Washing-free fluorescence detection system for simultaneously detecting PCSK9 and ApoB proteins and application of washing-free fluorescence detection system

Abstract

The invention discloses a washing-free fluorescence detection system for simultaneously detecting PCSK9 and ApoB proteins and application of the washing-free fluorescence detection system. The washing-free fluorescence detection system provided by the invention can be used for quantitatively detecting trace PCSK9 protein and trace ApoB protein in serum at the same time, and has the advantages of strong specificity (no interference or interference by other proteins in serum), high sensitivity (low detection limit), accuracy and reliability (the linearity of a standard curve is excellent; the repeatability, the stability and the precision are excellent. The washing-free fluorescence detection system enables fluorescence of trace PCSK9 protein and trace ApoB protein to achieve similar visual effect and intensity value level in imaging equipment, and solves the problem of simultaneous determination of two proteins with large content difference. Therefore, the washing-free fluorescence detection system can be used for simultaneously detecting the contents of the PCSK9 protein and the ApoB protein in the serum in vitro so as to realize multi-dimension evaluation of the lipid-lowering drug effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to simultaneous quantification of two proteins, in particular to a no-wash fluorescent detection system for simultaneously detecting PCSK9 and ApoB proteins and application thereof. BACKGROUND

[0002] Atherosclerotic cardiovascular disease (ASCVD) is closely related to dyslipidemia, which seriously affects people's health. In recent years, the European Society of Cardiology / European Society of Atherosclerosis Guidelines and the European Society of Atherosclerosis and European Clinical Chemistry and Laboratory Medicine Alliance Consensus Report point out that: because the concentration of apolipoprotein B (ApoB) contains the quantity information of all atherogenic cholesterol particles, and the concentration of low-density lipoprotein cholesterol (LDL-C) cannot contain all particle information, compared with the traditional ASCVD gold standard LDL-C, ApoB is a more accurate and comprehensive ASCVD risk marker, which can be better used to evaluate the efficacy of lipid-lowering therapy. Proprotein convertase subtilisin 9 (PCSK9) is a revolutionary target in the field of lipid-lowering in recent years. Abnormal elevation of PCSK9 levels can cause accumulation or secretion of low-density lipoprotein, lipoprotein (a), and triglyceride-rich lipoprotein particles in plasma, and these atherogenic lipoprotein particles all contain ApoB. The marketed PCSK9 inhibitors have been confirmed to have a strong effect on reducing the expression level of the above atherogenic lipoprotein particles in various related studies, and this effect is believed to be related to the reduced ApoB level.

[0003] That is, there is a strong correlation between PCSK9 and ApoB. Combined detection of PCSK9 and ApoB is expected to achieve multi-dimensionality of lipid-lowering drug efficacy evaluation, thereby playing more benefits in practical occasions such as assisting new drug development.

[0004] However, the current quantitative method of PCSK9 mainly relies on enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemical method, etc., which faces the bottleneck of complicated operation, low sensitivity and easy introduction of human error, etc. New analysis methods such as electrochemical sensor and homogeneous chemiluminescence have improved in sensitivity and operability, but are limited in single component evaluation. The current detection of ApoB mainly relies on immunoturbidimetry, immunohistochemical method, Western blotting, ELISA, etc., which faces similar problems as PCSK9 detection, and single-flow process can only detect single component. In actual application, the traditional method needs to be treated and reagent kits to be used separately for double-component combined detection of the same serum sample.

[0005] In view of the complicated operation, time-consuming and labor-consuming of traditional quantitative methods for PCSK9 and ApoB, a new method for simultaneous detection of two components is urgently needed. Literature research shows that the concentration of PCSK9 in serum is low, while the content of ApoB is abundant. How to solve the problem of such a large difference in the content level of the two proteins is a technical difficulty in the unified processing and simultaneous detection of two components.

[0006] In order to solve the problems existing in the prior art, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a no-wash fluorescent detection system for simultaneous detection of PCSK9 and ApoB protein and its application.

[0008] The above-mentioned purpose of the present application is achieved by the following technical solutions:

[0009] A no-wash fluorescent detection system for simultaneous detection of PCSK9 and ApoB protein, comprising:

[0010] (1) a fluorescent probe HMN-Cy5@DNA5 responsive to PCSK9 protein

[0011] The fluorescent probe HMN-Cy5@DNA5 uses mesoporous hollow silica as a carrier, the pores and hollow cavities of the carrier are filled with fluorescent dye Cy5, and the surface of the carrier is coated with DNA5 which blocks the fluorescent dye Cy5 in the pores and hollow cavities;

[0012] (2) a fluorescent probe HMN-Cy2@DNA6 responsive to ApoB protein

[0013] The fluorescent probe HMN-Cy2@DNA6 uses mesoporous hollow silica as a carrier, the pores and hollow cavities of the carrier are filled with fluorescent dye Cy2, and the surface of the carrier is coated with DNA6 which blocks the fluorescent dye Cy2 in the pores and hollow cavities;

[0014] (3) a pair of DNA-labeled antibody complexes Ab1-DNA1 and Ab2-DNA2 for capturing PCSK9 protein

[0015] Ab1 and Ab2 in the DNA-labeled antibody complexes Ab1-DNA1 and Ab2-DNA2 are two antibodies that can bind to different sites on PCSK9 protein, and DNA1 and DNA2 are connected to Ab1 and Ab2 respectively, DNA1 and DNA2 contain complementary binding regions, and DNA1 and DNA2 also contain complementary binding regions for attracting DNA5 to separate from the surface of the carrier and release the fluorescent dye Cy5 blocked in the carrier.

[0016] (4) the pair of DNA-labeled antibody complexes Ab3-DNA3, Ab4-DNA4 that capture ApoB protein

[0017] The Ab3 and Ab4 in the DNA-labeled antibody complexes Ab3-DNA3, Ab4-DNA4 are two antibodies that can bind to different sites on the ApoB protein, and the DNA3 and DNA4 are respectively connected to the Ab3 and Ab4, and the DNA3 and DNA4 contain complementary binding regions, and the DNA3 and DNA6 and the DNA4 and DNA6 also respectively contain complementary binding regions that can attract the DNA6 to separate from the carrier surface and release the fluorescent dye Cy2 blocked in the carrier.

