is to be understood that the terminology used in the instant specification is for the purpose of describing the particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values are based on the same run principle(s) as the recited values. For example, if a parameter is stated as having a range of values from 1 to 5, then it is intended that all stated intervening values of the parameter are also individually and specifically included within the scope of the present application. The same principle applies to ranges reciting an upper limit and a lower limit. In this disclosure and claims, the use of "or" means "and / or" unless stated otherwise. Furthermore, use of the term "including" as well as other forms such as "include", "includes" for claiming constancy indicates that the products or process steps are open-ended and do not exclude additional steps or components.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0039] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0040] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional steps or components.

[0041] The inventive concept is illustrated by Figure 1

[0042] ​In order to construct the ox-LDL particle direct detection system, the present application combines proximity ligation assay (PLA), hybridization chain reaction (HCR) and biomolecular template metallization to detect ox-LDL particles in samples with high specificity and high sensitivity.

[0043] The present application first designs a PLA-HCR system, which is a framework for subsequent ox-LDL metallization. The PLA-HCR system is mainly composed of two PLA probes (S1 chain and S2 chain) and two single-stranded hairpin loop oligonucleotide chains (H1 chain and H2 chain). Among them, the S1 chain includes three functional regions, namely: ox-LDL surface characteristic apolipoprotein aptamer region (Aptamer region, which is used for specific recognition of ox-LDL characteristic apolipoprotein ApoB-100), PLA complementary region and H1 chain partial complementary region; the S2 chain also includes three functional regions, namely: 3' end cholesterol region (used for recognizing the free cholesterol component on the surface of ox-LDL), PLA complementary region and H1 chain partial complementary region; the H1 chain and the H2 chain are both connected with thiol groups at both ends, which are subsequent metal ion binding sites.

[0044] When the target ox-LDL particle exists, the PLA double probe (S1 chain and S2 chain) recognizes the two main components at the same time. The Aptamer region of the S1 chain can specifically bind to the apolipoprotein on its surface, and at the same time the cholesterol region at the 3' end of the S2 chain can fuse with the cholesterol component on the surface of ox-LDL. At this time, the PLA double probe produces a proximity effect, and the PLA complementary regions of the S1 chain and the S2 chain are combined through base complementary pairing, and the exposed 3' end of the S1 chain and the H1 partial complementary region of the 5' end of the S2 chain together constitute a DNA single strand (Initiator, also known as I chain), which can bind to the H1 chain through base complementary pairing, open the hairpin structure of the H1 chain, thereby triggering the hybridization chain reaction of the H1 chain and the H2 chain, and in this process, a large number of metal binding sites are amplified, which builds a stable foundation for subsequent ox-LDL metallization and realizes signal amplification. When the target ox-LDL particle does not exist, the PLA double probe cannot trigger the proximity effect and combine with each other, so there is no opportunity to start the subsequent H1 chain and H2 chain hybridization chain reaction triggered by the I chain. Similarly, when only a single PLA probe exists or there is no complete ox-LDL particle, the HCR reaction cannot be triggered by the Initiator chain. The PLA-HCR system improves the specificity of the detection, making it possible to detect the concentration of ox-LDL particles itself, rather than just reflecting the particle concentration through part of the lipoprotein. In addition, the system realizes signal amplification through the HCR amplification reaction, further expanding the detection range of the method.

[0045] Further, due to the nanoscale size of ox-LDL particles, it is not conducive to the in-situ growth of metal nanomaterials on its membrane. Therefore, the application selects the high affinity of thiol groups to metal ions to introduce Au-Pt bimetallic nanoparticles with excellent catalytic activity, i.e. adding Au 3+ , Pt 4+ to the PLA-HCR system after its formation, so that it is combined with the metal ion attachment sites at both ends of the massively amplified H1 chain and H2 chain, forming an Au-Pt bimetallic nanoshell around the ox-LDL, endowing the ox-LDL particles with exogenous catalytic ability, and making them have high-efficiency peroxidase-like catalytic activity. Subsequently, the excess DNA chain, excess metal ions and non-specific interfering substances are removed by magnetic bead separation method. Finally, based on the catalytic H2O2-mediated 3,3',5,5'-tetramethylbenzidine (TMB) color reaction, the maximum absorption value of the color product at a specific wavelength is measured as the output detection signal by ultraviolet-visible spectroscopy, and the concentration of ox-LDL particles is accurately quantitatively detected.