[0018] Further, the nucleotide sequences of the DNA1, DNA2 and DNA5 are shown in Sequence NO. 1, Sequence NO. 2 and Sequence NO. 5, respectively.

[0019] Further, the nucleotide sequences of the DNA3, DNA4 and DNA6 are shown in Sequence NO. 3, Sequence NO. 4 and Sequence NO. 6, respectively.

[0020] In the preparation of the fluorescent probe HMN-Cy5@DNA5, the mesoporous hollow silica and the fluorescent dye Cy5 are dispersed in a solution and stirred at room temperature for 20-24 h.

[0021] Preferably, 20-40 μM of the fluorescent dye Cy5 corresponds to 1 mg of the mesoporous hollow silica in the solution.

[0022] In the preparation of the fluorescent probe HMN-Cy2@DNA6, the mesoporous hollow silica and the fluorescent dye Cy2 are dispersed in a solution and stirred at room temperature for 20-24 h.

[0023] Preferably, 20-40 μM of the fluorescent dye Cy2 corresponds to 1 mg of the mesoporous hollow silica in the solution.

[0024] In the above-mentioned no-wash fluorescent detection system, the mass ratio of the fluorescent probe HMN-Cy5@DNA5 to the fluorescent probe HMN-Cy2@DNA6 is 1:3-3:1.

[0025] The application of the above-mentioned fluorescent detection system in the in vitro simultaneous detection of the contents of PCSK9 protein and ApoB protein in serum.

[0026] The application of the above-mentioned fluorescent detection system in the in vitro evaluation of the efficacy of PCSK9 inhibitors.

[0027] Advantages:

[0028] The skilled in the art know that it is very difficult to simultaneously detect the strongly correlated PCSK9 protein and ApoB protein in serum, the former being a trace component and the latter being a micro component. The present application provides a no-wash fluorescent detection system for simultaneously detecting PCSK9 and ApoB protein (the detection method based on the detection system is named PAFLIA), which can simultaneously quantitatively detect trace PCSK9 protein and micro ApoB protein, and has strong specificity (not interfering with each other and not being interfered by other proteins in serum), high sensitivity (low detection limit), and accuracy and reliability (excellent linearity of standard curve; excellent repeatability, stability and precision). The no-wash fluorescent detection system makes the fluorescence of trace PCSK9 protein and micro ApoB protein in serum reach similar visual effects and intensity value levels in the imaging equipment, solving the problem of simultaneous detection of two proteins with large content difference. Therefore, the no-wash fluorescent detection system can be used for simultaneously detecting the contents of PCSK9 protein and ApoB protein in serum in vitro to realize the multi-dimensionality of lipid-lowering efficacy evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Fig. 1A is a preparation flow of probes HMN-Cy2@DNA6 and HMN-Cy5@DNA5; Fig. 1B is a schematic diagram of no-wash fluorescent imaging for simultaneous detection of PCSK9 and ApoB double components; Fig. 1C is a principle of the method by which the target protein PCSK9 induces the generation of long-chain DNA, opens the corresponding HMN probe, and releases the fluorescent signal molecule, taking the presence of target protein PCSK9 in the detection system as an example;

[0030] Figure 2 Fig. 2A is a TEM image (a), a STEM image (b), an EDX spectrum (c), and EDX-mapping images (d-i) of HMN; Fig. 2B is a TEM image (a), a STEM image (b), an EDX spectrum (c), and EDX-mapping images (d-i) of HMN-Cy5;

[0031] Figure 3 Fig. 3A is a TEM image (a), a STEM image (b), an EDX spectrum (c), and EDX-mapping images (d-i) of HMN-Cy5; Fig. 3B is a TEM image (a), a STEM image (b), an EDX spectrum (c), and EDX-mapping images (d-i) of HMN-Cy5@DNA5;

[0032] Figure 4 Fig. 4A is a TEM image (a), a STEM image (b, c), an EDX spectrum (d), and EDX-mapping images (e-i) of HMN-Cy2@DNA6;

[0033] Figure 5 Fig. 5A is an optical thermal map of Cy2 under different excitation wavelengths; Fig. 5B is a best excitation and emission spectrum diagram of Cy2;

[0034] Figure 6 A is the optical thermograph of Cy5 under different excitation wavelengths; B is the optimal excitation and emission spectrum of Cy5;

[0035] Figure 7 A is the dye Cy2 feeding concentration optimization result in the synthesis process of probe HMN-Cy2@DNA6; B is the dye Cy5 feeding concentration optimization result in the synthesis process of probe HMN-Cy5@DNA5 (n = 3);

[0036] Figure 8 A is the fluorescence (FL) intensity of Cy2 after mixing with Cy5 compared with the FL intensity of Cy2 alone; B is the FL intensity of Cy5 after mixing with Cy2 compared with the FL intensity of Cy5 alone; C is the FL intensity of both after mixing with Cy5 and Cy2 over time (n = 3);

[0037] Figure 9 It is the fluorescence spectrum of double probes that do not interfere with each other and have different intensities, wherein B1 is double probe (DP), B2 is DP+ApoB+PCSK9, B3 is DP+Ab-DNAs, S1 is B3+ApoB, S2 is B3+PCSK9, S3 is B3+ApoB+PCSK9, and the excitation parameter is λex=480nm / 640nm (n = 3);

[0038] Figure 10 It is the confocal imaging photo and the corresponding FL intensity of green channel and red channel for the feasibility verification of two-component scheme, wherein B1 is double probe (DP), B2 is DP+ApoB+PCSK9, B3 is DP+Ab-DNAs, S1 is B3+ApoB, S2 is B3+PCSK9, S3 is B3+ApoB+PCSK9; the error bar represents the standard deviation of three parallel experiments each with three repeated measurements (n = 3 x 3), and compared with B1 group (blank group), the green channel FL intensity *** P < 0.0001, red channel FL intensity #### P < 0.0001;

[0039] Figure 11 A is the confocal FL imaging photo of PAFLIA double probe HMN-Cy2@DNA6:HMN-Cy5@DNA5 ratio optimization; B is the FL intensity of green channel and red channel; the error bar represents the standard deviation of three parallel samples each with three repeated measurements (n = 3 x 3);

[0040] Figure 12A is the confocal FL imaging photo of PAFLIA time optimization; B is the FL intensity-time curve of the green channel; C is the FL intensity-time curve of the red channel; error bars represent the standard deviation of three parallel samples measured three times respectively (n=3x3);

[0041] Figure 13 A is the confocal FL imaging photo of PAFLIA for determination of different concentrations of PCSK9&ApoB; B is the ApoB standard curve; C is the PCSK9 standard curve; error bars represent the standard deviation of three parallel samples measured three times respectively (n=3x3);

[0042] Figure 14 A is the confocal FL imaging photo; B is the FL intensity of the green channel; C is the FL intensity of the red channel; error bars represent the standard deviation of three parallel experiments measured three times respectively (n=3x3); compared with the Blank group, the green channel FL intensity *** P<0.0001, red channel FL intensity #### P<0.0001;

[0043] Figure 15 A is the confocal FL imaging photo for investigating the repeatability and precision of the scheme, B is the FL intensity of the green channel; C is the FL intensity of the red channel; the within-batch column chart represents the FL intensity of three parallel samples measured three times respectively (n=3x3), and the between-batch column chart represents the FL intensity of six parallel samples (n=6);

[0044] Figure 16 A is the confocal FL imaging photo of PAFLIA for determination of 50μg·mL -1 ApoB and 25ng·mL -1 PCSK9 on day 0, 14, 31; B is the corresponding FL intensity; error bars represent the standard deviation of three parallel samples measured three times respectively (n=3x3);

[0045] Figure 17 A is the confocal FL imaging photo of PAFLIA method for detecting the consistency of PCSK9 and ApoB levels in mouse serum samples compared with ELISA method;

[0046] Figure 18 A is the 3D scatter plot and mapping between serum ApoB, LDL-C, and PCSK9; B is the correlation analysis of serum LDL-C concentration and ApoB concentration; C is the correlation analysis of serum PCSK9 concentration and LDL-C concentration; D is the correlation analysis between serum PCSK9 concentration and ApoB concentration;

[0047] Figure 19For PAFLIA applied to normal (NC) group of mice, high-fat model (HM) group of mice, berberine (BBR) administration group of mice and pseudo protodioscin (MPD) administration group of mice, the double-component protein was detected simultaneously, A was the confocal FL imaging map; B and D were the corresponding FL intensity; C and E were the converted PCSK9 concentration and ApoB concentration; the error bar represented the standard deviation of six parallel samples (n = 6); compared with the NC group, **** P<0.0001, compared with the HM group, ## P<0.01, ### P<0.001, #### P<0.0001. DETAILED DESCRIPTION

[0048] The substantial content of the application will be specifically introduced below in combination with examples, but the protection scope of the application is not limited by this.

[0049] In the following examples, room temperature refers to normal temperature.

[0050] Example 1: Establishing a no-wash type fluorescence detection system and method for simultaneous detection of PCSK9 and ApoB double components (the detection method based on the detection system is named as PAFLIA)

[0051] I. Reagents and instruments

[0052] PCSK9 antibodies (Ab1, Ab2) were purchased from Shanghai Aibio Trading Co., Ltd., ApoB antibodies (Ab3, Ab4) were purchased from Beijing Bioss Biotechnology Co., Ltd., oligonucleotides (see Table 1), TE buffer, 20x PBS buffer (pH 7.2-7.6), dimethyl sulfoxide (DMSO), fluorescent dyes Cy2 and Cy5 (purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.), HMN (purchased from Jiangsu Xianfeng Nanometer Material Technology Co., Ltd., particle size 100 nm). The Cy2 and Cy5 stock solutions were prepared with PBS-DMSO buffer (1:1).

[0053] Talos F200X transmission electron microscope (FEI, USA); automatic specific surface and porosity analyzer 3Flex (Micromeritics, USA); Colibri ultraviolet spectrometer (Titertek Berkinthold, Germany); FLS1000 fluorescence spectrometer (Edinburgh Instruments, UK); laser confocal microscope FV3000 (Olympus, Japan); EnVision multi-mode microplate detection system (PerkinElmer, USA).

[0054] Table 1 Oligonucleotide sequences

[0055]

[0056] The binding regions between DNA5 and DNA1, DNA2 are AAC ATG TCG and CG ACA TGT T, ATG CAG TGG and CCA CTG CAT, respectively; the binding regions between DNA6 and DNA3, DNA4 are AGC CGA GTT and AAC TCG GCT, TTCGTA GGA and TC CTA CGA A, respectively; the binding region between DNA1 and DNA2 is CAA GAC and GTC TTG, and the binding region between DNA3 and DNA4 is CAG CAC and GT GCT G. SH in Table 1 represents sulfhydryl.

[0057] II. Preparation method

[0058] 1. Preparation of HMN-Cy2@DNA6 and HMN-Cy5@DNA5

[0059] 1 mg of HMN was uniformly dispersed into 900 μL of 1 × PBS buffer by ultrasonic treatment, and 100 μL of Cy2 (300 μM) was added into the suspension, which was then wrapped with tin foil for light protection. The mixture was placed on a magnetic stirrer at room temperature (RT) for 20-24 h, and the solution was observed to be light yellow and transparent (or nearly colorless).