[0046] In summary, the metalization system based on PLA-HCR proposed by the application has the following core advantages. 1. High specificity: based on the proximity effect, a double-probe recognition system is designed to construct an “AND” logic gate, which overcomes the limitations of traditional methods that rely on a single component to indirectly reflect the concentration of lipoprotein complete particles; 2. Wide detection range: the gold-platinum bimetallic particles endow the ox-LDL particles with high-efficiency exogenous peroxidase-like catalytic activity, which is significantly superior to traditional peroxidase (HRP), achieving a wider detection range and lower detection limit; 3. Simple operation and clinical applicability: the application can directly detect high-concentration intact ox-LDL particles in patient serum without dilution and pretreatment, avoiding the cumbersome pretreatment process of traditional ELISA methods. The application has important significance for the further optimization of ox-LDL detection technology and the promotion of its detection method standardization process, and has wide application prospects in the early diagnosis, efficacy monitoring and prognosis evaluation of patients with atherosclerotic cardiovascular disease.

[0047] The nucleotide sequences of the PLA probe (S1 chain, S2 chain), 2 single-stranded hairpin oligonucleotide chains (H1 chain, H2 chain), I chain (Initiator) and C chain (Connector) used in the application are as follows:

[0048] The nucleotide sequence of the S1 chain is shown in SEQ ID NO. 1, specifically as follows:

[0049] CTTCGATGTAGTTTTTGTATGGGGTGCCCTGGTTCCTGCAtttttttttttatagagcCGTCCTTCATTTT-Biotin; wherein, the underlined part represents the ox-LDL surface-specific apolipoprotein aptamer region, the lower case part represents the PLA complementary region, and the non-underlined capital part represents the H1 chain partial complementary region, and the 3' end is modified with Biotin.

[0050] The nucleotide sequence of the S2 chain is shown in SEQ ID NO. 2, specifically:

[0051] gaccctaagcatacat GCTCTATATTTTTTTTTTTTTTTTTTTTTTTTT -Cholesterol; wherein, Cholesterol represents the cholesterol region, the underlined part represents the PLA complementary region, and the lower case part represents the H1 chain partial complementary region.

[0052] The nucleotide sequence of the H1 chain is shown in SEQ ID NO. 3, specifically:

[0053] Dithiol-TTTATGAAGGACGATGTATGCTTAGGGTCGACTTCCATAGACCCTAAGCATACATTTT-Dithiol; wherein, the 5' end and the 3' end are both modified with Dithiol.

[0054] The nucleotide sequence of the 6-FAM-H1-BHQ1 chain is shown in SEQ ID NO. 4, specifically:

[0055] TTTATGAAGGACGA / iBHQ1dT / GTATGCTTAGGGTCGACTTCCATAGACCCTAAGCATACATTTT-6-FAM; wherein, the 15th base is modified with BHQ1, and the 3' end is modified with 6-FAM.

[0056] The nucleotide sequence of the H2 chain is shown in SEQ ID NO. 5, specifically:

[0057] Dithiol-TTTGACCCTAAGCATACATCGTCCTTCATATGTATGCTTAGGGTCTATGGAAGTCTTT-Dithiol; wherein, the 5' end and the 3' end are both modified with Dithiol.

[0058] The nucleotide sequence of the I chain (Initiator) is shown in SEQ ID NO. 6, specifically:

[0059] GACCCTAAGCATACATCGTCCTTCATTTTT.

[0060] The nucleotide sequence of the C chain (Connector) is shown as SEQ ID NO. 7, specifically:

[0061] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCAGGAACCAGGGCACCCCATACAAAAACTACATCGAAG.