[0060] The obtained HMN-Cy2 was centrifuged (5000 rpm, 5 min) to remove the excess unloaded Cy2, and then was uniformly dispersed into 1 mL of 1 × PBS buffer. 100 μM of DNA6 610 μL was directly added into the above suspension and uniformly dispersed, and the reaction was carried out by a rotary mixer at RT for 1 h. After the reaction, the HMN-Cy2@DNA6 was collected by centrifugation at 5000 rpm for 5 min, and was washed with ultrapure water to remove the excess unloaded or blocked Cy2 and DNA6. Finally, the obtained yellow precipitate was uniformly dispersed in 1 mL of 1 × PBS, and was stored at 4 °C for subsequent experiments.

[0061] The preparation process of HMN-Cy5@DNA5 was the same as that of HMN-Cy2@DNA6, and the product was blue in color. The preparation process of the probes HMN-Cy2@DNA6 and HMN-Cy5@DNA5 is shown in Figure 1 A.

[0062] 2. Synthesis of two pairs of DNA-labeled antibody complexes (i.e., Ab1-DNA1, Ab2-DNA2, Ab3-DNA3 and Ab4-DNA4) for detection of PCSK9 and ApoB

[0063] Take 25 μL anti-PCSK9 (1 mg·mL -1 ), anti-ApoB (1 mg·mL -1 ) and 1.7 μL Sulfo-SMCC (4.8 mg·mL -1 ) respectively, and uniformly disperse them into 30 μL PBS1 (55 mM phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.4) solution, and react for 2 h at RT. Purify by ultrafiltration with a 50 kD ultrafiltration tube at 10,000 rpm for 10 min, retain the material in the inner tube, and discard the waste liquid.

[0064] Take 16 μL 100 mM dithiothreitol (DTT) PBS1 solution and 12 μL 100 μM thiol DNA (DNA1-DNA4), and disperse them in 28 μL PBS1 solution for reduction. Reduce DNA1, DNA2, DNA3, and DNA4 respectively at room temperature, and after 1 h, purify by centrifugation at 10,000 rpm for 10 min using a 10 kD ultrafiltration tube, retain the material in the inner tube, and discard the waste liquid.

[0065] Match each group of antibodies with the corresponding thiol DNA, and uniformly disperse them into 50 μL PBS2 (55 mM phosphate, 150 mM NaCl, 5 mM EDTA, pH 7.4), and incubate overnight at 4°C. The next day, remove the unreacted DNA using a 50 kD ultrafiltration tube (10,000 rpm for 10 min), and store the obtained DNA-labeled antibody complex in a 4°C refrigerator.

[0066] 3. PCSK9&ApoB no-wash two-component fluorescence imaging method detection process

[0067] Take 10 μL 1 mg·mL -1 HMN-Cy5@DNA5, 10 μL HMN-Cy2@DNA6, 1 μL mixed solution containing 100 nM of each of the two pairs of antibodies DNA, 3 μL 1×PBS buffer, and 1 μL standard solution (or serum sample) containing different concentrations of PCSK9&ApoB, and mix them in the corresponding centrifuge tube, and perform immunoreaction at RT. After one-step incubation for 60 min, centrifuge at 5,000 rpm for 3 min, and aspirate the supernatant for fluorescence imaging detection.

[0068] The method can perform parallel experiments simultaneously using different centrifuge tubes, and 7.5 μL of the supernatant can be taken from each sample, spotted on a hydrophobic paper chip (2.5 μL per spot, and each sample is spotted in triplicate), and green and red fluorescence images are collected simultaneously by setting different excitation lights on a laser confocal microscope.

[0069] To verify the reliability and repeatability of the proposed wash-free dual-component fluorescent imaging method, the same sample was detected every 3x3 array as a field of view (each sample was detected in triplicate and each sample was repeated three times). Finally, the gray scale values of the Red and Green channels were read respectively by the image analysis software Image J, and the fluorescence point map in the sensor array was converted into specific intensity numerical values.

[0070] The total volume of the imaging detection system was 25 μL. When detecting the fluorescence spectrum, each component was proportionally enlarged in volume, and the total volume was enlarged to 500 μL.

[0071] The schematic diagram of PCSK9&ApoB dual-component simultaneous detection fluorescent imaging is shown in Figure 1 B. The two pairs of antibody-labeled DNA of PCSK9 and ApoB were mixed with HMN-Cy2@DNA6 and HMN-Cy5@DNA5 in a homogeneous solution. When no target protein was present, the fluorescence signal was off; only when the target protein PCSK9 or ApoB was present, the specific DNA sequence could be formed to open the corresponding probe, triggering the release of the corresponding fluorescent signal molecule from the HMN, thereby turning on the respective corresponding fluorescence signal. The higher the content of PCSK9 or / and ApoB in the system, the more Cy5 or / and Cy2 released, and the stronger the supernatant signal. According to the fluorescence intensity, the quantification of PCSK9 or / and ApoB was realized. Figure 1 C in the presence of target protein PCSK9 in the detection system, the principle of the method is shown, which generates long-chain DNA after target protein induction, opens the corresponding HMN probe, and releases the fluorescent signal molecule.

[0072] III. Morphological characterization of HMN probe

[0073] The transmission electron microscopy (TEM) image of the HMN shows that the HMN is highly uniform monodisperse spherical, with a diameter of about 100 nm, and has a hollow structure, with a cavity diameter of about 70 nm Figure 2 a in the middle); the high-angle annular dark-field scanning transmission (STEM) image shows that the HMN sphere surface has mesoporous scattering Figure 2 b, the red arrow indicates); the energy dispersive X-ray spectroscopy (EDX) results show that the HMN contains C, O, Si, and N elements, indicating that there are amino groups in the HMN channel Figure 2 c-i in the middle).

[0074] The TEM image of HMN-Cy5 is similar to that of HMN, which is highly uniform hollow spherical, but the surface visibility of its STEM image is slightly lower than that of HMN, and the EDX element analysis results show that a new element S is added Figure 3 A in the middle), which corresponds to the element composition of Cy5 (C 33 H 40The presence of N2O8S2 indicates that Cy5 fills the pores in HMN.

[0075] After DNA5 was adsorbed onto the HMN-Cy5 surface, the characterization results are as follows: Figure 3 As shown in Figure B, EDX elemental analysis of HMN-Cy5@DNA5 added phosphorus (P) to the existing analysis, and the surface visibility of the material was further reduced, indicating successful DNA5 encapsulation. Furthermore, the presence of organic matter in the sample makes it susceptible to damage from the electron beam during mapping analysis, and the increased scanning time leads to the accumulation of carbon (C) elements, which also indirectly demonstrates the successful loading of the dye and DNA.

[0076] The characterization results of probe HMN-Cy2@DNA6 were similar to those of HMN-Cy5@DNA5, with TEM and STEM images ( Figure 4 The results showed that the surface mesopore visibility of DNA-coated HMN-Cy2@DNA6 decreased compared to unloaded HMN, and the addition of P element in EDX elemental analysis indicated that DNA6 was successfully coated on the HMN surface.

[0077] Example 2: Optimization of a wash-free fluorescence method for simultaneous detection of PCSK9 and ApoB in two components

[0078] 1. Investigation of the optical properties of dyes Cy2 & Cy5

[0079] By setting excitation light of different wavelengths, the optimal excitation and emission conditions for Cy2 and Cy5 were investigated, such as... Figure 5 and Figure 6 As shown, the fluorescence emission of Cy2 at different wavelengths indicates that Cy2 has the strongest fluorescence intensity under 480 nm excitation light; the fluorescence emission of Cy5 at different wavelengths indicates that Cy5 has the strongest fluorescence intensity under 640 nm excitation light.

[0080] 2. Optimization of Cy2 and Cy5 concentrations during probe preparation

[0081] During probe preparation, the higher the concentration of fluorescent dye, the more likely the background noise will be too strong during detection. Therefore, the concentrations of dyes Cy2 and Cy5 during the preparation of probes HMN-Cy2@DNA6 and HMN-Cy5@DNA5 were optimized.

[0082] Taking HMN-Cy2@DNA6 as an example, different concentrations of Cy2 solutions were mixed with 1 mg·mL⁻¹. -1 HMN was perfused at a 1:9 volume ratio to synthesize HMN-Cy2@DNA6 at different feed concentrations. The resulting probe was then administered at 1 mg / mL. -1The concentration of each probe solution was uniformly dispersed in 1 mL of PBS buffer. Each probe solution was divided in half, and TE buffer (background group) and C6 (the completely complementary strand of DNA6, signal group) were added to each half respectively for incubation. After complete reaction, the supernatant was centrifuged, and the fluorescence (FL) intensity was detected and compared. Results are as follows: Figure 7 As shown in Figure A, when the dye Cy2 concentration was 300 μM, the probe HMN-Cy2@DNA6 exhibited high fluorescence intensity and signal-to-noise ratio during detection. Therefore, 300 μM was chosen as the Cy2 concentration in the preparation of HMN-Cy2@DNA6.

[0083] The concentration of Cy5 added during the preparation of HMN-Cy5@DNA5 was optimized using a similar scheme, and the results are as follows: Figure 7 As shown in Figure B, when the dye Cy5 concentration was 300 μM, the probe HMN-Cy5@DNA5 exhibited high fluorescence intensity and signal-to-noise ratio during detection. Therefore, 300 μM was chosen as the Cy5 concentration in the preparation of HMN-Cy5@DNA5.

[0084] 3. Verification that the two dyes do not interfere with each other and have different intensities

[0085] The study investigated whether mixing dyes Cy2 and Cy5 interfered with each other's optical stability (e.g., leading to a decrease in fluorescence intensity). The results are as follows: Figure 8 As shown, the fluorescence intensity fluctuation of the mixture of Cy2 and Cy5 was negligible within 2 hours, indicating that the optical properties of the mixture were stable. Furthermore, it was found that at the same concentration (2 μM), Cy5 had a higher intensity than Cy2. Given the low concentration of PCSK9 in serum and the high abundance of ApoB, Cy5 was specifically used to design a probe signal molecule for detecting trace amounts of PCSK9, while Cy2 was used to design a probe signal molecule for detecting trace amounts of ApoB.

[0086] 4. Feasibility verification of two-component detection (signal differentiation and tuning)

[0087] At 20 μg·mL -1 ApoB and 10 ng / mL -1 Taking PCSK9 as an example, the feasibility of the scheme for simultaneous detection of two components was verified using a fluorescence spectrometer. Figure 9 The results showed that under excitation at 480 nm (left side of the coordinate axis), there was only background noise when the target protein ApoB was absent, and a significant signal appeared when only the target protein ApoB was present. The signal curves of ApoB alone and the mixture of PCSK9 and ApoB were basically fitted, indicating that the fluorescence intensity of ApoB was consistent in the single-target system and the dual-target system. A similar phenomenon was observed when PCSK9 was added under excitation at 640 nm (right side of the coordinate axis). These results demonstrate that ApoB and PCSK9 do not interfere with each other during detection, and their signals can be distinguished.

[0088] Figure 9 It can also be seen that although ApoB content is high and PCSK9 content is low, the specific ApoB response probe (HMN-Cy2@DNA6) filled with Cy2 used pulls down the fluorescence intensity of ApoB, while the specific PCSK9 response probe (HMN-Cy5@DNA5) filled with Cy5 pulls up the fluorescence intensity of PCSK9. After tuning, the signal of trace PCSK9 can be about an order of magnitude stronger than the signal of trace ApoB in the spectrometer.

[0089] 20μg·mL -1 ApoB and 10ng·mL -1 PCSK9, the feasibility of the two-component simultaneous detection scheme is verified using a confocal microscope. As shown in Figure 10 , this scheme only produces red and green fluorescent spots (Merge image shows yellow fluorescent spots) when both target proteins PCSK9 (Channel_R) and ApoB (Channel_G) are present in the system; only green fluorescent spots when only ApoB is present in the system; only red fluorescent spots when only PCSK9 is present in the system; and no obvious fluorescent spots when no target protein is present in the system. Figure 10 It is also shown that, after tuning, the signal of trace PCSK9 has a similar visual effect as the signal of trace ApoB in the confocal imaging device.