[0062] The above sequences are synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0063] Example 1 Construction of PLA-HCR system

[0064] 1. Preparation of PLA probe (S1 chain, S2 chain) and DNA hairpin chain (H1 chain, H2 chain): First, S1 chain, S2 chain, H1 chain (SEQ ID NO. 3) and H2 chain were dissolved in TM buffer (20 mM Tris-HCl, 40 mM NaCl and 50 mM MgCl2, pH 7.4) to prepare a solution with a final concentration of 100 μM. Incubate at 95°C for 5 min, then slowly cool to room temperature to form a stable secondary structure, and store at -20°C.

[0065] 2. Connection of S1 chain and streptavidin magnetic beads (MB): The connection of S1 chain and streptavidin magnetic beads (MB) is mainly based on the strong binding force between streptavidin and biotin. Before use, the streptavidin magnetic beads (10 mg / mL, particle size 1 μM) were repeatedly blown and washed in washing buffer (20 mM Tris-HCl, 1 M NaCl, 1 mM EDTA and 0.05% Tween-20) for 3 times, then resuspended in 20 mM Tris-HCl buffer. Take 12 μL S1 chain (100 μM) and 80 μL streptavidin magnetic beads (10 mg / mL), dilute to 100 μL reaction system with Tris-HCl buffer, incubate at 37°C for 4 h, and mix gently, then wash the unbound DNA chain with washing buffer to obtain MB-S1 chain (SEQ ID NO. 1), store at 4°C. The purpose of this step is to connect the streptavidin magnetic beads for subsequent magnetic separation and remove interfering substances.

[0066] 3. Construction of PLA-HCR system: 100 μL of Tris-HCl solution (20 mM, pH 7.4) was added to each of the four reaction strands MB-S1 strand, S2 strand, H1 strand, and H2 strand with a concentration of 1.6 μM, to obtain the PLA-HCR system. Different concentrations of ox-LDL were then added to initiate the PLA-HCR reaction, and the mixture was incubated at 37 °C for 90 min with gentle mixing.

[0067] Example 2: A method for detecting metalized ox-LDL based on proximity ligation HCR

[0068] 1. Construction of PLA-HCR system: The steps were the same as in Example 1.

[0069] 2. Construction of metalization system: 64 μL of the PLA-HCR system constructed in step 1 was mixed with 20 μL of the sample to be tested, and the mixture was diluted with Tris-HCl solution (final concentration of 20 mM, pH = 7.4) to 200 μL. L-Proline (L-proline) with a final concentration of 50 mM was added as a chelating agent, and the mixture was mixed gently. Then, 20 μL of HAuCl4 solution (original concentration of 20 mM) and 20 μL of H2PtCl6 solution (original concentration of 20 mM) were added in sequence, and the mixture was incubated at 37 °C for 1.5 h with gentle shaking. Then, 4 μL of freshly prepared ascorbic acid solution (original concentration of 500 mM) was added, and the mixture was incubated at 37 °C for 2 h with gentle shaking for metal ion reduction. Finally, the resulting solution was separated from excess interfering substances by magnetic bead separation, and resuspended with 100 μL of Tris-HCl solution to obtain the metalized ox-LDL solution.

[0070] 3. Detection

[0071] (1) Polyacrylamide gel electrophoresis: 12% native polyacrylamide gel (native-PAGE) was prepared with 5xTBE buffer, and the electrophoresis sample was prepared by mixing 15 μL of the reaction solution containing 3 μL of 6xloading buffer. The gel was electrophoresed at a constant voltage of 100 V in 1xTBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) for 60-90 min, then stained with 1xGel Red nucleic acid dye, and imaged.

[0072] (2) Fluorescence spectrum and fluorescence intensity determination experiment: take different DNA strands (S1 strand, S2 strand, H1 strand (SEQ ID NO. 4), H2 strand and I strand) with a concentration of 100 nM and 500 ng / mL of standard ox-LDL solution to co-react at 37°C for 90 min, and the fluorescence spectrum and fluorescence intensity of the sample solution after the reaction is completed are determined by a fluorescence spectrophotometer (F4500, Hitachi), the excitation wavelength is 492 nm, and the fluorescence spectrum of the reaction system is determined in the emission range of 505 nm-700 nm. In the fluorescence test, the excitation and emission slits are both set to 5 nm, the PMT voltage is set to 800 V, and the fluorescence emission intensity is determined at 520 nm. The fluorescence spectrum is to verify the feasibility of the previous PLA-HCR system, and does not involve the metallization process, so the system is triggered by the standard oxLDL solution.