[0090] The statistical analysis results of fluorescence imaging further verify that trace PCSK9 and trace ApoB do not interfere with each other's quantitative analysis in the same homogeneous system Figure 10 (column chart), i.e., each homogeneous system responds to and only responds to its own target protein. The above results show that the scheme has high feasibility and is expected to meet the dual-protein simultaneous quantification of actual samples under unified processing.

[0091] 5. Optimization of the probe ratio of PAFLIA

[0092] 25ng·mL -1 PCSK9 (Channel_R) and 50μg·mL -1 ApoB (Channel_G), the ratio of HMN probes is optimized. 1μL of a two-protein mixed standard solution, 1μL of 100nM of each of the two pairs of antibodies DNA, 3μL of PBS, and 20μL of different ratios of HMN-Cy5@DNA5 and HMN-Cy2@DNA6 are mixed. The immunoreaction is carried out under RT conditions, and after one-step incubation for 60min, centrifugation is performed at 5000rpm for 3min, and the supernatant is aspirated for fluorescence detection.

[0093] AsFigure 11 As shown, when the ratio of HMN-Cy2@DNA6 to HMN-Cy5@DNA5 is 3:1 (probe mass ratio), the green light in the confocal imaging device is brighter; when the ratio of HMN-Cy2@DNA6 to HMN-Cy5@DNA5 is 1:3 (probe mass ratio), the red light in the confocal imaging device is brighter; and when the ratio of HMN-Cy2@DNA6 to HMN-Cy5@DNA5 is 1:1 (probe mass ratio), the red and green light in the confocal imaging device have similar visual effects and similar fluorescence intensity values, indicating that under this probe ratio, the signal for dual-component detection can be optimally tuned.

[0094] 6. Optimization of PAFLIA immune response incubation time

[0095] The incubation time of the antibody-antigen reaction is an important parameter affecting the analytical performance of the method. 1 μL of a 50 μg / mL solution is used. -1 ApoB (Channel G) and 25 ng / mL -1 The PCSK9 (Channel R) mixed standard solution was uniformly dispersed into 24 μL of detection solution, which contained 10 μg HMN-Cy5@DNA5, 10 μg HMN-Cy2@DNA6, and 25 fmol of Ab1-DNA1, Ab2-DNA2, Ab3-DNA3, and Ab4-DNA4. After incubation at room temperature for different times, samples were centrifuged and subjected to laser confocal imaging. The results are as follows: Figure 12 As shown, the FL intensity of the green and red channels gradually increases with incubation time, reaching its maximum at 60 min. Therefore, the optimal incubation time for this method is determined to be 60 min.

[0096] 7. Fluorescence standard curves of PCSK9 & ApoB

[0097] The performance of the PAFLIA method for imaging detection was evaluated under optimal detection conditions. For example... Figure 13 As shown in Figure A, S1 to S7 correspond to 5, 10, 20, 50, 100, 150, and 200 μg·mL, respectively. -1 ApoB standards and in concentrations of 1, 5, 10, 25, 50, 75, and 100 ng / mL -1 For the PCSK9 standard, as the concentrations of PCSK9 (Channel R) and ApoB (Channel G) increase, the FL intensity of the red and green channels in the confocal imaging images increases in a concentration-dependent manner, which is consistent with the sandwich immunoassay principle of this method. Linear fitting revealed a good quantitative relationship between the FL intensity of the green and red channels in the image and the logarithms of the concentrations of ApoB and PCSK9 standards.

[0098] like Figure 13 As shown in Figure B, the linear range of ApoB is 5–200 μg·mL. -1 The linear equation is: y = 120.5logx - 72.72, R0 2 =0.9992, and the detection limit was calculated to be 4.08 μg·mL by adding 3 times the standard deviation to the blank signal. -1 PCSK9 linear range ( Figure 13 The concentration of C is 1–100 ng·mL. -1 The linear equation is: y = 85.9logx + 26.63, R 2 =0.9996, detection limit is 0.71 ng·mL -1 This method, compared to commercially available ELISA kits (ApoB detection limit: 21.2 μg / mL), offers a significant advantage. -1 PCSK9 detection limit: 1.2 ng / mL -1 It exhibits higher sensitivity and meets the need for simultaneous detection of multiple target proteins.

[0099] 8. Selection, repeatability and stability of the PAFLIA method

[0100] The PAFLIA method was used to analyze the serum levels of common interfering substances TC, TG, LDL-C, and HDL-C in a hyperlipidemic mouse model, assessing whether their presence affected the detection of ApoB (Channel G) and PCSK9 (Channel R), and examining the method's selectivity. Figure 14 As shown, the samples were: blank solution (Blank) and 100 μg / mL solution. -1 TC, 100 μg·mL -1 TG, 100 μg·mL -1 LDL-C, 100 μg·mL -1 HDL-C, 10 μg·mL -1 ApoB, 5 ng / mL -1 PCSK9 and mixtures include PCSK9, ApoB, TC, TG, LDL-C, and HDL-C. High concentrations of interfering components do not affect 10 μg / mL. -1 ApoB and 5 ng / mL -1 The imaging results for PCSK9 were affected. This result confirms that the proposed two-component fluorescence imaging analysis method only responds to the targets ApoB and PCSK9.

[0101] In addition, the repeatability and precision of FL imaging for single-batch and 6-batch parallel samples were investigated: 50 μg·mL -1 ApoB and 25 ng / mL -1The FL response of PCSK9 (n = 6) was obtained, and the results are shown in Figure 15 Figure 6, with the green channel FL intensity having an intra-batch precision RSD = 2.9% and an inter-batch precision RSD = 3.9%, and the red channel FL intensity having an intra-batch precision RSD = 3.4% and an inter-batch precision RSD = 3.8%. The above results show that the method has good repeatability and precision.

[0102] Under the confocal imaging microscope, the double-component fluorescence information of up to 9 samples can be photographed at the same time in a single field of view, and the detection of 6 parallel fields of view can be completed at the same time, which can be realized by simply moving the microscope stage. Considering that the immunization incubation time needs 1 h, the detection flux of the method is calculated to be 54 tests h -1 If combined with automatic spotting technology, the upper limit of the detection flux of the method can be further broken through.

[0103] The prepared HMN-Cy2@DNA6, HMN-Cy5@DNA5 probes and Ab1-DNA1, Ab2-DNA2, Ab3-DNA3 and Ab4-DNA4 were stored at 4°C when not in use, and repeated experiments were performed at 0 days, 14 days and 31 days, respectively, to investigate the stability of the method. The results are shown in Figure 16 Figure 7, after 31 days, the PAFLIA method for determining 25 ng mL -1 PCSK9 (R) and 50 μg mL -1 ApoB (G) respectively retained 95.55% and 90.02% of the initial fluorescence response, proving that the method has satisfactory stability. In summary, PAFLIA exhibits good analytical performance, laying a solid foundation for subsequent practical applications.

[0104] Example 3: Drug efficacy evaluation of the no-wash fluorescence method for simultaneous detection of PCSK9 and ApoB double components

[0105] I. Experimental materials

[0106] Mouse PCSK9 ELISA kit, mouse ApoB ELISA kit, commercial ELISA detection, berberine, methyl protodioscin.

[0107] 6-week-old male C57BL / 6J mice (body weight 18-21 grams, SPF level), purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. (Experimental Animal Production License No.: SCXK (Zhejiang) 2019-0004).

[0108] II. Experimental methods

[0109] 1. Animal feeding

[0110] Forty-eight 6-week-old male C57BL / 6J mice (body weight 18-21 g, SPF level) were used in this study. All mice were housed in an environment with a temperature controlled at 25 ± 2 °C, humidity constant at 60%, and a light / dark cycle of 12 h / 12 h, and free access to food and water. The experiment was carried out according to the protocol approved by the Institutional Animal Ethics Committee of China Pharmaceutical University (SYXK2024-01-006). In addition, all experimental animal procedures were carried out in accordance with the guidelines outlined in the Guidelines for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China.

[0111] 2. Mouse modeling and grouping

[0112] All animals were fed with normal feed one week before the experiment. After the adaptation period, the mice were randomly divided into 4 groups: ① control group (NC) (n = 6); ② high-fat model group (HM) (n = 6); ③ berberine administration group (BBR) (n = 6); ④ methyl protodioscin administration group (MPD) (n = 6). The control group of mice was fed with normal diet, and the other three groups of mice were fed with high-fat diet, and drug management was carried out synchronously, as shown in Table 2.

[0113] Table 2. Mouse grouping and administration

[0114]

[0115] 3. Sample processing

[0116] After 28 days, 24 mice were fasted overnight and euthanized. Blood samples from each group of mice were centrifuged at 3500 rpm for 10 min at 4 °C to obtain the corresponding serum samples, which were promptly stored in a -80 °C freezer for “efficacy evaluation application research”. Mouse serum was detected for PCSK9 and ApoB concentrations by PAFLIA.

[0117] The other 24 mice were maintained on the original regimen and continued to be fed, and after 56 days of administration, they were fasted overnight and euthanized. Blood samples from each group of mice were centrifuged at 3500 rpm for 10 min at 4 °C to obtain the corresponding serum samples, which were promptly stored in a -80 °C freezer for “consistency verification and correlation analysis experiment”. The PCSK9 and ApoB concentrations in the serum of mice were detected by PAFLIA and commercial kits, respectively.

[0118] III. Data statistics

[0119] Data were analyzed by Origin, GraphPad Prism and SPSS software. Data results were expressed as mean ± standard deviation (mean ± SD). Independent samples between two groups were analyzed by independent sample t test, differences among three or more groups were analyzed by one-way ANOVA and multiple comparison test, two paired samples were analyzed by paired sample t test. P < 0.05 was considered statistically significant.

[0120] IV. Experimental results

[0121] 1. Application of PAFLIA in two-component detection of mouse serum samples

[0122] In order to verify the reliability of the method for detecting serum PCSK9&ApoB levels, 24 mouse serum samples with longer modeling time (56 days) and larger theoretical content difference were selected for PCSK9&ApoB detection. The commercial ELISA detection method was used as a reference to verify the reliability of the method for detecting serum PCSK9&ApoB levels.

[0123] The results are shown in Table 3, Table 4, and Table 5. Figure 17 The results are shown in Table 3, Table 4, and Table 5.

[0124] Table 3 ELISA-PAFLIA detection of PCSK9&ApoB levels in the same batch of serum samples

[0125]

[0126] Table 4 ELISA-PAFLIA detection of PCSK9&ApoB levels in the same batch of serum samples

[0127]

[0128] 2. Application of PAFLIA in ApoB, PCSK9, LDL-C correlation analysis

[0129] Statistical correlation analysis of serum ApoB, PCSK9, and LDL-C levels revealed significant pairwise correlations between ApoB and LDL-C (r = 0.7160, P < 0.0001), PCSK9 and LDL-C (r = 0.6268, P = 0.0010), and PCSK9 and ApoB (r = 0.8019, P < 0.0001). Figure 18 This indicates that PCSK9 can serve as a biomarker for monitoring the efficacy of lipid-lowering drugs, and that ApoB can also serve as a biomarker for monitoring the efficacy of PCSK9 inhibitors. Furthermore, by comparing the correlation coefficients between PCSK9 and LDL-C (r = 0.6268) and between PCSK9 and ApoB (r = 0.8019), it was found that ApoB has a stronger correlation with PCSK9 than LDL-C, and can serve as a better indicator for predicting the efficacy of PCSK9 inhibitors.