[0073] Example 3 PLA-HCR system for feasibility verification

[0074] To verify the feasibility of the method, the present application first verifies the feasibility of the PLA-HCR system, and the specific steps are as follows:

[0075] 1. PLA system feasibility verification

[0076] Different systems are constructed:

[0077] (1) S1+oxLDL+H1: the same as the steps in Example 1, the difference is that only S1 strand and H1 strand (SEQ ID NO. 4) are used to obtain the PLA-HCR system, and 500 ng / mL of ox-LDL is added to start the PLA-HCR reaction, and incubated at 37°C for 90 min and gently mixed. After that, the fluorescence spectrum experiment is carried out to determine the fluorescence intensity.

[0078] (2) S2+oxLDL+H1: the same as the steps in Example 1, the difference is that only S2 strand and H1 strand (SEQ ID NO. 4) are used to obtain the PLA-HCR system, and 500 ng / mL of ox-LDL is added to start the PLA-HCR reaction, and incubated at 37°C for 90 min and gently mixed. After that, the fluorescence spectrum experiment is carried out to determine the fluorescence intensity.

[0079] (3) S1+S2+oxLDL+H1: the same as the steps in Example 1, the difference is that only S1 strand, S2 strand and H1 strand (SEQ ID NO. 4) are used to obtain the PLA-HCR system, and 500 ng / mL of ox-LDL is added to start the PLA-HCR reaction, and incubated at 37°C for 90 min and gently mixed. After that, the fluorescence spectrum experiment is carried out to determine the fluorescence intensity.

[0080] This invention simultaneously modifies the H1 hairpin chain with a 6-FAM fluorescent group and a BHQ1 quencher group. In this case, the fluorescence on the H1 hairpin is quenched, and it cannot emit light. For example... Figure 2 As shown in Figure A, when only a single probe (S1 chain or S2 chain) recognizes ox-LDL, the H1 hairpin chain (H1 chain) will not be opened, and the H1 chain remains in a state of fluorescence quenching with extremely low fluorescence intensity. However, only when both S1 and S2 chains recognize ox-LDL simultaneously, the proximity effect reduces the distance between the S1 and S2 chains, leading to a local increase in concentration and complementary base binding. At this point, the tail ends of both S1 and S2 probes open the H1 hairpin chain, restoring the fluorescence of the H1 chain and significantly increasing the fluorescence intensity, which is significantly different from the fluorescence intensity when only a single probe recognizes ox-LDL. This result demonstrates that only when the S1 chain, linked to the ox-ApoB-100 aptamer, and the S2 chain, linked to cholesterol, simultaneously recognize the main components on the ox-LDL surface can the subsequent HCR amplification reaction be initiated. The proximity reaction (i.e., PLA) is feasible, ensuring the high specificity of this method for ox-LDL recognition.

[0081] 2. Feasibility verification of the HCR system

[0082] Build different systems:

[0083] (1) H1: The procedure is the same as in Example 1, except that only the H1 chain (SEQ ID NO.4) is used to obtain the PLA-HCR system. 500 ng / mL of ox-LDL is added to start the PLA-HCR reaction. The mixture is incubated at 37°C for 90 min and then gently mixed. Fluorescence spectroscopy experiments are then performed to measure the fluorescence intensity.

[0084] (2) H1+H2: The steps are the same as in Example 1, except that only the H1 chain (SEQ ID NO.4) and the H2 chain are used to obtain the PLA-HCR system. 500 ng / mL of ox-LDL is added to initiate the PLA-HCR reaction. The mixture is incubated at 37°C for 90 min and then gently mixed. Fluorescence spectroscopy experiments are then performed to measure the fluorescence intensity.

[0085] (3) I+H1: The steps are the same as in Example 1, except that only the I chain and H1 chain (SEQ ID NO.4) are used to obtain the PLA-HCR system. 500 ng / mL of ox-LDL is added to start the PLA-HCR reaction. The mixture is incubated at 37°C for 90 min and then gently mixed. Fluorescence spectroscopy experiments are then performed to measure the fluorescence intensity.

[0086] (4) I+H1+H2: same as the step of example 1, except that only I chain, H1 chain (SEQ ID NO. 4) and H2 chain are used to obtain PLA-HCR system, and ox-LDL with a concentration of 500 ng / mL is added to start the PLA-HCR reaction, and incubated at 37°C for 90 min with gentle mixing. Then the fluorescence spectrum experiment is carried out to measure the fluorescence intensity.

[0087] Subsequently, the fluorescence spectrum of the HCR amplification reaction system of the present application is also verified. Since the HCR reaction in the present method requires the tail end of S1 chain and S2 chain (i.e. the partially complementary region of H1 chain) to jointly initiate, the present application forms an HCR reaction initiator chain (I chain, Initiator) with the tail end of the two probes for the subsequent HCR reaction system verification. As shown in the fluorescence spectrum of B in the present application, the fluorescence intensity of 6-FAM-H1-BHQ1 chain is used to represent the intensity of HCR reaction, when the initiator chain (I chain) is absent, H1 chain and H2 chain hardly have HCR reaction; when I chain exists, H1 chain is opened, thereby opening H2 chain, and initiating the subsequent HCR reaction. The results prove that the HCR amplification reaction is feasible, and has a very low background signal and a high reaction efficiency. Figure 2

[0088] 3. PLA-HCR system feasibility verification

[0089] (1) S1: same as the step of example 1, except that only S1 chain is used, and incubated at 37°C for 90 min with gentle mixing. Then the PAGE gel electrophoresis experiment is carried out.

[0090] (2) S2: same as the step of example 1, except that only S2 chain is used, and incubated at 37°C for 90 min with gentle mixing. Then the PAGE gel electrophoresis experiment is carried out.

[0091] (3) H1: same as the step of example 1, except that only H1 chain (SEQ ID NO. 3) is used, and incubated at 37°C for 90 min with gentle mixing. Then the PAGE gel electrophoresis experiment is carried out.

[0092] (4) H2: same as the step of example 1, except that only H2 chain is used, and incubated at 37°C for 90 min with gentle mixing. Then the PAGE gel electrophoresis experiment is carried out.

[0093] (5) C: C chain with the same concentration as the DNA chain in the step of example 1 is used to replace ox-LDL to start the PLA-HCR reaction, and incubated at 37°C for 90 min with gentle mixing. Then the PAGE gel electrophoresis experiment is carried out.

[0094] ​(6) S1+S2: same as the step of Example 1, except that only S1 strand and S2 strand are used, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0095] (7) S1+C: same as the step of Example 1, except that only S1 strand and C strand are used, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0096] (8) S2+C: same as the step of Example 1, except that only S2 strand and C strand are used, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0097] (9) S1+S2+C: same as the step of Example 1, except that only S1 strand, S2 strand and C strand are used, to obtain PLA system, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0098] (10) H1+H2: same as the step of Example 1, except that only H1 strand (SEQ ID NO. 3) and H2 strand are used, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0099] (11) S1+S2+H1+C: same as the step of Example 1, except that only S1 strand, S2 strand, H1 strand (SEQ ID NO. 3) and C strand are used, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0100] (12) S1+S2+H1+H2+C: same as the step of Example 1, except that only S1 strand, S2 strand, H1 strand (SEQ ID NO. 3), H2 strand and C strand are used, to start PLA-HCR reaction, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0101] (13) S1+S2+H1+H2: same as the step of Example 1, except that only S1 strand, S2 strand, H1 strand (SEQ ID NO. 3) and H2 strand are used, incubated at 37°C for 90 min and mixed gently. Then PAGE gel electrophoresis experiment is carried out.