[0130] 3. Application of PAFLIA in the efficacy evaluation of PCSK9 inhibitors

[0131] Taking berberine (BBR) and methyl prodiosgenin (MPD), two positively charged PCSK9 inhibitors, as examples, the established wash-free PCSK9 & ApoB PAFLIA method was used for efficacy evaluation. Twenty-four mouse serum samples with short modeling time (28 days) and theoretically small content differences were selected. Figure 19 As shown, the red, green, and yellow fluorescence of the high-fat model group (HM), normal group (NC), BBR-treated group, and MPD-treated group can be effectively distinguished. Statistical data show that compared with the NC group, PCSK9 and ApoB levels in the HM group increased by 355.81% and 101.02%, respectively; compared with the HM group, PCSK9 and ApoB levels in the BBR group decreased by 43.89% and 30.40%, respectively, and PCSK9 and ApoB levels in the MPD group decreased by 50.53% and 53.84%, respectively, indicating that the method can be applied to the pharmacodynamic feedback of PCSK9 inhibitors.

[0132] Using the established homogeneous fluorescence two-component imaging detection method, we first demonstrated that ApoB is more associated with PCSK9 than LDL-C. Subsequently, we investigated the effect of the method on the serum PCSK9 and ApoB expression levels of hyperlipidemic mice in each group, evaluated the PCSK9-lowering efficacy of PCSK9 inhibitors (taking positive control drugs berberine and methyl diosgenin as examples), and simultaneously reported the lipid-lowering (ApoB-lowering) efficacy.

[0133] In summary, the application discloses a no-wash fluorescent detection system for simultaneously detecting PCSK9 and ApoB proteins and application thereof. The no-wash PCSK9&ApoB double-component simultaneous detection method established by the application realizes simultaneous quantitative detection of trace PCSK9 and trace ApoB, and has application potential in the field of PCSK9 inhibitor drug efficacy evaluation. In addition, the method has the advantages of one-step reaction, simultaneous detection of double components with different abundances, no washing, simple operation, good selectivity, high stability, low sample consumption and the like, and provides strong assistance for drug discovery and life analysis.

[0134] The above examples serve to specifically introduce the essential content of the application, but those skilled in the art should know that the protection scope of the application should not be limited to the specific examples.

Claims

1. A no-wash fluorescent detection system for simultaneous detection of PCSK9 and ApoB proteins, characterized in that, Comprise: (1) fluorescent probe HMN-Cy5@DNA5 responding to PCSK9 protein The fluorescent probe HMN-Cy5@DNA5 takes mesoporous hollow silica as a carrier, the pores and hollow cavities of the carrier are filled with fluorescent dye Cy5, and the surface of the carrier is coated with DNA5 which blocks the fluorescent dye Cy5 in the pores and hollow cavities; (2) fluorescent probe HMN-Cy2@DNA6 responding to ApoB protein The fluorescent probe HMN-Cy2@DNA6 takes mesoporous hollow silica as a carrier, the pores and hollow cavities of the carrier are filled with fluorescent dye Cy2, and the surface of the carrier is coated with DNA6 which blocks the fluorescent dye Cy2 in the pores and hollow cavities; (3) paired DNA-labeled antibody complexes Ab1-DNA1 and Ab2-DNA2 capturing PCSK9 protein Ab1 and Ab2 in the DNA-labeled antibody complexes Ab1-DNA1 and Ab2-DNA2 are two antibodies that can bind to different sites on PCSK9 protein, DNA1 and DNA2 are connected to Ab1 and Ab2 respectively, and DNA1 and DNA2 contain complementary binding regions, and DNA1 and DNA5 and DNA2 and DNA5 also contain complementary binding regions that can attract DNA5 away from the surface of the carrier and release the fluorescent dye Cy5 blocked in the carrier; (4) paired DNA-labeled antibody complexes Ab3-DNA3 and Ab4-DNA4 capturing ApoB protein Ab3 and Ab4 in the DNA-labeled antibody complexes Ab3-DNA3 and Ab4-DNA4 are two antibodies that can bind to different sites on ApoB protein, DNA3 and DNA4 are connected to Ab3 and Ab4 respectively, and DNA3 and DNA4 contain complementary binding regions, and DNA3 and DNA6 and DNA4 and DNA6 also contain complementary binding regions that can attract DNA6 away from the surface of the carrier and release the fluorescent dye Cy2 blocked in the carrier.

2. The no rinse fluorescent detection system of claim 1, wherein, The nucleotide sequences of DNA1, DNA2 and DNA5 are shown in Sequence NO.1, Sequence NO.2 and Sequence NO.5 respectively.

3. The no rinse fluorescent detection system of claim 1, wherein, The nucleotide sequences of DNA3, DNA4 and DNA6 are shown in Sequence NO.3, Sequence NO.4 and Sequence NO.6 respectively.

4. The no-wait fluorescent detection system according to any one of claims 1 to 3, characterized in that: When preparing the fluorescent probe HMN-Cy5@DNA5, mesoporous hollow silica and fluorescent dye Cy5 are dispersed in a solution and stirred at room temperature for 20-24 hours.

5. The no rinse fluorescent detection system of claim 4, wherein: The solution contains 20-40 μM of fluorescent dye Cy5 per milligram of mesoporous hollow silica.

6. The no-wait fluorescent detection system according to any one of claims 1 to 3, wherein: When preparing the fluorescent probe HMN-Cy2@DNA6, mesoporous hollow silica and fluorescent dye Cy2 are dispersed in a solvent and stirred at room temperature for 20-24 hours.

7. The no-wait fluorescent detection system of claim 6, wherein: The solution contains 20-40 μM of fluorescent dye Cy2 per milligram of mesoporous hollow silica.

8. The no-wait fluorescent detection system according to any one of claims 1 to 3, wherein: The mass ratio of the fluorescent probe HMN-Cy5@DNA5 to the fluorescent probe HMN-Cy2@DNA6 is 1:3-3:

1.

9. The application of the fluorescent detection system according to any one of claims 1-8 to simultaneously detect the contents of PCSK9 protein and ApoB protein in serum in vitro.

10. The application of the fluorescent detection system according to any one of claims 1-8 to evaluate the efficacy of a PCSK9 inhibitor in vitro.

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