[0102] Finally, the PLA-HCR system is verified by PAGE gel experiment, and the application uses a connecting chain (C chain, Connector) which can simultaneously bind to S1 strand and S2 strand by base complementation to replace ox-LDL for verification. As shown in FIG. 1, the C chain is used to replace ox-LDL to verify the PLA-HCR system. Figure 3As shown, when the C chain is absent, S1 and S2 cannot bind (lane 6); when the C chain coexists with the S1 chain, a band with lower mobility than the S1 chain appears, proving that the S1 chain can complementarily bind with the C chain (lane 7); when the C chain coexists with the S2 chain, a band with lower mobility than the S2 chain appears, proving that the S2 chain can also complementarily bind with the C chain (lane 8), thus the C chain can play a role in simulating ox-LDL. When the C chain coexists with the S1 chain and the S2 chain, a band with lower mobility than the S1 chain+C chain (or the S2 chain+C chain) appears, proving the feasibility of the PLA system (lane 9), which is consistent with the result of A in Figure 2 In addition, when only the H1 chain coexists with the H2 chain (lane 10) or only the S1 chain, the S2 chain, the H1 chain and the H2 chain coexist (lane 13), the HCR reaction is hardly started, which is consistent with the result of B in Figure 2 When the C chain coexists, it can form the final PLA-HCR complex with the four chains (lane 12), which proves that the PLA-HCR overall system is feasible and has excellent specificity, greatly reducing the interference of background signals.

[0103] Example 4 Verification of ox-LDL peroxidase activity in metallization system

[0104] The present application verifies the ox-LDL peroxidase activity in the metallization system. The present application sets up a magnetic bead group, an unmetallized ox-LDL group as a control group of metallized ox-LDL, to exclude the influence of magnetic beads and compare the change of enzyme activity before and after ox-LDL metallization. The present application uses the classic TMB color reaction to verify the enzyme activity, and the absorbance of TMB color product represents the enzyme activity of each group.

[0105] Magnetic bead group (MB): Take 80 μL of streptavidin magnetic bead solution (10 mg / mL) and dilute to 100 μL with 20 mM Tris-HCl solution.

[0106] Unmetallized ox-LDL group (MB+DNA-anchored oxLDL): Same as the PLA-HCR system in Example 1.

[0107] Metallized ox-LDL group (MB+AuPt-metallized oxLDL): Same as the metallized ox-LDL solution in Example 2.

[0108] The peroxidase-like activity of oxLDL was verified by ultraviolet-visible spectroscopy. First, 2.5 μL of 1% TMB solution and 2.8 μL of 6% H2O2 solution were diluted to 50 μL in an acetic acid-sodium acetate buffer solution at pH=4, and 10 μL of each solution from the above three groups was added to the above solution, which was incubated at 37°C for 10 min to perform the redox reaction, and finally 50 μL of H2SO4 (2 M) was used to terminate the reaction. The absorption intensity of the colored product at 450 nm and the ultraviolet-visible spectroscopy in the wavelength range of 200 nm to 800 nm were measured using an ultraviolet spectrophotometer.

[0109] The results are shown in Figure 4 Fig. 4. The pure magnetic bead group and the non-metalized ox-LDL group both showed very weak absorbance, while the absorbance of the metalized ox-LDL was significantly increased. It is proved that the metalized ox-LDL has good enzyme activity, and the magnetic beads do not have significant interference with the metalized system.

[0110] Example 5: Optimization of metal ion concentration ratio conditions

[0111] Since the concentration ratio of metal ions has a great influence on the activity of metal nanoscale enzymes, the concentration ratio of bimetallic ions is optimized in the present application. Seven experimental groups of Au 3+ : Pt 4+ =4:1 to 1:4 are set, as follows:

[0112] Au 3+ : Pt 4+ =4:1: the detection method is the same as in Example 2, except that 20 μL of HAuCl4 solution (20 mM) and 5 μL of H2PtCl6 solution (20 mM) are sequentially added.

[0113] Au 3+ : Pt 4+ =3:1: the detection method is the same as in Example 2, except that 20 μL of HAuCl4 solution (20 mM) and 6.7 μL of H2PtCl6 solution (20 mM) are sequentially added.

[0114] Au 3+ : Pt 4+ =2:1: the detection method is the same as in Example 2, except that 20 μL of HAuCl4 solution (20 mM) and 10 μL of H2PtCl6 solution (20 mM) are sequentially added.

[0115] Au 3+ : Pt 4+=1:1: The detection method is the same as in Example 2, except that 20 μL of HAuCl4 solution (20 mM) and 20 μL of H2PtCl6 solution (20 mM) are added sequentially.

[0116] Au 3+ :Pt 4+ =1:2: The detection method is the same as in Example 2, except that 10 μL of HAuCl4 solution (20 mM) and 20 μL of H2PtCl6 solution (20 mM) are added sequentially.

[0117] Au 3+ :Pt 4+ =1:3: The detection method is the same as in Example 2, except that 6.7 μL of HAuCl4 solution (20 mM) and 20 μL of H2PtCl6 solution (20 mM) are added sequentially.

[0118] Au 3+ :Pt 4+ =1:4: The detection method is the same as in Example 2, except that 5 μL of HAuCl4 solution (20 mM) and 20 μL of H2PtCl6 solution (20 mM) are added sequentially.

[0119] like Figure 5 As shown, the peroxidase-like activity of metallized ox-LDL is best when the gold-platinum ion concentration ratio is 1:1. Therefore, this concentration ratio was selected for subsequent experiments.

[0120] Example 6: Verification of System Detection Performance

[0121] The detection method is the same as in Example 2, except that the final ox-LDL concentrations are 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL. Figure 6 and Figure 7 As shown, the absorbance of the chromogenic product at 450 nm increased with increasing ox-LDL concentration. Furthermore, within the range of 0.01-10 μg / mL, the detected absorbance showed a good linear relationship with the logarithmic concentration of ox-LDL, with a detection limit (LOD) of 4.14 ng / mL. The fitted linear equation was y = 0.80963x + 2.00638 (R²). 2 = 0.9906), where y is the absorbance intensity and x is the logarithm of the ox-LDL concentration. This result demonstrates that the proposed method can accurately detect the particle concentration of ox-LDL and exhibits higher specificity and a wider detection range compared to traditional ELISA methods, providing a new strategy for standardizing ox-LDL detection methods.

[0122] Example 7 Actual sample detection

[0123] Human serum samples (15 ASCVD patients, 12 healthy people) were collected, centrifuged at 3000 rpm for 10 min, and serum was collected. The detection method was the same as Example 2. As shown in Table 2, the oxLDL concentrations of the two groups had a significant difference (p<0.001), and the method could effectively distinguish the oxLDL content in the serum of ASCVD patients and healthy people, and had a certain actual diagnostic ability for ASCVD. Figure 8

[0124] The above-described examples are only to describe the preferred modes of the present application, and not to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.​

Claims

1. A PLA-HCR metallization system for detecting oxidized low-density lipoprotein, characterized in that, The PLA-HCR metallization system includes a PLA-HCR system and a metallization system; the PLA-HCR system includes an S1 chain, an S2 chain, an H1 chain, an H2 chain, and streptavidin magnetic beads; the nucleotide sequence of the S1 chain is shown in SEQ ID NO.1, wherein the 3' end is modified with biotin; the nucleotide sequence of the S2 chain is shown in SEQ ID NO.2, wherein the 3' end is modified with cholesterol, where cholesterol represents the cholesterol region; the nucleotide sequence of the H1 chain is shown in SEQ ID NO.3 or SEQ ID NO.4, wherein the 15th base of the nucleotide sequence described in SEQ ID NO.4 is modified with BHQ1, and the 3' end is modified with 6-FAM; the nucleotide sequence of the H2 chain is shown in SEQ ID NO.5, wherein both the 5' and 3' ends are modified with Dithiol; The metallization system includes L-Proline, HAuCl4 solution, H2PtCl6 solution and ascorbic acid solution.

2. The application of the PLA-HCR metallization system according to claim 1 in the preparation of products for detecting oxidized low-density lipoprotein.

3. The application according to claim 2, characterized in that, The products include reagents, kits, chips, or biosensors.

4. A product for detecting oxidized low-density lipoprotein, characterized in that, The product includes the PLA-HCR metallization system as described in claim 1.

5. The product according to claim 4, characterized in that, The products include reagents, kits, chips, or biosensors.

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